**Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship**

Daniela Pedicino, Ada Francesca Giglio, Vincenzo Alessandro Galiffa, Francesco Trotta and Giovanna Liuzzo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50611

## **1. Introduction**

44 Pathophysiology and Complications of Diabetes Mellitus

2005; 76:suppl.3:59-62.

diabetes mellitus. Science 1978; 200: 21.

Philadelphia: Saunders 1999.p 798-801.

[80] Diabetic Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications

[81] Bookchin RM, Gallop PM. Structure of hemoglobin A,c: Nature of the N-terminal ,8

[82] Gallop, P. M., and M. A. Paz. Posttranslational protein modifications, with special

[83] Bunn HF, Haney DM, Kamnin S, et al. The biosynthesis of hemoglobin: A Slow

[84] Lorenza calisti, Simona tognetti. Measure of glycosylated hemoglobin. Acta Biomed

[85] Bunn HF, Gabbay KH, Gallop PM. The glycosylation of hemoglobin: relevance to

[86] Brownlee M, Cerami A, Vlassara H. Advanced products glycosylation and the pathogenesis of diabetic vascular disease. Diabetes/Metabolism Reviews 1988; 4: 437-51. [87] David BS, Burtis AC, Ashwood RE editors. Tietz text book of clinical chemistry 3rd ed:

[88] Agarwal S, Sandeep KB, Anuradha R, Vasudha K, Chadha KH, Doli P, et al.

[89] Salah R, Saleh Ben Hamed, Pajica Pavkovic, Zeljko Metelko. Microalbuminuria and

[90] Little RR, et al. The National glycoemoglobin standardization program: a five year

[91] Goldstein DE, Peth SB, England JD, Hess RL, Da Costa J. Effects of acute changes in

[92] Chandrashekar M , Sultanpur , Deepa K , S.Vijay Kumar. Comprehensive review on

[93] Amin A, Nanji, Morris R, Pudek. Glycosylated hemoglobins: A review.Can Fam

in insulin dependent diabetes mellitus. N. Engl J Med 1993; 329:977-986.

chain blocking group. Biochem Biophys Res Commun 1968; 32:86-93.

glycosylation of hemoglobin in vivo. J Clin Invest 1976; 57: 1652-1659.

attention to collagen and elastin. Physiol. Rev 1975; 55:418-487.

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blood glucose on glycated hemoglobin. Diabetes 1980; 29:623-8.

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Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 1997, 2003)

## **2. Epidemiology**

Diabetes is one of the most common chronic diseases in the world. It is thought that more than 360 million persons will be affected by this disease in 2030 (Wild et al., 2004). Prevalence of diabetes is higher in western countries because of the increasing of population age, physical inactivity and obesity, however it is rapidly spreading also in developing countries due to the socio-economic growth with progressive urbanization and changes in lifestyle.

Cardiovascular disease (CVD) in diabetic patients is characterized by microvascular damage, associated with the development of diabetic retinopathy, nephropathy, and neuropathy, and macrovascular complications linked to the accelerated course of atherosclerosis shown in these patients. Coronary heart disease (CHD) remains the principal cause of morbidity and mortality, in association with an increased risk of developing cerebrovascular disease, peripheral vascular disease and heart failure.

© 2012 Liuzzo et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Liuzzo et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **3. Classification and pathogenesis (Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 1997)**

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 47

Since the isolated treatment of hyperglycemia has not been associated to a reduction of CV risk in diabetic people, more aggressive primary and secondary prevention measures are needed in these patients (ADVANCE Collaborative Group, 2008; UKPDS Group, 1998).

The early onset and the burden of macroangiopathy in diabetic patients have a multifactorial pathogenesis and are the result of very complex mechanisms including the coexistence of multiple risk factors, such as obesity, hypertension and dyslipidemia. Moreover hyperglycemia, insulin resistance, hyperinsulinemia and the presence of Advanced Glycation End-products (AGE) in plasma and vascular wall are all mechanisms involved in the establishment of a pro-inflammatory state characterized by the activation of inflammatory cells and cytokine production, leading to immune dysregulation and pro-

On the other hand, inflammation can be considered a common link between these factors, being involved in each step of atherothrombosis, from the formation to the complications of

Several studies have demonstrated a correlation between T2DM, inflammation and innate immunity system. These evidences, together with more recent findings on inflammation and immune mechanisms, could pave the way to a new etiopathogenic hypothesis of Metabolic Syndrome and T2DM, firstly proposed by Pickup in 1997 (Pickup, 2004), and suggesting that activation of innate immunity, together with a chronic inflammatory response, could also play a pivotal and early role in *causing* diabetes, instead of being a mere *consequence* of

Recent evidences have also shown that adaptive immunity and autoreactivity could play a

Established T2DM is associated with elevated circulating biomarkers of innate immunity activation, including C-reactive protein (CRP) and interleukin (IL)-6 and these alterations are also present in patients with pre-diabetes and metabolic syndrome. In fact several crosssectional studies in non-diabetic subjects, in the general population (Festa et al., 2000; Ford, 1999a, 1999b; Frohlich et al., 2000; Hak et al., 2001; Sakkinen et al., 2000; Yudkin et al., 1999; Visser et al., 1999; Weyer et al., 2002)23-31, or in individuals with impaired glucose tolerance (IGT)/impaired fasting glucose (IFG) (Muller et al. 2002;, 2002b, Sriharan et al., 2002), have confirmed that acute-phase reactants are positively correlated with measures of insulin resistance, plasma insulin concentration, BMI, waist circumference, and circulating

Additional cross-sectional studies in newly diagnosed (Temelkova-Kurktschiev et al., 2002) or established T2DM patients (Arnalich et al., 2000; Leinonen et al., 2003; Richarsdon & Tayek, 2002; Rodriguez-Moran & Guerrero-Romero, 1999) have confirmed that acute-phase

the plaque, and in the metabolic dysregulation characterizing diabetes.

role in the pathogenesis of T2DM and in its complications (Figure 1).

triglyceride, and negatively correlated with HDL cholesterol concentration.

hyperglycemia, hyperinsulinemia and obesity.

**5. Diabetes and innate immunity** 

**5.1. Systemic markers of inflammation** 

thrombotic state.

DM is classified on the basis of pathogenetic mechanisms leading to hyperglycemia:


Immune system and autoimmunity play a pivotal role in the pathogenesis of type 1 diabetes mellitus (T1DM) (Atkinson & Maclaren, 1994), however inflammation may play a crucial intermediary role also in type 2 diabetes mellitus (T2DM) (Mykkänen, 2000) and in the development of its complications, including cardiovascular disease, thus linking it with several coexisting conditions thought to originate through inflammatory mechanisms.

## **4. Inflammation, diabetes and cardiovascular risk**

Epidemiological studies conducted at the end of 1970 described diabetes as a major independent risk factor for cardiovascular disease, causing 2-4 folds increase in cardiovascular risk (Kannel & McGee, 1979). Atherosclerosis is responsible for the 80% of deaths in diabetic patients (Gu K et al., 1998)7, and diabetes is considered a "coronary disease equivalent", since several studies pointed out that diabetes-associated CV risk is similar to that observed among non-diabetic patients with prior myocardial infarction (MI) (Haffner et al., 1998; Schramm et al., 2008).

Diabetes is associated with an increased risk of MI and affects more than 30% of patients with acute coronary syndromes (ACS) (Fang & Alderman, 2006). Diabetic patients show a worse outcome after ACS events (Malmberg et al., 2000; Murcia et al., 2004), a more complicated course of the disease and a higher incidence of ischemic recurrences (Cantrill et al., 1995; Miettinem et al., 1998; Shindler et al., 2000). Moreover, if undergoing revascularization procedures, they have a worse prognosis than patients without diabetes (Banning et al., 2010; Hlatky et al., 2009).

Several angiographic studies highlighted a greater spread and progression of atherosclerotic disease in diabetes patients. Moreover, histological specimens of atherosclerotic plaques obtained in diabetic patients exhibit larger lipid core, a higher inflammatory cell infiltration and increased neovascularization (Burke et al., 2004; Moreno & Fuster, 2004).

Since the isolated treatment of hyperglycemia has not been associated to a reduction of CV risk in diabetic people, more aggressive primary and secondary prevention measures are needed in these patients (ADVANCE Collaborative Group, 2008; UKPDS Group, 1998).

The early onset and the burden of macroangiopathy in diabetic patients have a multifactorial pathogenesis and are the result of very complex mechanisms including the coexistence of multiple risk factors, such as obesity, hypertension and dyslipidemia. Moreover hyperglycemia, insulin resistance, hyperinsulinemia and the presence of Advanced Glycation End-products (AGE) in plasma and vascular wall are all mechanisms involved in the establishment of a pro-inflammatory state characterized by the activation of inflammatory cells and cytokine production, leading to immune dysregulation and prothrombotic state.

On the other hand, inflammation can be considered a common link between these factors, being involved in each step of atherothrombosis, from the formation to the complications of the plaque, and in the metabolic dysregulation characterizing diabetes.

Several studies have demonstrated a correlation between T2DM, inflammation and innate immunity system. These evidences, together with more recent findings on inflammation and immune mechanisms, could pave the way to a new etiopathogenic hypothesis of Metabolic Syndrome and T2DM, firstly proposed by Pickup in 1997 (Pickup, 2004), and suggesting that activation of innate immunity, together with a chronic inflammatory response, could also play a pivotal and early role in *causing* diabetes, instead of being a mere *consequence* of hyperglycemia, hyperinsulinemia and obesity.

Recent evidences have also shown that adaptive immunity and autoreactivity could play a role in the pathogenesis of T2DM and in its complications (Figure 1).

## **5. Diabetes and innate immunity**

46 Pathophysiology and Complications of Diabetes Mellitus

Gestational DM.

**and Classification of Diabetes Mellitus, 1997)** 

**3. Classification and pathogenesis (Expert Committee on the Diagnosis** 

 Type 1, due to a virtually complete lack of endogenous pancreatic insulin production caused by an immune-mediated destruction of pancreatic beta cells (Immunomediated

 Type 2, accounting for ∼90–95% of diabetic patients. Its complex pathogenesis, resulting from a combination of genetic predisposition, unhealthy diet, physical inactivity, and increasing weight with a central distribution of the adipose tissue leads to insulin

Other specific types of diabetes, related to genetic defects of insulin secretion and/or

Immune system and autoimmunity play a pivotal role in the pathogenesis of type 1 diabetes mellitus (T1DM) (Atkinson & Maclaren, 1994), however inflammation may play a crucial intermediary role also in type 2 diabetes mellitus (T2DM) (Mykkänen, 2000) and in the development of its complications, including cardiovascular disease, thus linking it with several coexisting conditions thought to originate through inflammatory mechanisms.

Epidemiological studies conducted at the end of 1970 described diabetes as a major independent risk factor for cardiovascular disease, causing 2-4 folds increase in cardiovascular risk (Kannel & McGee, 1979). Atherosclerosis is responsible for the 80% of deaths in diabetic patients (Gu K et al., 1998)7, and diabetes is considered a "coronary disease equivalent", since several studies pointed out that diabetes-associated CV risk is similar to that observed among non-diabetic patients with prior myocardial infarction (MI)

Diabetes is associated with an increased risk of MI and affects more than 30% of patients with acute coronary syndromes (ACS) (Fang & Alderman, 2006). Diabetic patients show a worse outcome after ACS events (Malmberg et al., 2000; Murcia et al., 2004), a more complicated course of the disease and a higher incidence of ischemic recurrences (Cantrill et al., 1995; Miettinem et al., 1998; Shindler et al., 2000). Moreover, if undergoing revascularization procedures, they have a worse prognosis than patients without diabetes

Several angiographic studies highlighted a greater spread and progression of atherosclerotic disease in diabetes patients. Moreover, histological specimens of atherosclerotic plaques obtained in diabetic patients exhibit larger lipid core, a higher inflammatory cell infiltration

and increased neovascularization (Burke et al., 2004; Moreno & Fuster, 2004).

DM is classified on the basis of pathogenetic mechanisms leading to hyperglycemia:

Type I diabetes), or by unknown mechanisms (Idiopatic Type I diabetes);

resistance and usually relative (rather than absolute) insulin deficiency;

action in peripheral tissues, endocrinophaties, or infections;

**4. Inflammation, diabetes and cardiovascular risk** 

(Haffner et al., 1998; Schramm et al., 2008).

(Banning et al., 2010; Hlatky et al., 2009).

## **5.1. Systemic markers of inflammation**

Established T2DM is associated with elevated circulating biomarkers of innate immunity activation, including C-reactive protein (CRP) and interleukin (IL)-6 and these alterations are also present in patients with pre-diabetes and metabolic syndrome. In fact several crosssectional studies in non-diabetic subjects, in the general population (Festa et al., 2000; Ford, 1999a, 1999b; Frohlich et al., 2000; Hak et al., 2001; Sakkinen et al., 2000; Yudkin et al., 1999; Visser et al., 1999; Weyer et al., 2002)23-31, or in individuals with impaired glucose tolerance (IGT)/impaired fasting glucose (IFG) (Muller et al. 2002;, 2002b, Sriharan et al., 2002), have confirmed that acute-phase reactants are positively correlated with measures of insulin resistance, plasma insulin concentration, BMI, waist circumference, and circulating triglyceride, and negatively correlated with HDL cholesterol concentration.

Additional cross-sectional studies in newly diagnosed (Temelkova-Kurktschiev et al., 2002) or established T2DM patients (Arnalich et al., 2000; Leinonen et al., 2003; Richarsdon & Tayek, 2002; Rodriguez-Moran & Guerrero-Romero, 1999) have confirmed that acute-phase

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 49

**(years)** 

3-4

5

20

6

2.3

7.2

**marker(s) analized Subjects Follow-up** 

women

women

women

German men and women

aged men and women

Multiethnic subjects

predict the development of T2DM and this has been confirmed by several follow-up studies

**Pradhan et al.** 2001 CRP and IL-6 US women 4

**Vozarova et al.** 2002 White blood count Pima Indians 5,5

**Freeman et al.** 2002 CRP Scottish men 5

**Nakanishi et al.** 2002 White blood count Japanese men 6

risk of IL-6 and IL-1

The association between altered levels of acute-phase reactants and the development of diabetes is generally independent of age, sex, blood glucose concentration, family history of diabetes, physical activity, smoking, and baseline atherosclerosis, while it seems to be

It has been shown that treatment with high doses of aspirin is associated with a 25% reduction in fasting plasma glucose, a 50% reduction in triglycerides and a 15% decrease of total cholesterol and CRP, even if no change in body weight occurs (Hundal et al., 2002).

Recent studies have shown a role played by genetic variations in influencing the innate immune response and the risk of developing T2DM, obesity and atherosclerosis (Fernandez-Real & Pickup, 2008). These variations can relate to genes encoding proteins like

inflammatory markers, cytokines and cellular pattern-recognition receptors (PRR).

**Snijder et al.** 2001 CRP Dutch men and

combined

**Thorand et al.** 2003 CRP German middle-

Legend: CRP, C-reactive protein; IL, interleukin; PAI, plasminogen activator inhibitor. **Table 1.** Inflammatory markers and the prediction of T2DM development

**Barzilay et al.** 2001 CRP US men and

PAI-1

**Ford et al.** 2002 White blood count US men and

in different populations (Table 1).

**Authors Year Inflammatory** 

**Festa et al.** 2002 CRP, fibrinogen, and

**Spranger et al.** 2003 IL-6, with additional

weaker if adjusted for obesity (Pickup, 2004).

**Figure 1.** Schematic representation of the principal mechanisms linking diabetes, vascular injury and atherosclerotic disease. Hyperglycemia induces formation of advanced glycation end products (AGEs) that bind to their receptors (RAGE) present on endothelial cells, smooth muscle cells, monocytes and macrophages, thus promoting vascular inflammation, endothelial dysfunction, and prothrombotic state. Hyperglycemia and AGEs also cause generation of reactive oxygen species (ROS), which in turn increase AGE and oxidized low-density lipoproteins (ox-LDL) formation. These pathways are all involved in the development of atherosclerosis and plaque progression/destabilization in diabetic patients.

markers such as CRP and IL-6 are elevated in these subjects compared with non-diabetic controls (Katsuki et al., 1998; Pickup et al., 2000; Winkler et al., 1998).

On the other side it has been shown how abnormal circulating levels of acute-phase reactants, in particular CRP and serum amyloid A, and inflammatory cytokines like IL-6, are good predictor of the development of T2DM in nondiabetic subjects. Schmidt and colleagues (Duncan et al., 1999; Schmidt et al., 1999), using data from the Atherosclerosis Risk in Communities study, showed for the first time that inflammatory markers, such as white blood cell count, low serum albumin, α1-acid glycoprotein, fibrinogen, and sialic acid,


predict the development of T2DM and this has been confirmed by several follow-up studies in different populations (Table 1).

48 Pathophysiology and Complications of Diabetes Mellitus

patients.

**Figure 1.** Schematic representation of the principal mechanisms linking diabetes, vascular injury and atherosclerotic disease. Hyperglycemia induces formation of advanced glycation end products (AGEs) that bind to their receptors (RAGE) present on endothelial cells, smooth muscle cells, monocytes and macrophages, thus promoting vascular inflammation, endothelial dysfunction, and prothrombotic state. Hyperglycemia and AGEs also cause generation of reactive oxygen species (ROS), which in turn increase AGE and oxidized low-density lipoproteins (ox-LDL) formation. These pathways are all involved in the development of atherosclerosis and plaque progression/destabilization in diabetic

markers such as CRP and IL-6 are elevated in these subjects compared with non-diabetic

On the other side it has been shown how abnormal circulating levels of acute-phase reactants, in particular CRP and serum amyloid A, and inflammatory cytokines like IL-6, are good predictor of the development of T2DM in nondiabetic subjects. Schmidt and colleagues (Duncan et al., 1999; Schmidt et al., 1999), using data from the Atherosclerosis Risk in Communities study, showed for the first time that inflammatory markers, such as white blood cell count, low serum albumin, α1-acid glycoprotein, fibrinogen, and sialic acid,

controls (Katsuki et al., 1998; Pickup et al., 2000; Winkler et al., 1998).

Legend: CRP, C-reactive protein; IL, interleukin; PAI, plasminogen activator inhibitor.

**Table 1.** Inflammatory markers and the prediction of T2DM development

The association between altered levels of acute-phase reactants and the development of diabetes is generally independent of age, sex, blood glucose concentration, family history of diabetes, physical activity, smoking, and baseline atherosclerosis, while it seems to be weaker if adjusted for obesity (Pickup, 2004).

It has been shown that treatment with high doses of aspirin is associated with a 25% reduction in fasting plasma glucose, a 50% reduction in triglycerides and a 15% decrease of total cholesterol and CRP, even if no change in body weight occurs (Hundal et al., 2002).

Recent studies have shown a role played by genetic variations in influencing the innate immune response and the risk of developing T2DM, obesity and atherosclerosis (Fernandez-Real & Pickup, 2008). These variations can relate to genes encoding proteins like inflammatory markers, cytokines and cellular pattern-recognition receptors (PRR).

Genetic predisposition to high transcription rate of TNF-α and IL-6 genes is associated with an increased risk of developing obesity, insulin-resistance and diabetes (Fernandez-Real & Ricart, 2003).

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 51

(Holland et al., 2007). However, the previously described studies have not adequately eliminated potential contamination of the reagents used in the experimental condition with bacterial products. Therefore, the direct stimulation of TLRs in various cell types attributed

The expression in the vessel wall of both TLR2 and TLR4 has a synergistic effect on the progression of atherosclerotic plaque (Monaco et al., 2009; Shinoara et al., 2007). TLR4, whose endogenous ligand is ox-LDL (Xu et al., 2001), is highly expressed in SMC of atherosclerotic vessels, where it has been associated with the induction of a proinflammatory phenotype (Loppnow et al., 2008; Otsui et al., 2007). Furthermore, TLR4 has been found in atherosclerotic lesions and at the site of plaque rupture in patients with MI (Ishikawa et al., 2008), and its expression is increased in thrombi from patients with acute coronary syndromes (Wyss et al., 2010; Yonekawa et al., 2011). Moreover, several studies showed that circulating monocytes of patients with atherosclerotic disease exhibit higher expression of TLR2 and TLR4 as compared to healthy individuals (Geng et al., 2006; Kuwahata et al., 2010; Mizoguchi et al., 2007; Shiraki et al., 2006), and an enhanced TLR signaling has been demonstrated in monocytes of patients with ACS (Ashida et al., 2005;

To date, the mechanisms linking high-fat diets with TLR-signaling and associated pathologies, such as atherosclerosis and insulin resistance, remain to be discovered. As an alternative TLR-dependent mechanism, currently under investigation, the large quantities of lipopeptide and LPS derived from the commensal organisms of the mammalian intestine may contribute to systemic stimulation of TLR2 or TLR4 signaling. Administration of LPS in mice has been associated with an increase of hepatic insulin resistance and a decrease of glucose tolerance (Arkan et al., 2005; Cani et al., 2007). It has been shown that blood levels of LPS are higher in T2DM patients than in healthy controls and correlate with insulin levels and glucose ( Al-Attas et al., 2009; Creely et al., 2007; Harte et al., 2010). Hence, an increased level of PAMPs like LPS may play an important role in the development of the

Main sources of PAMPs are represented by infections, commensals and diet (Erridge, 2011). It's difficult to assess the quantitative contribution of each of them to PAMPs burden in humans, but increasing evidences are demonstrating that, under certain conditions like high fat meals, PAMPs derived from commensals and diet can effectively translocate from the intestinal lumen to the circulation (Erridge et al, 2007; Laugerette et al., 2010). Indeed, it has been widely demonstrated that oral microorganisms and human periodontitis are associated with an increased risk of developing atherosclerosis and T2DM (Bahekar et al., 2007). The small intestine seems to be the main contributor of the global circulating PAMPs burden, mostly due to the absorption of PAMPs swallowed from the oral cavity. This is probably due to the bigger surface area compared to large intestine and the fat-soluble nature of PAMPs such as LPS, accounting for their easier absorption in chylomicrons with dietary fat, a process taking place only in the small intestine (Ghoshal et al., 2009). Moreover, it is reasonable that the most part of PAMPs absorbed in the large intestine firstly reach liver through the portal system, being there effectively removed from circulation; on the other

inflammatory status characterizing metabolic diseases like T2DM.

to saturated FAs might be due to LPS contamination (Erridge & Samani, 2009).

Methe et al., 2005; Versteeg et al., 2008).

Increased levels of inflammatory markers and insulin resistance have been also connected to a genetically determined reduction of serum levels of soluble CD14, a molecule expressed by macrophages able to bind lipopolysaccharide (LPS), and Bactericidal and Permeability Increasing protein (BPI), produced by neutrophils (Fernandez-Real et al., 2003; Gubern, 2006).

Moreover, decreased levels of mannose-binding lectine (MBL), a protein involved in the clearance of infectious pathogens through the induction of complement activation and macrophage phagocytosis, have been associated both with a raised risk of infections (Summerfield et al., 1997), CHD (Best et al., 2004), obesity and insulin resistance (Fernandez-Real et al, 2006).

## **5.2. Toll like receptors as link between inflammation and metabolic diseases**

Mechanisms by which the activation of the innate immunity can cause insulin resistance have been clarified recently; many studies have revealed how TNF-α could activate the c-Jun NH2-terminal kinase, a stress-induced kinase which serinephosphorylates many signaling proteins, including insulin receptor substrate (IRS)-1 and IRS-2, thereby inhibiting insulin signaling (Morris et al., 2003).

A crucial role, in this setting, is probably played by Toll-like receptors (TLR). TLR are key receptors of innate immunity recognizing a huge number of molecules usually expressed by pathogen microorganisms but absent in mammal tissues, named pathogen-associated molecular patterns (PAMPs), and other molecules called damage-activated molecular patterns (DAMPs); therefore TLR belong to the family of PRR (Kawai & Akira, 2010).

To date, 13 TLRs have been described, both located on the extracellular surface or in the intracellular compartment (Takeda & Akira, 2004). Among them, TLR2 and TLR4 have been associated with metabolic disorders, as well as with atherosclerosis and its clinical manifestations. TLR2 and TLR4 loss-of-function, absence or inhibition in high-fat diet murine models has been related to a decrease in weight gain, insulin resistance and betacells dysfunction (Caricilli et al., 2008; Ehses et al., 2010; Tsukumo et al., 2007). TLR4 is highly conserved and selectively activated by lipopolysaccharides (LPS), a constituent of Gram-negative bacterial cell-wall (Kawai & Akira, 2010). Some authors have demonstrated how the lauric acid, a medium-chain fatty acid (FA) component of LPS, trigger TLR4 signaling in macrophages and have revealed how saturated FAs, but not unsaturated, activate inflammatory signals in adipose cells and macrophages (Lee et al., 2001, 2003). Other studies have proposed that the sphingolipid ceramide, synthesized from FAs, might represent a possible link between high-fat diet intake and TLR pathways. Indeed, sphingolipid ceramide is able to activate TLR4 signaling (Fischer et al., 2007; Schwartz et al, 2010), and the inibition of its biosynthesis improves glucose tolerance in murine models (Holland et al., 2007). However, the previously described studies have not adequately eliminated potential contamination of the reagents used in the experimental condition with bacterial products. Therefore, the direct stimulation of TLRs in various cell types attributed to saturated FAs might be due to LPS contamination (Erridge & Samani, 2009).

50 Pathophysiology and Complications of Diabetes Mellitus

Ricart, 2003).

Real et al, 2006).

insulin signaling (Morris et al., 2003).

2006).

Genetic predisposition to high transcription rate of TNF-α and IL-6 genes is associated with an increased risk of developing obesity, insulin-resistance and diabetes (Fernandez-Real &

Increased levels of inflammatory markers and insulin resistance have been also connected to a genetically determined reduction of serum levels of soluble CD14, a molecule expressed by macrophages able to bind lipopolysaccharide (LPS), and Bactericidal and Permeability Increasing protein (BPI), produced by neutrophils (Fernandez-Real et al., 2003; Gubern,

Moreover, decreased levels of mannose-binding lectine (MBL), a protein involved in the clearance of infectious pathogens through the induction of complement activation and macrophage phagocytosis, have been associated both with a raised risk of infections (Summerfield et al., 1997), CHD (Best et al., 2004), obesity and insulin resistance (Fernandez-

**5.2. Toll like receptors as link between inflammation and metabolic diseases** 

Mechanisms by which the activation of the innate immunity can cause insulin resistance have been clarified recently; many studies have revealed how TNF-α could activate the c-Jun NH2-terminal kinase, a stress-induced kinase which serinephosphorylates many signaling proteins, including insulin receptor substrate (IRS)-1 and IRS-2, thereby inhibiting

A crucial role, in this setting, is probably played by Toll-like receptors (TLR). TLR are key receptors of innate immunity recognizing a huge number of molecules usually expressed by pathogen microorganisms but absent in mammal tissues, named pathogen-associated molecular patterns (PAMPs), and other molecules called damage-activated molecular

To date, 13 TLRs have been described, both located on the extracellular surface or in the intracellular compartment (Takeda & Akira, 2004). Among them, TLR2 and TLR4 have been associated with metabolic disorders, as well as with atherosclerosis and its clinical manifestations. TLR2 and TLR4 loss-of-function, absence or inhibition in high-fat diet murine models has been related to a decrease in weight gain, insulin resistance and betacells dysfunction (Caricilli et al., 2008; Ehses et al., 2010; Tsukumo et al., 2007). TLR4 is highly conserved and selectively activated by lipopolysaccharides (LPS), a constituent of Gram-negative bacterial cell-wall (Kawai & Akira, 2010). Some authors have demonstrated how the lauric acid, a medium-chain fatty acid (FA) component of LPS, trigger TLR4 signaling in macrophages and have revealed how saturated FAs, but not unsaturated, activate inflammatory signals in adipose cells and macrophages (Lee et al., 2001, 2003). Other studies have proposed that the sphingolipid ceramide, synthesized from FAs, might represent a possible link between high-fat diet intake and TLR pathways. Indeed, sphingolipid ceramide is able to activate TLR4 signaling (Fischer et al., 2007; Schwartz et al, 2010), and the inibition of its biosynthesis improves glucose tolerance in murine models

patterns (DAMPs); therefore TLR belong to the family of PRR (Kawai & Akira, 2010).

The expression in the vessel wall of both TLR2 and TLR4 has a synergistic effect on the progression of atherosclerotic plaque (Monaco et al., 2009; Shinoara et al., 2007). TLR4, whose endogenous ligand is ox-LDL (Xu et al., 2001), is highly expressed in SMC of atherosclerotic vessels, where it has been associated with the induction of a proinflammatory phenotype (Loppnow et al., 2008; Otsui et al., 2007). Furthermore, TLR4 has been found in atherosclerotic lesions and at the site of plaque rupture in patients with MI (Ishikawa et al., 2008), and its expression is increased in thrombi from patients with acute coronary syndromes (Wyss et al., 2010; Yonekawa et al., 2011). Moreover, several studies showed that circulating monocytes of patients with atherosclerotic disease exhibit higher expression of TLR2 and TLR4 as compared to healthy individuals (Geng et al., 2006; Kuwahata et al., 2010; Mizoguchi et al., 2007; Shiraki et al., 2006), and an enhanced TLR signaling has been demonstrated in monocytes of patients with ACS (Ashida et al., 2005; Methe et al., 2005; Versteeg et al., 2008).

To date, the mechanisms linking high-fat diets with TLR-signaling and associated pathologies, such as atherosclerosis and insulin resistance, remain to be discovered. As an alternative TLR-dependent mechanism, currently under investigation, the large quantities of lipopeptide and LPS derived from the commensal organisms of the mammalian intestine may contribute to systemic stimulation of TLR2 or TLR4 signaling. Administration of LPS in mice has been associated with an increase of hepatic insulin resistance and a decrease of glucose tolerance (Arkan et al., 2005; Cani et al., 2007). It has been shown that blood levels of LPS are higher in T2DM patients than in healthy controls and correlate with insulin levels and glucose ( Al-Attas et al., 2009; Creely et al., 2007; Harte et al., 2010). Hence, an increased level of PAMPs like LPS may play an important role in the development of the inflammatory status characterizing metabolic diseases like T2DM.

Main sources of PAMPs are represented by infections, commensals and diet (Erridge, 2011). It's difficult to assess the quantitative contribution of each of them to PAMPs burden in humans, but increasing evidences are demonstrating that, under certain conditions like high fat meals, PAMPs derived from commensals and diet can effectively translocate from the intestinal lumen to the circulation (Erridge et al, 2007; Laugerette et al., 2010). Indeed, it has been widely demonstrated that oral microorganisms and human periodontitis are associated with an increased risk of developing atherosclerosis and T2DM (Bahekar et al., 2007). The small intestine seems to be the main contributor of the global circulating PAMPs burden, mostly due to the absorption of PAMPs swallowed from the oral cavity. This is probably due to the bigger surface area compared to large intestine and the fat-soluble nature of PAMPs such as LPS, accounting for their easier absorption in chylomicrons with dietary fat, a process taking place only in the small intestine (Ghoshal et al., 2009). Moreover, it is reasonable that the most part of PAMPs absorbed in the large intestine firstly reach liver through the portal system, being there effectively removed from circulation; on the other

hand, PAMPs from the small intestine, through chylomicrons absorption, can reach lymphatic system and general circulation bypassing the liver. Finally a quote of PAMPs may come from diet. Interestingly, it has been demonstrated that PAMPs are nearly absent in fresh food, but they can be copious in a number of processed food typical of Western diet, such as meat and dairy products (Erridge, 2010, 2011).

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 53

concentration (Maedler et al., 2002). Several models have been proposed to explain the inflammasome mediated pancreatic islets dysfunction and particularly the role of ROS induced inflammasome activation has been highlighted. Hyperglycemia stimulates mitochondrial ROS production by increasing the activity of the electron transport chain, leading to the activation of NLRP3. Thioredoxin-interacting protein (TXNIP) is usually bound to oxidoreductase thioredoxin, however, when intracellular ROS increase, it seems to act as an upstream specific activating lingand for NLRP3. TXNIP expression is induced by glucose (Oka et al., 2009) and repressed by insulin (Parikh et al., 2007). Moreover, glucose induces the expression of TXNIP in pancreatic islets but not in macrophages(Zhou et al., 2010) and glucose dependent IL-1β secretion in pancreatic islets is inhibited in TXNIP- and NLRP3-knockout mice and antagonized by ROS-blockers. Taken together, these evidences suggest that a chronic condition of high plasmatic glucose levels induces pancreatic islets dysfunction through a mechanisms involving TXNIP-dependent NLRP3 inflammasome and that, once activated, this inflammasome could represent an adjunctive and self-maintaining

Hystopathological studies recently showed deposition of islet amyloid polypeptide (IAPP, also known as amylin) in pancreatic islets of T2DM patients (Seino et al., 2001), that seems to be able to specifically activate the NLRP3 inflammasome through a mechanism that involves

Additional support for a pathological role of inflammasomes in T2DM comes from human clinical trials in which blockade of IL-1β signaling by Anakinra, a recombinant human IL-1 receptor antagonist (IL-1RA) demonstrated sustained reduction of inflammation, improved glycaemic control and β-cell function in T2DM patients (Dinarello et al., 2010; Larsen et al.,

Moreover inflammatory cytokines such as TNF-α, IL-1β, and IL-6 also downregulate peroxisome proliferator activated receptor-γ (PPAR-γ) expression (Tanaka et al., 1999). PPAR-γ is a ligand-activated transcription factor highly expressed in adipose tissue, where it controls adipocyte differentiation and lipid storage, and modulates insulin action. It represents the target of thiazolidinediones (TZDs) pioglitazone and rosiglitazone, which are demonstrated to improve glycemic control and insulin-sensitivity and to reduce T2DM-

As noted above, much evidence suggests an intimate relationship among IL-1β, the NLRP3 inflammasome and the metabolism of lipids and carbohydrates. This occurs at the level of enhanced NLRP3 inflammasome activation and processing of IL-1β to the mature cytokine in response to saturated fatty acids and also at the level of glucose metabolism through the requirement of glycolysis for induction of IL-1β mRNA. The pathogenic role of IL-1β in atherosclerotic plaque formation and in insulin resistance in T2DM attests to the importance of inflammasome-mediated pathways as link between inflammation, T2DM and CVD. The exacerbation of NLRP3 inflammasome activation by cholesterol crystals in atherosclerosis (Duewell et al., 2010; Rajamäki et al, 2010) and by IAPP in type 2 diabetes (Masters et al.,

disruption of the phagolysosomal pathway (Masters et al., 2010).

associated inflammation (Miyazaki et al., 2001a, 2001b)120,121.

2010), provides a positive feedback loop to promote disease pathogenesis.

immune-metabolic stressor.

2007).

## **5.3. Role of inflammasomes in peripheral insulin resistance**

Recent studies also highlighted a crucial role of inflammasomes pathways both in insulin production and in insulin sensitivity.

Inflammasomes are group of protein complexes which recognize a diverse set of inflammation-inducing stimuli, including PAMPs, and DAMPs (Strowig et al., 2012). The activation of these complexes lead to the proteolitic activation of caspase-1 and, finally, to the production and release of important pro-inflammatory cytokines such as IL-1β and IL-18 (Davis et al., 2011; Schroder & Tschopp, 2010). The most widely studied inflammasome is the NLRP3 inflammasome, which could be activated by a large variety of signals, included PAMPs, DAMPs and bacterial toxins.

A two-step process is required to induce NLRP3 inflammasome activation. A first priming step is usually mediated by PRRs, such as TLR, or cytokines receptors known to induce activation of NFkB, and leads to production and intracellular release of inactive forms of NLRP3. A subsequent activation step induces the inflammasome assembly; it starts in response to a variety of stimuli, such as potassium efflux, extracellular ATP, reactive oxygen species (ROS) and rupture of lysosomal membrane integrity, and leads to caspase-1 activation and cleavage of pro-IL1β. Recent evidences suggest that NLRP3 play a pivotal role both in the early stages and in the chronic progression of T2DM (Kahn et al., 2006). Vandanmagsar et al. found that NLRP3 inflammasome is largely expressed in adipose-tissue-infiltrating macrophages, and it is activated by obesity–associated 'danger–signals', such as the saturated fatty acid palmitate and lipotoxicity–associated ceramide (Vandanmagscar et al., 2011). They also demonstrated how the expression of NLPR3 in the adipose tissue is directly correlated to insulin resistance both in mice and humans and that blockade of NLRP3 could reduce inflammation and improve insulin sensitivity (Vandanmagscar et al., 2011). Other studies demonstrated that during obesity, circulating free fatty acids are scavenged by adipose tissue macrophages to produce ceramide ( Shah et al., 2008) and confirmed the role of this lipid molecule in inducing NLRP3 inflammasome activation (Boden & Ceramide, 2008). IL-1β, produced as a result of inflammasome activation, inhibits insulin signaling (Wen et al., 2011) by direct serine phosphorylation of IRS-1 and induces the expression of TNF-α (Strowig et al, 2012), an insulin-resistance-promoting cytokine as discussed above. IL-1β and IL-18 also induce type 1 CD4+T-helper cells differentiation in adipose tissue (Vandanmagscar et al., 2011). Moreover, the activation of caspase-1 seems to be related also to adipocytes differentiation and adipokines production (Stienstra et al., 2011).

Inflammasome activation is also involved in impaired insulin secretion associated with overt T2DM. Human β-cells are capable to produce IL-1β when exposed to elevated glucose concentration (Maedler et al., 2002). Several models have been proposed to explain the inflammasome mediated pancreatic islets dysfunction and particularly the role of ROS induced inflammasome activation has been highlighted. Hyperglycemia stimulates mitochondrial ROS production by increasing the activity of the electron transport chain, leading to the activation of NLRP3. Thioredoxin-interacting protein (TXNIP) is usually bound to oxidoreductase thioredoxin, however, when intracellular ROS increase, it seems to act as an upstream specific activating lingand for NLRP3. TXNIP expression is induced by glucose (Oka et al., 2009) and repressed by insulin (Parikh et al., 2007). Moreover, glucose induces the expression of TXNIP in pancreatic islets but not in macrophages(Zhou et al., 2010) and glucose dependent IL-1β secretion in pancreatic islets is inhibited in TXNIP- and NLRP3-knockout mice and antagonized by ROS-blockers. Taken together, these evidences suggest that a chronic condition of high plasmatic glucose levels induces pancreatic islets dysfunction through a mechanisms involving TXNIP-dependent NLRP3 inflammasome and that, once activated, this inflammasome could represent an adjunctive and self-maintaining immune-metabolic stressor.

52 Pathophysiology and Complications of Diabetes Mellitus

production and in insulin sensitivity.

PAMPs, DAMPs and bacterial toxins.

adipokines production (Stienstra et al., 2011).

such as meat and dairy products (Erridge, 2010, 2011).

**5.3. Role of inflammasomes in peripheral insulin resistance** 

hand, PAMPs from the small intestine, through chylomicrons absorption, can reach lymphatic system and general circulation bypassing the liver. Finally a quote of PAMPs may come from diet. Interestingly, it has been demonstrated that PAMPs are nearly absent in fresh food, but they can be copious in a number of processed food typical of Western diet,

Recent studies also highlighted a crucial role of inflammasomes pathways both in insulin

Inflammasomes are group of protein complexes which recognize a diverse set of inflammation-inducing stimuli, including PAMPs, and DAMPs (Strowig et al., 2012). The activation of these complexes lead to the proteolitic activation of caspase-1 and, finally, to the production and release of important pro-inflammatory cytokines such as IL-1β and IL-18 (Davis et al., 2011; Schroder & Tschopp, 2010). The most widely studied inflammasome is the NLRP3 inflammasome, which could be activated by a large variety of signals, included

A two-step process is required to induce NLRP3 inflammasome activation. A first priming step is usually mediated by PRRs, such as TLR, or cytokines receptors known to induce activation of NFkB, and leads to production and intracellular release of inactive forms of NLRP3. A subsequent activation step induces the inflammasome assembly; it starts in response to a variety of stimuli, such as potassium efflux, extracellular ATP, reactive oxygen species (ROS) and rupture of lysosomal membrane integrity, and leads to caspase-1 activation and cleavage of pro-IL1β. Recent evidences suggest that NLRP3 play a pivotal role both in the early stages and in the chronic progression of T2DM (Kahn et al., 2006). Vandanmagsar et al. found that NLRP3 inflammasome is largely expressed in adipose-tissue-infiltrating macrophages, and it is activated by obesity–associated 'danger–signals', such as the saturated fatty acid palmitate and lipotoxicity–associated ceramide (Vandanmagscar et al., 2011). They also demonstrated how the expression of NLPR3 in the adipose tissue is directly correlated to insulin resistance both in mice and humans and that blockade of NLRP3 could reduce inflammation and improve insulin sensitivity (Vandanmagscar et al., 2011). Other studies demonstrated that during obesity, circulating free fatty acids are scavenged by adipose tissue macrophages to produce ceramide ( Shah et al., 2008) and confirmed the role of this lipid molecule in inducing NLRP3 inflammasome activation (Boden & Ceramide, 2008). IL-1β, produced as a result of inflammasome activation, inhibits insulin signaling (Wen et al., 2011) by direct serine phosphorylation of IRS-1 and induces the expression of TNF-α (Strowig et al, 2012), an insulin-resistance-promoting cytokine as discussed above. IL-1β and IL-18 also induce type 1 CD4+T-helper cells differentiation in adipose tissue (Vandanmagscar et al., 2011). Moreover, the activation of caspase-1 seems to be related also to adipocytes differentiation and

Inflammasome activation is also involved in impaired insulin secretion associated with overt T2DM. Human β-cells are capable to produce IL-1β when exposed to elevated glucose Hystopathological studies recently showed deposition of islet amyloid polypeptide (IAPP, also known as amylin) in pancreatic islets of T2DM patients (Seino et al., 2001), that seems to be able to specifically activate the NLRP3 inflammasome through a mechanism that involves disruption of the phagolysosomal pathway (Masters et al., 2010).

Additional support for a pathological role of inflammasomes in T2DM comes from human clinical trials in which blockade of IL-1β signaling by Anakinra, a recombinant human IL-1 receptor antagonist (IL-1RA) demonstrated sustained reduction of inflammation, improved glycaemic control and β-cell function in T2DM patients (Dinarello et al., 2010; Larsen et al., 2007).

Moreover inflammatory cytokines such as TNF-α, IL-1β, and IL-6 also downregulate peroxisome proliferator activated receptor-γ (PPAR-γ) expression (Tanaka et al., 1999). PPAR-γ is a ligand-activated transcription factor highly expressed in adipose tissue, where it controls adipocyte differentiation and lipid storage, and modulates insulin action. It represents the target of thiazolidinediones (TZDs) pioglitazone and rosiglitazone, which are demonstrated to improve glycemic control and insulin-sensitivity and to reduce T2DMassociated inflammation (Miyazaki et al., 2001a, 2001b)120,121.

As noted above, much evidence suggests an intimate relationship among IL-1β, the NLRP3 inflammasome and the metabolism of lipids and carbohydrates. This occurs at the level of enhanced NLRP3 inflammasome activation and processing of IL-1β to the mature cytokine in response to saturated fatty acids and also at the level of glucose metabolism through the requirement of glycolysis for induction of IL-1β mRNA. The pathogenic role of IL-1β in atherosclerotic plaque formation and in insulin resistance in T2DM attests to the importance of inflammasome-mediated pathways as link between inflammation, T2DM and CVD. The exacerbation of NLRP3 inflammasome activation by cholesterol crystals in atherosclerosis (Duewell et al., 2010; Rajamäki et al, 2010) and by IAPP in type 2 diabetes (Masters et al., 2010), provides a positive feedback loop to promote disease pathogenesis.

Taken together, these findings support a crucial role of different molecules and pathways of innate immunity in the complex metabolic imbalance underlining T2DM, and possible contributing to the disease-associated cardiovascular risk. Insight into the above described molecular pathways could help in the design of new therapeutic strategies.

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 55

differentiation in T2DM patients. In recent years, a higher percentage of a particular T-cell type, CD4+ CD28null T lymphocytes, has been found in diabetic patients undergoing microvascular complications, e.g. proliferative retinopathy (Canton et al., 2004). An expansion of this particular T-cell population, which is infrequent in healthy young people and slightly expanded in the elderly, has been detected in patients with unstable angina (Liuzzo et al., 1999, 2000); in this population, a percentage of CD4+CD28null T-cells >4%, representing the 90th percentile of distribution in healthy individuals, is associated with a poor outcome (Liuzzo et al., 2007). These cells have particular aggressive features, showing an increased IFN-γ production and anti-apoptotic factors expression (Liuzzo et al., 2001), and could be involved in abrupt atherosclerotic plaque destabilization through several mechanisms. In fact, CD4+CD28null T-lymphocytes exert cytolitic effects on endothelial cells and express high levels of TNF-related apoptosis-inducing ligand (TRAIL), thus promoting smooth muscle cells apoptosis within the atherosclerotic plaque (Nakajima et al., 2002; Sato et al., 2006). With these premises, the recent finding of an expansion of CD4+ CD28null T-cells in diabetic patients is extremely interesting, suggesting a possible role of adaptive immune disregulation, either primary or induced by the altered metabolic status and the inflammatory environment characterizing the disease, in the increased cardiovascular risk which is one of the most relevant clinical features of T2DM, accounting for the majority of disease-related mortality and morbidity (Giubilato et al., 2011). Consistently, in the same study CD4+ CD28null T-lymphocytes expansion was closely related to a poor glycaemic control, and was associated with a higher incidence of cardiovascular events during follow-

Other fingerprints of adaptive immunity activation have been investigated in T2DM

Increased activity of adenosine-deaminase (ADA) has been described in this population (Prakash et al., 2006). ADA is an enzyme that converts adenosine into inosine through an irreversible deamination reaction, and it is involved in T-cell proliferation and activation (Kather, 1990). Moreover, since adenosine increases glucose uptake into cells, an effect of ADA in reducing tissutal insulin sensitivity has been described (Gorrell et al., 2001). A recent study has confirmed an increased ADA activity in T2DM patients, underlining also an association between enzyme function and fasting glucose levels, as well as HbA1c. Thus, inflammation, T-lymphocytes activation and glucose metabolism seem to be tightly related

Tregs are another important T-cell type widely involved in autoreactive processes and in the modulation of the inflammatory environment associated with various diseases and pathological conditions. In the setting of diabetes mellitus, Tregs have been extensively investigated both in animal models and human patients with T1DM (Chatenoud et al., 2005, Randolph & Fathman, 2006), while less studies have been performed on Tregs in T2DM. Interestingly, a recent study in mice demonstrated that Treg induction was associated to a reduction of adipose tissue inflammation and insulin resistance, with a concomitant improvement of metabolic parameters of lipid metabolism and glycaemic control (Ilan et al, 2010). Consistently, a subsequent study proved an inverse relation between Treg expression

up.

patients.

in the complex setting of T2DM (Lee et al., 2011).

## **6. Diabetes and adaptive immunity**

In the past years, a possible role of adaptive immunity and autoreactive mechanisms in the pathogenesis of T2DM has probably been underestimated and, therefore, poorly investigated. However, increasing evidences support the role of autoimmunity and adaptive immune system in the pathogenesis of T2DM and its vascular complications (Brooks-Worrell & Palmer, 2012; Nikolajczyk et al, 2011). It has been recently demonstrated that Tlymphocytes of patients with T2DM produce large amounts of pro-inflammatory cytokines, such as IL-8, showing in contrast a decreased production of anti-inflammatory cytokines, such as IL-10 (Jagannathan et al., 2010). These functional alterations are consistent with those previously demonstrated in monocytes of T2DM patients (Giulietti et al., 2007; Hatanaka et al., 2006; Pitocco et al., 2009), and result in an imbalance of cytokines network and in a strongly pro-inflammatory environment. High pro-inflammatory cytokines production has been associated in several studies with insulin-resistance and DM development, while the inhibition of some pro-inflammatory mediators prevented insulinresistance in mice (Arkan et al., 2005; Cai et al., 2005; de Roos et al., 2009; Ehses et al., 2009; Reimers, 1998.

Moreover, the role of a perturbation of T-cell repertoire has been demonstrated in murine models of T2DM. Particularly, regulatory T-cells (Treg) are significantly diminished in the adipose tissue of obese insulin-resistant mice compared to non-obese animals. Treg cells isolated and expanded ex-vivo, in these models, were found able to exert an antiinflammatory activity and lessen insulin-resistance (Feuerer et al., 2009). On the other hand, Interferon (IFN)-γ-producing cells in the adipose tissue of obese mice may cause an imbalance in glucose homeostasis. The alterations mediated by T-cells with a Th1 phenotype, characterized by IFN- γ production, can be counterbalanced by CD4+T-cells with an anti-inflammatory phenotype, such as Treg and Th2 lymphocytes producing IL-10 (Winer et al., 2009), thus underlining the importance of a physiological balance between different T-cells subset in the metabolic homeostasis of adipose tissue, which has a crucial role in the pathogenesis of insulin resistance and T2DM onset. Another cellular type possibly involved in inflammation and insulin-resistance in T2DM are IL-17 producing Tcells, so called Th17. This aggressive, pro-inflammatory T-cell subset has been found at high levels following IL-6 stimulation in the spleen of obese mice, and could contribute to the inflammatory environment strongly related to insulin resistance development and maintenance in T2DM (Winer et al., 2009). Consistently with this hypothesis, high levels of cytokines conditioning T-cell differentiation toward a Th17 phenotype, such as IL-6, IL-1β and Tranforming Growth Factor (TGF)-β, have been measured in diabetic patients (Acosta-Rodriguez et al., 2007; Andriankaja et., 2009; Osborn et al., 2008; Yang et al., 2008). These pro-inflammatory cytokines could promote Th17 cells expansion and inhibit Treg differentiation in T2DM patients. In recent years, a higher percentage of a particular T-cell type, CD4+ CD28null T lymphocytes, has been found in diabetic patients undergoing microvascular complications, e.g. proliferative retinopathy (Canton et al., 2004). An expansion of this particular T-cell population, which is infrequent in healthy young people and slightly expanded in the elderly, has been detected in patients with unstable angina (Liuzzo et al., 1999, 2000); in this population, a percentage of CD4+CD28null T-cells >4%, representing the 90th percentile of distribution in healthy individuals, is associated with a poor outcome (Liuzzo et al., 2007). These cells have particular aggressive features, showing an increased IFN-γ production and anti-apoptotic factors expression (Liuzzo et al., 2001), and could be involved in abrupt atherosclerotic plaque destabilization through several mechanisms. In fact, CD4+CD28null T-lymphocytes exert cytolitic effects on endothelial cells and express high levels of TNF-related apoptosis-inducing ligand (TRAIL), thus promoting smooth muscle cells apoptosis within the atherosclerotic plaque (Nakajima et al., 2002; Sato et al., 2006). With these premises, the recent finding of an expansion of CD4+ CD28null T-cells in diabetic patients is extremely interesting, suggesting a possible role of adaptive immune disregulation, either primary or induced by the altered metabolic status and the inflammatory environment characterizing the disease, in the increased cardiovascular risk which is one of the most relevant clinical features of T2DM, accounting for the majority of disease-related mortality and morbidity (Giubilato et al., 2011). Consistently, in the same study CD4+ CD28null T-lymphocytes expansion was closely related to a poor glycaemic control, and was associated with a higher incidence of cardiovascular events during followup.

54 Pathophysiology and Complications of Diabetes Mellitus

**6. Diabetes and adaptive immunity** 

Reimers, 1998.

Taken together, these findings support a crucial role of different molecules and pathways of innate immunity in the complex metabolic imbalance underlining T2DM, and possible contributing to the disease-associated cardiovascular risk. Insight into the above described

In the past years, a possible role of adaptive immunity and autoreactive mechanisms in the pathogenesis of T2DM has probably been underestimated and, therefore, poorly investigated. However, increasing evidences support the role of autoimmunity and adaptive immune system in the pathogenesis of T2DM and its vascular complications (Brooks-Worrell & Palmer, 2012; Nikolajczyk et al, 2011). It has been recently demonstrated that Tlymphocytes of patients with T2DM produce large amounts of pro-inflammatory cytokines, such as IL-8, showing in contrast a decreased production of anti-inflammatory cytokines, such as IL-10 (Jagannathan et al., 2010). These functional alterations are consistent with those previously demonstrated in monocytes of T2DM patients (Giulietti et al., 2007; Hatanaka et al., 2006; Pitocco et al., 2009), and result in an imbalance of cytokines network and in a strongly pro-inflammatory environment. High pro-inflammatory cytokines production has been associated in several studies with insulin-resistance and DM development, while the inhibition of some pro-inflammatory mediators prevented insulinresistance in mice (Arkan et al., 2005; Cai et al., 2005; de Roos et al., 2009; Ehses et al., 2009;

Moreover, the role of a perturbation of T-cell repertoire has been demonstrated in murine models of T2DM. Particularly, regulatory T-cells (Treg) are significantly diminished in the adipose tissue of obese insulin-resistant mice compared to non-obese animals. Treg cells isolated and expanded ex-vivo, in these models, were found able to exert an antiinflammatory activity and lessen insulin-resistance (Feuerer et al., 2009). On the other hand, Interferon (IFN)-γ-producing cells in the adipose tissue of obese mice may cause an imbalance in glucose homeostasis. The alterations mediated by T-cells with a Th1 phenotype, characterized by IFN- γ production, can be counterbalanced by CD4+T-cells with an anti-inflammatory phenotype, such as Treg and Th2 lymphocytes producing IL-10 (Winer et al., 2009), thus underlining the importance of a physiological balance between different T-cells subset in the metabolic homeostasis of adipose tissue, which has a crucial role in the pathogenesis of insulin resistance and T2DM onset. Another cellular type possibly involved in inflammation and insulin-resistance in T2DM are IL-17 producing Tcells, so called Th17. This aggressive, pro-inflammatory T-cell subset has been found at high levels following IL-6 stimulation in the spleen of obese mice, and could contribute to the inflammatory environment strongly related to insulin resistance development and maintenance in T2DM (Winer et al., 2009). Consistently with this hypothesis, high levels of cytokines conditioning T-cell differentiation toward a Th17 phenotype, such as IL-6, IL-1β and Tranforming Growth Factor (TGF)-β, have been measured in diabetic patients (Acosta-Rodriguez et al., 2007; Andriankaja et., 2009; Osborn et al., 2008; Yang et al., 2008). These pro-inflammatory cytokines could promote Th17 cells expansion and inhibit Treg

molecular pathways could help in the design of new therapeutic strategies.

Other fingerprints of adaptive immunity activation have been investigated in T2DM patients.

Increased activity of adenosine-deaminase (ADA) has been described in this population (Prakash et al., 2006). ADA is an enzyme that converts adenosine into inosine through an irreversible deamination reaction, and it is involved in T-cell proliferation and activation (Kather, 1990). Moreover, since adenosine increases glucose uptake into cells, an effect of ADA in reducing tissutal insulin sensitivity has been described (Gorrell et al., 2001). A recent study has confirmed an increased ADA activity in T2DM patients, underlining also an association between enzyme function and fasting glucose levels, as well as HbA1c. Thus, inflammation, T-lymphocytes activation and glucose metabolism seem to be tightly related in the complex setting of T2DM (Lee et al., 2011).

Tregs are another important T-cell type widely involved in autoreactive processes and in the modulation of the inflammatory environment associated with various diseases and pathological conditions. In the setting of diabetes mellitus, Tregs have been extensively investigated both in animal models and human patients with T1DM (Chatenoud et al., 2005, Randolph & Fathman, 2006), while less studies have been performed on Tregs in T2DM. Interestingly, a recent study in mice demonstrated that Treg induction was associated to a reduction of adipose tissue inflammation and insulin resistance, with a concomitant improvement of metabolic parameters of lipid metabolism and glycaemic control (Ilan et al, 2010). Consistently, a subsequent study proved an inverse relation between Treg expression

and function and insulin resistance in mice; Treg expansion was also associated with a reduction of signs of diabetes-related end-organ damage, such as nephropathy (Eller et al, 2011).

Type 2 Diabetes, Immunity and Cardiovascular Risk: A Complex Relationship 57

Type 2 diabetes is a complex disease involving the whole metabolic profile of the organism and exerting pathological effects on several organs and systems. The disease is associated with a chronic low-grade inflammation predictive of, and possibly responsible for, many of the clinical signs and complications of T2DM. The diabetes-associated inflammatory status can be the consequence of the metabolic abnormalities characterizing the disease, but increasing evidences are proposing also an important role of immune system disregulation, involving both innate and adaptive immunity, in the pathogenesis of T2DM. Cellular homeostasis is strictly dependent on the cross talk between immune system and metabolic regulators. Hence, any imbalances between them could represent a trigger for metabolic dysfunctions such those related to diabetes. Despite the huge number of evidences at our disposal highlighting the role of TLRs' and inflammasomes' pathways in pancreatic islets dysfunction and T2DM, to date no drugs directly targeting TLRs or the NLRP3 inflammasome have been developed. However, clinical trials have been addressed, with positive results, at evaluating the efficacy of downstream products' blockers, such as

Further studies are warranted in unraveling the complex relationship between T2DM and

Daniela Pedicino, Ada Francesca Giglio, Vincenzo Alessandro Galiffa, Francesco Trotta and

Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, & Sallusto F. (2007). Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. *Nat. Immunol*, 8, 9, (Sep 2007), 942–949,

ADVANCE Collaborative Group, Patel A, MacMahon S, Chalmers J, Neal B, Billot L, Woodward M, Marre M, Cooper M, Glasziou P, Grobbee D, Hamet P, Harrap S, Heller S, Liu L, Mancia G, Mogensen CE, Pan C, Poulter N, Rodgers A, Williams B, Bompoint S, de Galan BE, Joshi R & Travert F. (2008). Intensive blood-glucose control and cardiovascular outcomes in patients with type 2 diabetes. *N Engl J Med,*358, 24, (Jun

Al-Attas OS, Al-Daghri NM, Al-Rubeaan K, da Silva NF, Sabico SL, Kumar S, McTernan PG & Harte AL. (2009). Changes in endotoxin levels in T2DM subjects on anti-diabetic

immune system, and its implication for cardiovascular diseases.

**8. Conclusions** 

Anakinra, a recombinant IL-1RA.

*Institute of Cardiology, Catholic University, Rome, Italy* 

therapies. *Cardiovasc Diabetol* , 8, (Apr 2009),20

**Author details** 

Giovanna Liuzzo\*

**9. References** 

1529-2908

2008), 2560-2572

Corresponding Author

 \*

Finally, B-lymphocytes function has been poorly investigated in T2DM, but some data seem to indicate a role of these cells in the establishment and/or maintenance of a chronic proinflammatory state in this setting. For example, an altered B-cell activity related to cellular TLR dysfunction and leading to increased IL-8 and decreased IL-10 production has been recently demonstrated (Jagannathan et al, 2010).

Overall, these evidences suggest a diabetes-associated alteration of all components of adaptive immunity; these alterations could be implicated in the pathogenesis of the disease and, on the other hand, triggered and maintained by the disease itself, thus creating a proinflammatory, pro-atherosclerotic, vascular-damaging environment strongly associated with cardiovascular complications of T2DM.

## **7. Treating T2DM by targeting immunity**

As a role of inflammation has been suggested in the development of diabetes and its vascular complications, TLRs and inflammasome could represent attractive drug targets. Several drugs currently adopted to control hyperglycemia and inflammation and improve prognosis in T2DM patients may also exert their effects on TLR-mediated pathways. For example, it has been shown that statin therapy reduces TLR2 and TLR4 expression (Methe et al., 2005; Niessner et al., 2006; Stoll et al., 2006). The role of PPAR-γ agonists in inhibiting TLR activation both in vitro and in vivo has also been investigated (Dasu et al., 2009; Ji et al., 2009), as well as the ability of some angiotensin receptor blockers to decrease mRNA and protein levels of TLR2 and TLR4 (Dasu et al., 2009). However, although several molecules and drugs could potentially reduce inflammation associated with TLR signaling, studies on humans have to date shown a clear beneficial effect only related to statin therapy. Moreover, no drugs directly targeting TLRs have been developed.

For what concerns inflammasome's related pathways, the role of IL-1β in the impairment of pancreatic β -cell function, leading to apoptosis and decompensated insulin secretion, has prompted the use of anakinra in a double-blind clinical trial in patients with T2DM, that showed an improvement in β -cell secretory function, glycemia and inflammatory markers both during treatment and after drug withdrawal (Larsen et al., 2007, 2009).

A recent study tested in mice the efficacy of a high affinity monoclonal antibody to IL-1β, XOMA 052, showing an inhibition of atherosclerotic plaques formation (Bhaskar et al., 2011). Although clinical trials testing this antibody in T2DM patients failed in demonstrating an improvement in glycemic control, XOMA 052 potentially might reduce cardiovascular risk, since its administration in diabetic patients was associated with a reduction of inflammatory markers and increased levels of high-density lipoprotein.

Furthermore, drugs directly targeting caspase-1 have been tested in mice with promising results in reducing obesity and improving insulin sensitivity (Stienstra et al., 2010).

## **8. Conclusions**

56 Pathophysiology and Complications of Diabetes Mellitus

been recently demonstrated (Jagannathan et al, 2010).

**7. Treating T2DM by targeting immunity** 

no drugs directly targeting TLRs have been developed.

markers and increased levels of high-density lipoprotein.

cardiovascular complications of T2DM.

2011).

and function and insulin resistance in mice; Treg expansion was also associated with a reduction of signs of diabetes-related end-organ damage, such as nephropathy (Eller et al,

Finally, B-lymphocytes function has been poorly investigated in T2DM, but some data seem to indicate a role of these cells in the establishment and/or maintenance of a chronic proinflammatory state in this setting. For example, an altered B-cell activity related to cellular TLR dysfunction and leading to increased IL-8 and decreased IL-10 production has

Overall, these evidences suggest a diabetes-associated alteration of all components of adaptive immunity; these alterations could be implicated in the pathogenesis of the disease and, on the other hand, triggered and maintained by the disease itself, thus creating a proinflammatory, pro-atherosclerotic, vascular-damaging environment strongly associated with

As a role of inflammation has been suggested in the development of diabetes and its vascular complications, TLRs and inflammasome could represent attractive drug targets. Several drugs currently adopted to control hyperglycemia and inflammation and improve prognosis in T2DM patients may also exert their effects on TLR-mediated pathways. For example, it has been shown that statin therapy reduces TLR2 and TLR4 expression (Methe et al., 2005; Niessner et al., 2006; Stoll et al., 2006). The role of PPAR-γ agonists in inhibiting TLR activation both in vitro and in vivo has also been investigated (Dasu et al., 2009; Ji et al., 2009), as well as the ability of some angiotensin receptor blockers to decrease mRNA and protein levels of TLR2 and TLR4 (Dasu et al., 2009). However, although several molecules and drugs could potentially reduce inflammation associated with TLR signaling, studies on humans have to date shown a clear beneficial effect only related to statin therapy. Moreover,

For what concerns inflammasome's related pathways, the role of IL-1β in the impairment of pancreatic β -cell function, leading to apoptosis and decompensated insulin secretion, has prompted the use of anakinra in a double-blind clinical trial in patients with T2DM, that showed an improvement in β -cell secretory function, glycemia and inflammatory markers

A recent study tested in mice the efficacy of a high affinity monoclonal antibody to IL-1β, XOMA 052, showing an inhibition of atherosclerotic plaques formation (Bhaskar et al., 2011). Although clinical trials testing this antibody in T2DM patients failed in demonstrating an improvement in glycemic control, XOMA 052 potentially might reduce cardiovascular risk, since its administration in diabetic patients was associated with a reduction of inflammatory

Furthermore, drugs directly targeting caspase-1 have been tested in mice with promising

results in reducing obesity and improving insulin sensitivity (Stienstra et al., 2010).

both during treatment and after drug withdrawal (Larsen et al., 2007, 2009).

Type 2 diabetes is a complex disease involving the whole metabolic profile of the organism and exerting pathological effects on several organs and systems. The disease is associated with a chronic low-grade inflammation predictive of, and possibly responsible for, many of the clinical signs and complications of T2DM. The diabetes-associated inflammatory status can be the consequence of the metabolic abnormalities characterizing the disease, but increasing evidences are proposing also an important role of immune system disregulation, involving both innate and adaptive immunity, in the pathogenesis of T2DM. Cellular homeostasis is strictly dependent on the cross talk between immune system and metabolic regulators. Hence, any imbalances between them could represent a trigger for metabolic dysfunctions such those related to diabetes. Despite the huge number of evidences at our disposal highlighting the role of TLRs' and inflammasomes' pathways in pancreatic islets dysfunction and T2DM, to date no drugs directly targeting TLRs or the NLRP3 inflammasome have been developed. However, clinical trials have been addressed, with positive results, at evaluating the efficacy of downstream products' blockers, such as Anakinra, a recombinant IL-1RA.

Further studies are warranted in unraveling the complex relationship between T2DM and immune system, and its implication for cardiovascular diseases.

## **Author details**

Daniela Pedicino, Ada Francesca Giglio, Vincenzo Alessandro Galiffa, Francesco Trotta and Giovanna Liuzzo\* *Institute of Cardiology, Catholic University, Rome, Italy* 

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**Chapter 4** 

© 2012 Vujičić et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Vujičić et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Decline in glomerular filtration rate (ml/min/year)

**Diabetic Nephropathy** 

Gordana Đorđević and Sanjin Rački

http://dx.doi.org/10.5772/50115

**1. Introduction** 

Božidar Vujičić, Tamara Turk, Željka Crnčević-Orlić,

American physician Paul Kimmelstiel (1900-1970) in 1936 [3].

Diabetes Type 1 Type 2 Normoalbuminuria 1,2 - 3,6 0,96 Microalbuminuria 1,2 - 3,6 2,4 Proteinuria 9,6 - 12 5,4 - 7,2

http://emedicine.medscape.com/article/238946-overview. Accessed 2012 May 14

albuminuria and blood pressure remain within the normal range.

insulin) [4]. There are five stages in the development of diabetic nephropathy.

Diabetes mellitus (DM) is the most frequent cause of chronic kidney failure in both developed and developing countries [1]. Diabetic nephropathy, also known as Kimmelstiel-Wilson syndrome or nodular diabetic glomerulosclerosis or intercapillary glomerulonephritis, is a clinical syndrome characterized by albuminuria (>300 mg/day or >200 mcg/min) confirmed on at least two occasions 3-6 months apart, permanent and irreversible decrease in glomerular filtration rate (GFR) (Table 1), and arterial hypertension [2]. The syndrome was first described by a British physician Clifford Wilson (1906-1997) and

**Table 1.** Decline in glomerular filtration rate in various stages of type 1 and type 2 diabetes. Available:

Diabetic nephropathy is a chronic complication of both type 1 DM (beta cell destruction – absolute lack of insulin) and type 2 DM (insulin resistance and/or decreased secretion of

**Stage I:** Hypertrophic hyper filtration. In this stage, GFR is either normal or increased. Stage I lasts approximately five years from the onset of the disease. The size of the kidneys is increased by approximately 20% and renal plasma flow is increased by 10%-15%, while

Additional information is available at the end of the chapter


## **Chapter 4**

## **Diabetic Nephropathy**

Božidar Vujičić, Tamara Turk, Željka Crnčević-Orlić, Gordana Đorđević and Sanjin Rački

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50115

## **1. Introduction**

70 Pathophysiology and Complications of Diabetes Mellitus

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Diabetes mellitus (DM) is the most frequent cause of chronic kidney failure in both developed and developing countries [1]. Diabetic nephropathy, also known as Kimmelstiel-Wilson syndrome or nodular diabetic glomerulosclerosis or intercapillary glomerulonephritis, is a clinical syndrome characterized by albuminuria (>300 mg/day or >200 mcg/min) confirmed on at least two occasions 3-6 months apart, permanent and irreversible decrease in glomerular filtration rate (GFR) (Table 1), and arterial hypertension [2]. The syndrome was first described by a British physician Clifford Wilson (1906-1997) and American physician Paul Kimmelstiel (1900-1970) in 1936 [3].


**Table 1.** Decline in glomerular filtration rate in various stages of type 1 and type 2 diabetes. Available: http://emedicine.medscape.com/article/238946-overview. Accessed 2012 May 14

Diabetic nephropathy is a chronic complication of both type 1 DM (beta cell destruction – absolute lack of insulin) and type 2 DM (insulin resistance and/or decreased secretion of insulin) [4]. There are five stages in the development of diabetic nephropathy.

**Stage I:** Hypertrophic hyper filtration. In this stage, GFR is either normal or increased. Stage I lasts approximately five years from the onset of the disease. The size of the kidneys is increased by approximately 20% and renal plasma flow is increased by 10%-15%, while albuminuria and blood pressure remain within the normal range.

© 2012 Vujičić et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Vujičić et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Stage II**: The quiet stage. This stage starts approximately two years after the onset of the disease and is characterized by kidney damage with basement membrane thickening and mesangial proliferation. There are still no clinical signs of the disease. GFR returns to normal values. Many patients remain in this stage until the end of their life.

Diabetic Nephropathy 73

In the United States, the occurrence of diabetic nephropathy in patients beginning kidney replacement therapy doubled in the 1991-2001 period [12]. Fortunately, the trend has been decreasing, most likely due to the better prevention and earlier diagnosis and treatment of

Glomerular filtration barrier functions as a complex biological sieve. As opposed to other capillaries in the body, glomerular capillaries are highly permeable to water (hydraulic conductivity) and relatively impermeable to large molecules. Such permeability is possible because of the unique three-layer structure of glomerular filtration membrane consisting of endothelial glycocalyx, glomerular basement membrane, and podocytes (glomerular visceral epithelial cells). Pathological changes develop in the glomeruli of patients with

The severity of glomerular damage is proportional to GFR value, DM duration, and blood glucose regulation [16,17]. The main pathohystological changes in diabetic nephropathy include the thickening of the glomerular basement membrane (GBM), mesangial expansion, nodular sclerosis – Kimmelstiel-Wilson change, diffuse glomerular sclerosis, tubular interstitial fibrosis, and arteriosclerosis and hyalinosis of kidney blood vessels

**Figure 1.** Photography shows delicate structure of normal glomerulus with thin glomerular basement

membrane and unrecognizable mesangium. HE stain, X 400.

long-duration DM before the appearance of microalbuminuria.

DM [15].

**3. Pathology** 

(Figures 1-3).

**Stage III:** The microalbuminuria stage (albumin 30-300 mg/dU) or initial nephropathy. This is the first clinically detectable sign of glomerular damage. It usually occurs five to ten years after the onset of the disease. Blood pressure may be increased or normal. Approximately 40% of patients reach this stage.

**Stage IV:** Chronic kidney failure (CKF) is the irreversible stage. Proteinuria develops (albumin > 300 mg/dU), GFR decreases below 60 mL/min/1.73 m2, and blood pressure increases above normal values.

**Stage V:** Terminal kidney failure (TKF) (GFR < 15 mL/min/1.73 m2). Approximately 50% of the patients with TKF require kidney replacement therapy (peritoneal dialysis, hemodialysis, kidney transplantation) [5].

In the initial stages of diabetic nephropathy, increased kidney size and changed Doppler indicators may be the early morphological signs of renal damage, while proteinuria and GFR are the best indicators of the degree of the damage [6].

## **2. Epidemiology**

The prognostic value of a small amount of albumin in urine for the development of kidney damage in patients with type 1 or 2 DM was confirmed in the early 1980's. This stage of kidney damage was called the microalbuminuria stage or initial nephropathy [7]. Approximately 20-30% of the patients develop microalbuminuria after 15 years of disease duration and less than half develop real nephropathy [8]. The European Diabetes (EURODIAB) Prospective Complications Study Group [9] and 18-year Danish study [10] showed that the overall occurrence of microalbuminuria in patients with type 1 and 2 DM is 12.6% (after 7.3 years) and 33%, respectively. According to the United Kingdom Prospective Diabetes Study (UKPDS), the annual incidence of microalbuminuria in patients with type 2 DM in Great Britain is 2% and the prevalence is 25% ten years after the diagnosis [2]. Proteinuria develops in approximately 15-40% patients with type 1 DM, usually after 15-20 years of DM duration [11]. In patients with type 2 DM, the prevalence varies between 5% and 20% on average [2]. Diabetic nephropathy is more frequent in African Americans, Asian Americans, and Native Americans [12]. In Caucasians, the progressive kidney disease is more frequent in patients with type 1 than type 2 DM, although its overall prevalence in the diabetic population is higher in patients with type 2 DM because this type of DM is more prevalent [13]. The occurrence of diabetic nephropathy in Pima Indians is very interesting, indeed. According to a study published in 1990, around 50% of Pima Indians with type 2 DM developed nephropathy after 20 years of the disease, and 15% of them were already in the terminal stage of kidney failure [14].

In the United States, the occurrence of diabetic nephropathy in patients beginning kidney replacement therapy doubled in the 1991-2001 period [12]. Fortunately, the trend has been decreasing, most likely due to the better prevention and earlier diagnosis and treatment of DM [15].

## **3. Pathology**

72 Pathophysiology and Complications of Diabetes Mellitus

40% of patients reach this stage.

increases above normal values.

**2. Epidemiology** 

hemodialysis, kidney transplantation) [5].

the terminal stage of kidney failure [14].

GFR are the best indicators of the degree of the damage [6].

**Stage II**: The quiet stage. This stage starts approximately two years after the onset of the disease and is characterized by kidney damage with basement membrane thickening and mesangial proliferation. There are still no clinical signs of the disease. GFR returns to normal

**Stage III:** The microalbuminuria stage (albumin 30-300 mg/dU) or initial nephropathy. This is the first clinically detectable sign of glomerular damage. It usually occurs five to ten years after the onset of the disease. Blood pressure may be increased or normal. Approximately

**Stage IV:** Chronic kidney failure (CKF) is the irreversible stage. Proteinuria develops (albumin > 300 mg/dU), GFR decreases below 60 mL/min/1.73 m2, and blood pressure

**Stage V:** Terminal kidney failure (TKF) (GFR < 15 mL/min/1.73 m2). Approximately 50% of the patients with TKF require kidney replacement therapy (peritoneal dialysis,

In the initial stages of diabetic nephropathy, increased kidney size and changed Doppler indicators may be the early morphological signs of renal damage, while proteinuria and

The prognostic value of a small amount of albumin in urine for the development of kidney damage in patients with type 1 or 2 DM was confirmed in the early 1980's. This stage of kidney damage was called the microalbuminuria stage or initial nephropathy [7]. Approximately 20-30% of the patients develop microalbuminuria after 15 years of disease duration and less than half develop real nephropathy [8]. The European Diabetes (EURODIAB) Prospective Complications Study Group [9] and 18-year Danish study [10] showed that the overall occurrence of microalbuminuria in patients with type 1 and 2 DM is 12.6% (after 7.3 years) and 33%, respectively. According to the United Kingdom Prospective Diabetes Study (UKPDS), the annual incidence of microalbuminuria in patients with type 2 DM in Great Britain is 2% and the prevalence is 25% ten years after the diagnosis [2]. Proteinuria develops in approximately 15-40% patients with type 1 DM, usually after 15-20 years of DM duration [11]. In patients with type 2 DM, the prevalence varies between 5% and 20% on average [2]. Diabetic nephropathy is more frequent in African Americans, Asian Americans, and Native Americans [12]. In Caucasians, the progressive kidney disease is more frequent in patients with type 1 than type 2 DM, although its overall prevalence in the diabetic population is higher in patients with type 2 DM because this type of DM is more prevalent [13]. The occurrence of diabetic nephropathy in Pima Indians is very interesting, indeed. According to a study published in 1990, around 50% of Pima Indians with type 2 DM developed nephropathy after 20 years of the disease, and 15% of them were already in

values. Many patients remain in this stage until the end of their life.

Glomerular filtration barrier functions as a complex biological sieve. As opposed to other capillaries in the body, glomerular capillaries are highly permeable to water (hydraulic conductivity) and relatively impermeable to large molecules. Such permeability is possible because of the unique three-layer structure of glomerular filtration membrane consisting of endothelial glycocalyx, glomerular basement membrane, and podocytes (glomerular visceral epithelial cells). Pathological changes develop in the glomeruli of patients with long-duration DM before the appearance of microalbuminuria.

The severity of glomerular damage is proportional to GFR value, DM duration, and blood glucose regulation [16,17]. The main pathohystological changes in diabetic nephropathy include the thickening of the glomerular basement membrane (GBM), mesangial expansion, nodular sclerosis – Kimmelstiel-Wilson change, diffuse glomerular sclerosis, tubular interstitial fibrosis, and arteriosclerosis and hyalinosis of kidney blood vessels (Figures 1-3).

**Figure 1.** Photography shows delicate structure of normal glomerulus with thin glomerular basement membrane and unrecognizable mesangium. HE stain, X 400.

Diabetic Nephropathy 75

Among other pathological lesions, we should mention hyalinosis, the so-called fibrin cap, which consists of accumulated hyaline material between endothelial cells and glomerular basement membrane (Figure 4) [18]. Fibrin cap is present in approximately 60% of the cases

**Figure 4.** Fibrin cap (arrow) is characteristic for diabetic nephropathy. It is caused by insudation and accumulation of glycosilated plasma proteins between the glomerular endothelium and the glomerular basement membrane. Diffuse expansion of mesangium is designated by four point star. PAS stain,

There is a significant overlap between the described changes in patients in different stages of albuminuria, independent of their type of DM [16]. All histological patterns have identical prognostic significance (Figures 5,6). However, the fact that the expansion of mesangium and glomerular sclerosis do not occur simultaneously indicates their different pathogenesis within diabetic nephropathy [20]. Under light microscopy, the reduction in the podocyte

Since histological changes in both types of DM overlap to a great extent, the Scientific Committee of the Society for Pathological Anatomy established the Pathologic Classification of Diabetic Nephropathy, where diabetic nephropathy is histologically divided into four

number is easily noticed in patients with type 1 DM and 2 [21].

stages of glomerular damage (Table 2).

X 200.

and is believed to be associated with chronic ischemia [19].

**Figure 2.** Class II b diabetic nephropathy. Diffuse expansion of mesangium (star) and diffuse thickening of the glomerular basement membrane (arrow). PAS stain, X400.

**Figure 3.** Class III diabetic nephropathy. Sclerotic nodule (Kimmelstiel–Wilson) in nodular diabetic nephropathy (arrow). Afferent and efferent arteriolar hyalinosis is characteristic for diabetic nephropathy (star). The arrow in the lower right corner indicates thickening of the tubular basement membrane. Mallory stain, X 100.

Among other pathological lesions, we should mention hyalinosis, the so-called fibrin cap, which consists of accumulated hyaline material between endothelial cells and glomerular basement membrane (Figure 4) [18]. Fibrin cap is present in approximately 60% of the cases and is believed to be associated with chronic ischemia [19].

74 Pathophysiology and Complications of Diabetes Mellitus

**Figure 2.** Class II b diabetic nephropathy. Diffuse expansion of mesangium (star) and diffuse thickening

**Figure 3.** Class III diabetic nephropathy. Sclerotic nodule (Kimmelstiel–Wilson) in nodular diabetic nephropathy (arrow). Afferent and efferent arteriolar hyalinosis is characteristic for diabetic nephropathy (star). The arrow in the lower right corner indicates thickening of the tubular basement

of the glomerular basement membrane (arrow). PAS stain, X400.

membrane. Mallory stain, X 100.

**Figure 4.** Fibrin cap (arrow) is characteristic for diabetic nephropathy. It is caused by insudation and accumulation of glycosilated plasma proteins between the glomerular endothelium and the glomerular basement membrane. Diffuse expansion of mesangium is designated by four point star. PAS stain, X 200.

There is a significant overlap between the described changes in patients in different stages of albuminuria, independent of their type of DM [16]. All histological patterns have identical prognostic significance (Figures 5,6). However, the fact that the expansion of mesangium and glomerular sclerosis do not occur simultaneously indicates their different pathogenesis within diabetic nephropathy [20]. Under light microscopy, the reduction in the podocyte number is easily noticed in patients with type 1 DM and 2 [21].

Since histological changes in both types of DM overlap to a great extent, the Scientific Committee of the Society for Pathological Anatomy established the Pathologic Classification of Diabetic Nephropathy, where diabetic nephropathy is histologically divided into four stages of glomerular damage (Table 2).


Diabetic Nephropathy 77

In addition to diabetic nephropathy, glomerular sclerosis can also develop in other

d. Idiopathic diseases mostly associated with smoking and increased blood pressure [23]. It means that pathological findings in the urine of patients with DM (proteinuria and erythrocyturia) are not necessarily the result of diabetic nephropathy and should not be considered as such. This finding is a diagnostic challenge for a clinician as well as pathologist [24]. Therefore, in case of hematuria, more severe nephrotic syndrome, and/or rapidly advancing deterioration of renal function without concomitant diabetic nephropathy in patients with DM, we should consider an underlying non-diabetic kidney disease. Kidney biopsy with a complete analysis of the sample (light, immunofluorescent, and electron microscopies) represents the gold standard in the diagnostic workup of patients with non-diabetic renal disease. Always correlate the biopsy findings with the clinical history. If the patient is not diabetic, consider the diagnosis of idiopathic nodular

**Figure 5.** There was marked thickening, irregularity of the basement membrane of the capillary wall

with lamellation (electron microscopy, arrow, 2.8 k)

pathological conditions in patients with DM. These are:

c. chronic membranoproliferative glomerulonephritis

glomerulosclerosis

a. dysproteinemia (amyloidosis and other deposit diseases)

b. conditions with chronic ischemia (cyanotic congenital heart disease)

**Table 2.** Four classes of glomerular lesions in diabetic nephropathy. Adapted from [22].

LM, light microscopy. EM, electronic microscopy. GBM, glomerular basement membrane.

a On the basis of direct measurement of GBM width by EM, these individual cutoff levels may be considered indicative when other GBM measurements are used.

The same group of international experts established the histological scoring system for the changes in the interstitium and relevant blood vessels (Table 3) [22].


**Table 3.** Interstitial and vascular lesions of diabetic nephropaty. Adapted from [22]. IFTA, interstitial fibrosis and tubular athrophy.

In addition to diabetic nephropathy, glomerular sclerosis can also develop in other pathological conditions in patients with DM. These are:


76 Pathophysiology and Complications of Diabetes Mellitus

changes and EM-proven GBM

(Kimmelstiel – Wilson lesion)

considered indicative when other GBM measurements are used.

changes in the interstitium and relevant blood vessels (Table 3) [22].

I Mild or nonspecific LM

thickening

III Nodular sclerosis

IV Advanced diabetic

Interstitial lesions

Vascular lesions

artery)

arteriosclerosis (score worst

IFTA, interstitial fibrosis and tubular athrophy.

glomerulosclerosis

Class Description Inclusion criteria

Biopsy does not meet any of the criteria

Mild mesangial expansion in 25% of the

Severe mesangial expansion in 25% of the

Biopsy does not meet criteria for class IV At least one convincing Kimmelstiel –Wilson lesion

Lesions from classes I through III

< 25% 1 25% - 50% 2 > 50% 3

Infiltration only in relation to IFTA 1 Infiltration in areas without IFTA 2

At least one area of arteriolar hyalinosis 1 More than one area of arteriolar hyalinosis 2

No intimal thickening 0

Intimal thickening less than thickness of media 1 Intimal thickening greater than thickness of media 2

Global glomerular sclerosis in 50% of Glomeruli

years of age and oldera

observed mesangium

observed mesangium

II a Mild mesangial expansion Biopsy does not meet criteria for class III or IV

II b Severe mesangial expansion Biopsy does not meet criteria for class III or IV

The same group of international experts established the histological scoring system for the

Lesion Criteria Score

IFTA No IFTA 0

interstitial inflammation Absent 0

arteriolar hyalinosis Absent 0

**Table 3.** Interstitial and vascular lesions of diabetic nephropaty. Adapted from [22].

presence of large vessels Yes/No

**Table 2.** Four classes of glomerular lesions in diabetic nephropathy. Adapted from [22]. LM, light microscopy. EM, electronic microscopy. GBM, glomerular basement membrane. a On the basis of direct measurement of GBM width by EM, these individual cutoff levels may be

mentioned below for class II, III, or IV GBM 395 nm in female and 430 nm in male individuals 9

d. Idiopathic diseases mostly associated with smoking and increased blood pressure [23].

It means that pathological findings in the urine of patients with DM (proteinuria and erythrocyturia) are not necessarily the result of diabetic nephropathy and should not be considered as such. This finding is a diagnostic challenge for a clinician as well as pathologist [24]. Therefore, in case of hematuria, more severe nephrotic syndrome, and/or rapidly advancing deterioration of renal function without concomitant diabetic nephropathy in patients with DM, we should consider an underlying non-diabetic kidney disease. Kidney biopsy with a complete analysis of the sample (light, immunofluorescent, and electron microscopies) represents the gold standard in the diagnostic workup of patients with non-diabetic renal disease. Always correlate the biopsy findings with the clinical history. If the patient is not diabetic, consider the diagnosis of idiopathic nodular glomerulosclerosis

**Figure 5.** There was marked thickening, irregularity of the basement membrane of the capillary wall with lamellation (electron microscopy, arrow, 2.8 k)

Diabetic Nephropathy 79

The role of hormones was experimentally demonstrated in the study by Serri et al, who showed that the infusion of somatostatin analogues (octreotide) partly led to the decrease in hyperfiltration and kidney size. In their study, glycemic regulation, plasma glucagon, and growth hormone levels remained unchanged, but the concentration of insulin-like growth factor-1 (IGF-1) decreased [28]. Pathogenetic role of IGF-1 has not been completely elucidated, but it is known that exogenous administration of his hormone in non-DM patients leads to afferent arteriolar dilation and GFR increase, which are the changes also observed in initial diabetic nephropathy [29]. The identical hemodynamic changes, along with the increase in kidney size, occur in experimental animal models after the infusion of IGF-1 [30]. Sex hormones may also influence hyperfiltration. Cherney et al. [31] observed a decrease in kidney blood flow and vascular resistance in response to hyperglycemia in women, but not in men. The same study showed that the addition of angiotensin-converting enzyme inhibitor (ACEI) resulted in a decrease in blood pressure in both men and women,

The enzyme aldose reductase converts intracellular glucose to sorbitol, which remains in the cell. Although research in patients with type 1 DM and known hyperfiltration has shown that the infusion of aldose reductase inhibitor (tolrestat) decreases GFR to normal values, a

Increased renal tubular sodium reabsorption due to increased sodium-glucose cotransport leads to the increase in extracellular fluid volume, which then increases GFR [33]. In an experimental DM model, it was shown that hyperinsulinemia and mild hyperglycemia stimulate reabsorption of sodium in the proximal tubules, resulting in the decreased fluid flow to distal tubules, which then activates the so-called tubuloglomerular feedback mechanism in the macula densa [34]. This causes the afferent arteriole dilation and leads to an increase in the GFR. In this case, the renal hyper filtration response to the imbalance caused by increased sodium reabsorption in the proximal tubules consequently increases fluid retention. Although the role of glomerular hyperfiltration in the pathogenesis of diabetic nephropathy is unquestionable, it itself is not sufficient to cause

Part of the excess glucose in chronic hyperglycemia binds to free amino acids of circulating or tissue proteins. This non-enzymatic process produces reversible early glycation products, and later, irreversible advanced glycation end products (AGEs), which accumulate in the tissues and contribute to the development of microvascular complications of DM[35].

possible therapeutic use of this agent should be confirmed in more studies [32].

**Increased sodium reabsorption and tubuloglomerular feedback**

**Hormones** 

**Sorbitol** 

kidney damage.

*Glycation end-products* 

**Poor control of metabolic factors** 

but GFR decreased only in women [31].

**Figure 6.** Mesangial regions are also expanded by excess mesangial matrix-like material (electron microscopy, arrow, 7.1 k)

If primary glomerular disease is found in a patient with DM, with or without diabetic nephropathy, the therapeutic approach changes as well as the course and outcome of the renal disease [25].

## **4. Pathogenesis**

Pathogenesis of diabetic nephropathy is very complicated and results from the interaction of hemodynamic and metabolic factors.

### **Glomerular hyper filtration**

Increased intraglomerular pressure and hyper filtration as early changes in the development of diabetic nephropathy were described by Stadler and Schmidt in 1959 [26]. In the 1970's, Mogensen emphasized that as many as 40% newly found DM cases had increased glomerular filtration [27].

Although the mechanism of development of hyper filtration is not completely understood, several factors have been found to play a role in its development.

#### **Hormones**

78 Pathophysiology and Complications of Diabetes Mellitus

microscopy, arrow, 7.1 k)

renal disease [25].

**4. Pathogenesis** 

hemodynamic and metabolic factors.

**Glomerular hyper filtration** 

glomerular filtration [27].

**Figure 6.** Mesangial regions are also expanded by excess mesangial matrix-like material (electron

If primary glomerular disease is found in a patient with DM, with or without diabetic nephropathy, the therapeutic approach changes as well as the course and outcome of the

Pathogenesis of diabetic nephropathy is very complicated and results from the interaction of

Increased intraglomerular pressure and hyper filtration as early changes in the development of diabetic nephropathy were described by Stadler and Schmidt in 1959 [26]. In the 1970's, Mogensen emphasized that as many as 40% newly found DM cases had increased

Although the mechanism of development of hyper filtration is not completely understood,

several factors have been found to play a role in its development.

The role of hormones was experimentally demonstrated in the study by Serri et al, who showed that the infusion of somatostatin analogues (octreotide) partly led to the decrease in hyperfiltration and kidney size. In their study, glycemic regulation, plasma glucagon, and growth hormone levels remained unchanged, but the concentration of insulin-like growth factor-1 (IGF-1) decreased [28]. Pathogenetic role of IGF-1 has not been completely elucidated, but it is known that exogenous administration of his hormone in non-DM patients leads to afferent arteriolar dilation and GFR increase, which are the changes also observed in initial diabetic nephropathy [29]. The identical hemodynamic changes, along with the increase in kidney size, occur in experimental animal models after the infusion of IGF-1 [30]. Sex hormones may also influence hyperfiltration. Cherney et al. [31] observed a decrease in kidney blood flow and vascular resistance in response to hyperglycemia in women, but not in men. The same study showed that the addition of angiotensin-converting enzyme inhibitor (ACEI) resulted in a decrease in blood pressure in both men and women, but GFR decreased only in women [31].

#### **Sorbitol**

The enzyme aldose reductase converts intracellular glucose to sorbitol, which remains in the cell. Although research in patients with type 1 DM and known hyperfiltration has shown that the infusion of aldose reductase inhibitor (tolrestat) decreases GFR to normal values, a possible therapeutic use of this agent should be confirmed in more studies [32].

#### **Increased sodium reabsorption and tubuloglomerular feedback**

Increased renal tubular sodium reabsorption due to increased sodium-glucose cotransport leads to the increase in extracellular fluid volume, which then increases GFR [33]. In an experimental DM model, it was shown that hyperinsulinemia and mild hyperglycemia stimulate reabsorption of sodium in the proximal tubules, resulting in the decreased fluid flow to distal tubules, which then activates the so-called tubuloglomerular feedback mechanism in the macula densa [34]. This causes the afferent arteriole dilation and leads to an increase in the GFR. In this case, the renal hyper filtration response to the imbalance caused by increased sodium reabsorption in the proximal tubules consequently increases fluid retention. Although the role of glomerular hyperfiltration in the pathogenesis of diabetic nephropathy is unquestionable, it itself is not sufficient to cause kidney damage.

#### **Poor control of metabolic factors**

#### *Glycation end-products*

Part of the excess glucose in chronic hyperglycemia binds to free amino acids of circulating or tissue proteins. This non-enzymatic process produces reversible early glycation products, and later, irreversible advanced glycation end products (AGEs), which accumulate in the tissues and contribute to the development of microvascular complications of DM[35].

AGEs modulate the cell activation, signal transduction, and cytokine and growth factor expression through the activation of R-dependent and R-independent signal pathways. Bonding to their podocyte receptors, AGEs may induce expression of some factors considered to play the key role in the pathogenesis of diabetic nephropathy, such as transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) [36]. In non-diabetic mice, the infusion of early products of glycation up to the concentration seen in diabetic mice increases the kidneys blood flow, GFR, and intraglomerular pressure, which are characteristic of untreated DM [37].

Diabetic Nephropathy 81

**Cytokines and Growth Factors** 

of fibrosis in kidney injury [48].

TGF-beta is increased [50,51].

**Nephrine Expression** 

**5. Risk factors** 

**Genetic Predisposition** 

the development of this disease.

**Race** 

Hyperglycemia stimulates increased expression of different growth factors and activation of

In the kidney biopsy samples from patients with type 2 DM, a significant increase in platelet derived growth factor (PDGF) expression was found. Moreover, the site of expression of this factor is adjacent to the areas of interstitial fibrosis, which is important in the pathogenesis

Hyperglycemia also increases the glomerular expression of TGF-beta; matrix proteins are specifically stimulated by this growth factor [49]. Furthermore, the expression of bone morphogenic protein 7 (BMP-7) in DM is decreased, and the expression of profibrinogenic

Nephrine is a transmembrane protein, the main structural element in *slit* diaphragm and as such, it is important for the maintenance of filtration membrane integrity. More recent studies have shown the association between the decreased expression of nephrine and

There are several risk factors for the development of diabetic nephropathy. They can be divided into those that cannot be altered (genetic factors, age, and race) and those that can

Genetic predisposition substantially determines the occurrence and severity of diabetic nephropathy [18,40]. The likeliness of diabetic nephropathy is higher in siblings and children of parents with diabetic nephropathy, independently of the type of DM [54]. There is a 14% probability for a child of the parents without proteinuria to develop clinical proteinuria, 23% probabilities in cases where one of the parents has proteinuria, and 46% probability in case that both parents have proteinuria. This increased risk cannot be explained by the duration of DM, increased blood pressure or gycemic regulation. However, genetic predisposition for excessive salt intake and arterial hypertension could play a role. Although likeliness of chromosomes 3, 7, 18, and 20 to be associated with diabetic nephropathy is relatively high, we still cannot confirm the role of particular predisposing genetic determinants due to inconsistent results of the studies of genetic factors important in

The incidence of diabetic nephropathy is increased in African American, Mexican American, and Asian Indian ethnic groups. Occurrence and severity of the disease are higher in Blacks

albuminuria progression in the model of human diabetic nephropathy [52,53].

and must be changed (hyperglycemia, hypertension, dyslipidemia, and GFR)[53].

cytokines, which overall contributes to further kidney damage [46,47].

#### *Hyperglycemia*

The evidence from *in vitro* studies shows that hyperglycemia has a direct effect on mesangial cell proliferation, matrix expansion, and glycosylation of glomerular proteins [38,39].

#### *Protein kinase C*

The activation of protein kinase C (PKC) is one of the main mediators of hyperglycemiainduced tissue injury. PCK activation leads to increased vascular permeability, increased synthesis of extracellular matrix components, and increased production of reactive oxygen species (ROS), which are important mediators of kidney injury [40].

#### **Heparanase Expression**

The regulation of heparanase expression plays an important role in the pathogenesis of diabetic nephropathy. The reduction in heparin sulfate on the surface of endothelial cell changes the negative charge of glycocalyx and consequently increases albumin permeability of the glomerular filtration membrane [41].

#### **Reactive Oxygen Species**

Increasing evidence shows the importance of reactive oxygen species (ROS) in the pathogenesis of diabetic nephropathy. Although the ROS production may be influenced by numerous mechanisms, the most important role in their production is played by superoxide produced by glycolysis and oxidative phosphorylation in the mitochondria. ROS activate all important pathogenetic mechanisms, such as increased production of AGEs, increased glucose entry into the polyol pathway, and PKC activation [42]. In addition, ROS directly damage endothelial glycocalyx, which leads to albuminuria without the concurrent damage to the GBM itself.

#### **Prorenin**

Increased serum prorenin plays a role in the development of diabetic nephropathy in children and adolescents [43]. Prorenin binds to a specific tissue receptor, leading to the activation of the signal pathway of mitogen-activating protein kinases (MAPK), which potentiate the development of kidney damage [44]. Using an experimental model of diabetic nephropathy, Ichihara et al. [45] indicated a possible role of prorenin in the development of diabetic nephropathy. In their study, a prolonged prorenin receptor blockade cancelled the activation of MAPK, which prevented the development of diabetic nephropathy despite the increased activity of angiotensine II.

#### **Cytokines and Growth Factors**

80 Pathophysiology and Complications of Diabetes Mellitus

which are characteristic of untreated DM [37].

of the glomerular filtration membrane [41].

increased activity of angiotensine II.

*Hyperglycemia* 

*Protein kinase C* 

**Heparanase Expression** 

**Reactive Oxygen Species** 

**Prorenin** 

AGEs modulate the cell activation, signal transduction, and cytokine and growth factor expression through the activation of R-dependent and R-independent signal pathways. Bonding to their podocyte receptors, AGEs may induce expression of some factors considered to play the key role in the pathogenesis of diabetic nephropathy, such as transforming growth factor-beta (TGF-beta) and connective tissue growth factor (CTGF) [36]. In non-diabetic mice, the infusion of early products of glycation up to the concentration seen in diabetic mice increases the kidneys blood flow, GFR, and intraglomerular pressure,

The evidence from *in vitro* studies shows that hyperglycemia has a direct effect on mesangial

The activation of protein kinase C (PKC) is one of the main mediators of hyperglycemiainduced tissue injury. PCK activation leads to increased vascular permeability, increased synthesis of extracellular matrix components, and increased production of reactive oxygen

The regulation of heparanase expression plays an important role in the pathogenesis of diabetic nephropathy. The reduction in heparin sulfate on the surface of endothelial cell changes the negative charge of glycocalyx and consequently increases albumin permeability

Increasing evidence shows the importance of reactive oxygen species (ROS) in the pathogenesis of diabetic nephropathy. Although the ROS production may be influenced by numerous mechanisms, the most important role in their production is played by superoxide produced by glycolysis and oxidative phosphorylation in the mitochondria. ROS activate all important pathogenetic mechanisms, such as increased production of AGEs, increased glucose entry into the polyol pathway, and PKC activation [42]. In addition, ROS directly damage endothelial glycocalyx, which leads to albuminuria without the concurrent damage to the GBM itself.

Increased serum prorenin plays a role in the development of diabetic nephropathy in children and adolescents [43]. Prorenin binds to a specific tissue receptor, leading to the activation of the signal pathway of mitogen-activating protein kinases (MAPK), which potentiate the development of kidney damage [44]. Using an experimental model of diabetic nephropathy, Ichihara et al. [45] indicated a possible role of prorenin in the development of diabetic nephropathy. In their study, a prolonged prorenin receptor blockade cancelled the activation of MAPK, which prevented the development of diabetic nephropathy despite the

cell proliferation, matrix expansion, and glycosylation of glomerular proteins [38,39].

species (ROS), which are important mediators of kidney injury [40].

Hyperglycemia stimulates increased expression of different growth factors and activation of cytokines, which overall contributes to further kidney damage [46,47].

In the kidney biopsy samples from patients with type 2 DM, a significant increase in platelet derived growth factor (PDGF) expression was found. Moreover, the site of expression of this factor is adjacent to the areas of interstitial fibrosis, which is important in the pathogenesis of fibrosis in kidney injury [48].

Hyperglycemia also increases the glomerular expression of TGF-beta; matrix proteins are specifically stimulated by this growth factor [49]. Furthermore, the expression of bone morphogenic protein 7 (BMP-7) in DM is decreased, and the expression of profibrinogenic TGF-beta is increased [50,51].

#### **Nephrine Expression**

Nephrine is a transmembrane protein, the main structural element in *slit* diaphragm and as such, it is important for the maintenance of filtration membrane integrity. More recent studies have shown the association between the decreased expression of nephrine and albuminuria progression in the model of human diabetic nephropathy [52,53].

## **5. Risk factors**

There are several risk factors for the development of diabetic nephropathy. They can be divided into those that cannot be altered (genetic factors, age, and race) and those that can and must be changed (hyperglycemia, hypertension, dyslipidemia, and GFR)[53].

#### **Genetic Predisposition**

Genetic predisposition substantially determines the occurrence and severity of diabetic nephropathy [18,40]. The likeliness of diabetic nephropathy is higher in siblings and children of parents with diabetic nephropathy, independently of the type of DM [54]. There is a 14% probability for a child of the parents without proteinuria to develop clinical proteinuria, 23% probabilities in cases where one of the parents has proteinuria, and 46% probability in case that both parents have proteinuria. This increased risk cannot be explained by the duration of DM, increased blood pressure or gycemic regulation. However, genetic predisposition for excessive salt intake and arterial hypertension could play a role. Although likeliness of chromosomes 3, 7, 18, and 20 to be associated with diabetic nephropathy is relatively high, we still cannot confirm the role of particular predisposing genetic determinants due to inconsistent results of the studies of genetic factors important in the development of this disease.

#### **Race**

The incidence of diabetic nephropathy is increased in African American, Mexican American, and Asian Indian ethnic groups. Occurrence and severity of the disease are higher in Blacks

(3- to 6-fold in comparison with Caucasians), American Mexicans, and especially in Pima Indians in the North West part of the United States [55]. This observation in genetically incongruent populations suggests that socioeconomic factors, such as nutrition and poor control of glycemia, blood pressure, and body weight, play the key role.

Diabetic Nephropathy 83

approximately 25-50% above normal range. These patients have a higher risk of developing

Dynamics of structural and hemodynamic changes is influenced by increased intraglomerular pressure, with the resulting glomerular hyperfiltration and hypertrophy and damage to the endothelial wall. Strict glycemic control, limited protein intake, and blood pressure control may slow down the progress of renal disease in type 1 DM [62]. The situation with type 2 DM is somewhat different. More than 45% of patients with type 2 DM at diagnosis have GFR that is two standard deviations higher than that in their age-matched no-DM or overweight controls [63]. Granted, the hyper filtration rate (117-133 mL/min on average) is lower than that in type 1 DM. Patients with type 2 DM are older and, therefore, have greater likelihood of developing atherosclerotic vascular changes that influence GFR and glomerular size [64]. The role of intraglomerular hypertension in the pathogenesis of diabetic nephropathy explains why systemic hypertension is such an important risk factor for the development of this kidney disease [65]. Studies on animal models showed that DM is associated with damage of renal autoregulation. As a result, increased blood pressure does not induce the expected vasoconstriction in the afferent arteriole, which would reduce

Diabetic nephropathy often develops in patients with poor glycemic control. The degree of glycemic control is an important predictor of terminal kidney failure [67]. In Krolewski et al's [68] study, the prevalence of terminal kidney failure was 36% in patients with the worst glycemic control in comparison with 9% in the group with well-controlled glycaemia.

It is generally accepted that the degree of glycemic control is a very important risk factor for

High body mass index (BMI) increases the risk of development of chronic kidney disease in patients with DM [53]. Furthermore, adequate diet and reduction in body weight decrease proteinuria and improve kidney function in these patients [69]. The role of overweight as a risk factor for diabetic nephropathy (independent of DM and glycemic control) has not been

Although recent studies have shown the association between smoking and progression of diabetic nephropathy, a large prospective study by Hovind et al. [70] did not confirm the association between smoking and decreased GFR rate in patients with DM with or without

Ahmed et al. [71] showed the association between the use of oral contraceptives and

the influence of systemic hypertension on intraglomerular pressure [66].

diabetic nephropathy [61].

**Glycemic Regulation** 

**Overweight** 

clearly confirmed.

**Smoking** 

ACEI therapy.

**Oral Contraception** 

development of diabetic nephropathy.

the development diabetic nephropathy.

#### **Age**

In patients with type 2 DM, age and duration of DM increase the risk for albuminuria [53]. In the population study of 1586 Pima Indians with type 2 DM, subjects diagnosed with DM before age 20 had a higher risk of developing terminal kidney failure (25 vs. 5 patients in 1000 incident patients). According to Svensson et al. [56] the risk of terminal kidney failure in patients with type 1 DM was low if the disease was diagnosed by the age of 5.

#### **Increased Blood Pressure**

There is a high prevalence rate of hypertension in patients with type 1 DM (40%) and type 2 DM (70%), even before albuminuria can be found.

Evidence from several large clinical studies (UKPDS, ADVANCE) indicates a causal relationship between the increased arterial pressure and diabetic nephropathy [57]. Moreover, at least three factors have been shown to contribute to the development of increased arterial pressure in this metabolic disorder including hyperinsulinemia, excessive extracellular fluid volume, and increased arterial rigidity. Hyperinsulinemia contributes to the development of increased arterial pressure via insulin resistance in type 2 DM or via administration of insulin per *se*. Randeree et al. study in 80 patients with type 2 DM who started treatment with exogenous insulin showed an increase in their blood pressure from 132/81 mm Hg to 149/89 mm Hg [58]. This hypertensive response, although not reported in all clinical studies, is most likely mediated by weight gain combined with pro-hypertensive effect of insulin. Hyperinsulinemia could be the link between overweight and increased blood pressure in patients with or without DM, since it increases sympathetic activity and retention of sodium in the kidneys.

Sodium and water retention are induced by insulin itself, while the increased filtration of glucose is induced by hyperglycemia. The excess filtered glucose is reabsorbed (as long as there is a moderate hyperglycemia) in the proximal tubule via sodium-glucose co-transport, which concurrently leads to the increase in sodium reabsorption [59]. Sodium reabsorption increases blood pressure, which may be prevented and regulated by salt-free diet.

Patients with DM have increased arterial stiffness, which develops due to the increased glycation of proteins and consequent development of arteriosclerosis. Decreased arterial elasticity in patients with glucose intolerance or DM contributes to the increased systolic pressure as an independent mortality risk factor [60].

#### **Glomerular Filtration Rate**

Increased GFR at diagnosis is a risk factor for the development of diabetic nephropathy. In approximately half of the patients with type 1 DM lasting up to five years, GFR value is approximately 25-50% above normal range. These patients have a higher risk of developing diabetic nephropathy [61].

Dynamics of structural and hemodynamic changes is influenced by increased intraglomerular pressure, with the resulting glomerular hyperfiltration and hypertrophy and damage to the endothelial wall. Strict glycemic control, limited protein intake, and blood pressure control may slow down the progress of renal disease in type 1 DM [62]. The situation with type 2 DM is somewhat different. More than 45% of patients with type 2 DM at diagnosis have GFR that is two standard deviations higher than that in their age-matched no-DM or overweight controls [63]. Granted, the hyper filtration rate (117-133 mL/min on average) is lower than that in type 1 DM. Patients with type 2 DM are older and, therefore, have greater likelihood of developing atherosclerotic vascular changes that influence GFR and glomerular size [64]. The role of intraglomerular hypertension in the pathogenesis of diabetic nephropathy explains why systemic hypertension is such an important risk factor for the development of this kidney disease [65]. Studies on animal models showed that DM is associated with damage of renal autoregulation. As a result, increased blood pressure does not induce the expected vasoconstriction in the afferent arteriole, which would reduce the influence of systemic hypertension on intraglomerular pressure [66].

#### **Glycemic Regulation**

82 Pathophysiology and Complications of Diabetes Mellitus

DM (70%), even before albuminuria can be found.

**Age** 

**Increased Blood Pressure** 

retention of sodium in the kidneys.

**Glomerular Filtration Rate** 

pressure as an independent mortality risk factor [60].

(3- to 6-fold in comparison with Caucasians), American Mexicans, and especially in Pima Indians in the North West part of the United States [55]. This observation in genetically incongruent populations suggests that socioeconomic factors, such as nutrition and poor

In patients with type 2 DM, age and duration of DM increase the risk for albuminuria [53]. In the population study of 1586 Pima Indians with type 2 DM, subjects diagnosed with DM before age 20 had a higher risk of developing terminal kidney failure (25 vs. 5 patients in 1000 incident patients). According to Svensson et al. [56] the risk of terminal kidney failure

There is a high prevalence rate of hypertension in patients with type 1 DM (40%) and type 2

Evidence from several large clinical studies (UKPDS, ADVANCE) indicates a causal relationship between the increased arterial pressure and diabetic nephropathy [57]. Moreover, at least three factors have been shown to contribute to the development of increased arterial pressure in this metabolic disorder including hyperinsulinemia, excessive extracellular fluid volume, and increased arterial rigidity. Hyperinsulinemia contributes to the development of increased arterial pressure via insulin resistance in type 2 DM or via administration of insulin per *se*. Randeree et al. study in 80 patients with type 2 DM who started treatment with exogenous insulin showed an increase in their blood pressure from 132/81 mm Hg to 149/89 mm Hg [58]. This hypertensive response, although not reported in all clinical studies, is most likely mediated by weight gain combined with pro-hypertensive effect of insulin. Hyperinsulinemia could be the link between overweight and increased blood pressure in patients with or without DM, since it increases sympathetic activity and

Sodium and water retention are induced by insulin itself, while the increased filtration of glucose is induced by hyperglycemia. The excess filtered glucose is reabsorbed (as long as there is a moderate hyperglycemia) in the proximal tubule via sodium-glucose co-transport, which concurrently leads to the increase in sodium reabsorption [59]. Sodium reabsorption

Patients with DM have increased arterial stiffness, which develops due to the increased glycation of proteins and consequent development of arteriosclerosis. Decreased arterial elasticity in patients with glucose intolerance or DM contributes to the increased systolic

Increased GFR at diagnosis is a risk factor for the development of diabetic nephropathy. In approximately half of the patients with type 1 DM lasting up to five years, GFR value is

increases blood pressure, which may be prevented and regulated by salt-free diet.

control of glycemia, blood pressure, and body weight, play the key role.

in patients with type 1 DM was low if the disease was diagnosed by the age of 5.

Diabetic nephropathy often develops in patients with poor glycemic control. The degree of glycemic control is an important predictor of terminal kidney failure [67]. In Krolewski et al's [68] study, the prevalence of terminal kidney failure was 36% in patients with the worst glycemic control in comparison with 9% in the group with well-controlled glycaemia.

It is generally accepted that the degree of glycemic control is a very important risk factor for the development diabetic nephropathy.

### **Overweight**

High body mass index (BMI) increases the risk of development of chronic kidney disease in patients with DM [53]. Furthermore, adequate diet and reduction in body weight decrease proteinuria and improve kidney function in these patients [69]. The role of overweight as a risk factor for diabetic nephropathy (independent of DM and glycemic control) has not been clearly confirmed.

#### **Smoking**

Although recent studies have shown the association between smoking and progression of diabetic nephropathy, a large prospective study by Hovind et al. [70] did not confirm the association between smoking and decreased GFR rate in patients with DM with or without ACEI therapy.

#### **Oral Contraception**

Ahmed et al. [71] showed the association between the use of oral contraceptives and development of diabetic nephropathy.

Each of the above-described factors increases the risk of diabetic nephropathy, but none is predictive enough for the development of diabetic nephropathy in an individual patient.

Diabetic Nephropathy 85

membrane nephropathy, minimal change disease, IgA nephropathy, focal glomerulosclerosis, Henoch-Schönlein purpura, proliferative glomerulonephritis, and so on.

a. Proteinuria, which started in the first five years after the diagnosis of type 1 DM. Latent nephropathy, is present between 10 and 15 years after the onset of type 1 DM. This period is probably the same in type 2 DM, but the exact time of the onset of the disease

b. Acute onset of kidney disease. Diabetic nephropathy is a slowly developing disease. c. The presence of erythrocytes (mostly acanthocytes) and rouleaux formations in urine sediment. Patients with microscopic hematuria may have a benign familial hematuria, which is present in approximately 9% of population with or without diabetic

d. The absence of diabetic retinopathy or neuropathy in patients with type 1 DM. As opposed to that, the absence of retinopathy in patients with type 2 DM does not exclude

f. A significant decrease in GFR (>30%) within two to three months after the introduction

Proteinuria and kidney failure in patients with DM may also be caused by other diseases apart from primary glomerular diseases. The most frequent cause is atherosclerotic vascular disease (nephrosclerosis) in older patients with type 2 DM [82]. This disease cannot be clinically discerned from diabetic nephropathy without kidney biopsy. However, kidney biopsy is not necessary in most cases, because the correct diagnosis in this patient group is not clinically important. What speaks in favor of nephrosclerosis is the significant increase in serum creatinine after the introduction of ACEI or ARB for the treatment of hypertension or slowing down the progress of chronic kidney disease. The same occurs when there is a

The effect of strict glycemic control depends on the DM stage in which it was started and consequent normalization of glucose metabolism. Intensified insulin therapy has the

a. It partly decreases glomerular hypertrophy and hyperfiltration (in fasting state and after protein-rich meal), both of which are important risk factors for permanent

b. It postpones the development of albuminuria [83]. Intensified insulin therapy that keeps glucose values within normal ranges decreases the development or progress of

The main clinical signs of primary glomerular disease are as follows:

is difficult to determine.

the presence of diabetic retinopathy.

e. Signs and/or symptoms of other systemic disease.

of ACEI or angiotensin II receptor blockers (ARB) therapy.

nephropathy [81]

bilateral renal artery stenosis.

following effects on the kidney:

glomerular damage.

diabetic nephropathy.

**Strict Glycemic Control** 

**Nephrosclerosis** 

**9. Treatment** 

## **6. Association between diabetic nephropathy and retinopathy**

Patients with type 1 DM and nephropathy almost always have other complications related to the underlying disease, such as retinopathy and neuropathy [9]. Retinopathy has easily recognizable clinical manifestations and always precedes the clinically manifest signs of nephropathy in the same patient. The vice versa is not the case. A small number of patients with advanced retinopathy have glomerular histological changes and microalbuminuria, but most have no biopsy evidence of kidney disease [72]. The association between diabetic nephropathy and retinopathy is weaker in patients with type 2 DM. In a study carried out by Parving et al. [73] in 35 patients with type 2 DM and proteinuria (> 300 mg/day), 27 of these patients had biopsy evidence of nephropathy. Diabetic retinopathy was present in 15 of these 27 patients and in none of the eight patients without diabetic nephropathy. Further analysis showed that approximately one-third of patients without retinopathy had no biopsy evidence of diabetic nephropathy [74].

Thus, patients with type 2 DM and significant proteinuria and retinopathy were most likely to develop diabetic nephropathy, whereas those with proteinuria but without retinopathy had a greater likelihood of having an underlying non-diabetic kidney disease [75]. In the study by Schwartz et al, biopsy was performed in 36 patients with type 2 DM and nephropathy. In 17 of them, biopsy showed visible glomerulosclerosis with Kimmelstiel-Wilson nodules, whereas in the remaining 15 patients, biopsy showed changes characteristic of diabetic nephropathy (mesangial sclerosis), but with no classical nodules present. There was no difference in the duration of disease and glycemic regulation between patients with and those without nodules. A strong association was found between severe retinopathy and presence of Kimmelstiel-Wilson nodules. The reason is still unknown[76].

According to the K/DOQI 2007 Guidelines, etiology of kidney disease in most patients with DM should be ascribed to DM if pathologic proteinuria and retinopathy are present [77]. In case that no retinopathy is present, non-diabetic causes of kidney disease should be investigated.

## **7. Biomarkers of diabetic nephropaty**

Albuminuria remains the only biomarker acceptable for diagnostic purposes, although some growth factors are expected to replace albuminuria in future. It is known that values of TGF beta, vascular endothelial growth factor (VEGF), and CTGF are increased in the plasma and urine of patients with diabetic nephropathy [78-80].

## **8. Non-diabetic kidney disease**

Proteinuria is sometimes present in DM because of the primary glomerular disease rather than diabetic nephropathy. In that case, possible caused of kidney damage may include membrane nephropathy, minimal change disease, IgA nephropathy, focal glomerulosclerosis, Henoch-Schönlein purpura, proliferative glomerulonephritis, and so on. The main clinical signs of primary glomerular disease are as follows:


#### **Nephrosclerosis**

84 Pathophysiology and Complications of Diabetes Mellitus

biopsy evidence of diabetic nephropathy [74].

**7. Biomarkers of diabetic nephropaty** 

urine of patients with diabetic nephropathy [78-80].

**8. Non-diabetic kidney disease** 

investigated.

Each of the above-described factors increases the risk of diabetic nephropathy, but none is predictive enough for the development of diabetic nephropathy in an individual patient.

Patients with type 1 DM and nephropathy almost always have other complications related to the underlying disease, such as retinopathy and neuropathy [9]. Retinopathy has easily recognizable clinical manifestations and always precedes the clinically manifest signs of nephropathy in the same patient. The vice versa is not the case. A small number of patients with advanced retinopathy have glomerular histological changes and microalbuminuria, but most have no biopsy evidence of kidney disease [72]. The association between diabetic nephropathy and retinopathy is weaker in patients with type 2 DM. In a study carried out by Parving et al. [73] in 35 patients with type 2 DM and proteinuria (> 300 mg/day), 27 of these patients had biopsy evidence of nephropathy. Diabetic retinopathy was present in 15 of these 27 patients and in none of the eight patients without diabetic nephropathy. Further analysis showed that approximately one-third of patients without retinopathy had no

Thus, patients with type 2 DM and significant proteinuria and retinopathy were most likely to develop diabetic nephropathy, whereas those with proteinuria but without retinopathy had a greater likelihood of having an underlying non-diabetic kidney disease [75]. In the study by Schwartz et al, biopsy was performed in 36 patients with type 2 DM and nephropathy. In 17 of them, biopsy showed visible glomerulosclerosis with Kimmelstiel-Wilson nodules, whereas in the remaining 15 patients, biopsy showed changes characteristic of diabetic nephropathy (mesangial sclerosis), but with no classical nodules present. There was no difference in the duration of disease and glycemic regulation between patients with and those without nodules. A strong association was found between severe retinopathy and

According to the K/DOQI 2007 Guidelines, etiology of kidney disease in most patients with DM should be ascribed to DM if pathologic proteinuria and retinopathy are present [77]. In case that no retinopathy is present, non-diabetic causes of kidney disease should be

Albuminuria remains the only biomarker acceptable for diagnostic purposes, although some growth factors are expected to replace albuminuria in future. It is known that values of TGF beta, vascular endothelial growth factor (VEGF), and CTGF are increased in the plasma and

Proteinuria is sometimes present in DM because of the primary glomerular disease rather than diabetic nephropathy. In that case, possible caused of kidney damage may include

presence of Kimmelstiel-Wilson nodules. The reason is still unknown[76].

**6. Association between diabetic nephropathy and retinopathy** 

Proteinuria and kidney failure in patients with DM may also be caused by other diseases apart from primary glomerular diseases. The most frequent cause is atherosclerotic vascular disease (nephrosclerosis) in older patients with type 2 DM [82]. This disease cannot be clinically discerned from diabetic nephropathy without kidney biopsy. However, kidney biopsy is not necessary in most cases, because the correct diagnosis in this patient group is not clinically important. What speaks in favor of nephrosclerosis is the significant increase in serum creatinine after the introduction of ACEI or ARB for the treatment of hypertension or slowing down the progress of chronic kidney disease. The same occurs when there is a bilateral renal artery stenosis.

## **9. Treatment**

#### **Strict Glycemic Control**

The effect of strict glycemic control depends on the DM stage in which it was started and consequent normalization of glucose metabolism. Intensified insulin therapy has the following effects on the kidney:


c. It stabilizes or decreases the elimination of proteins in patients with pronounced proteinuria. This effect is not apparent in patients who are not relatively normogycemic during two years. Furthermore, re-established normoglycemia after combined kidney and pancreas transplantation in patients with type 1 DM has preventive effects on recurrence of nephropathy in kidney transplant [84].

Diabetic Nephropathy 87

**Figure 7.** The RAAS and examples of RAAS inhibitors that are available for oral treatment.

angiotensin-aldosterone system.

ACE, angiotensin-converting enzyme; Ang I, angiotensin I; Ang II, angiotensin II; ARB, angiotensin-IIreceptor blocker; AT 1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; RAAS, renin-

only to vasodilatation, growth inhibition, and antiatherogenic effects, but also to heart hypertrophy and poorer revascularization after the obstruction of coronary or peripheral artery [90]. In 1977, Ondetti et al. [91] started a new era in the research of pathophysiological role of the RAAS in kidney disease by developing the first ACEI (captopril) for the treatment of renovascular hypertension. In 1986, Zatz et al. [92] provided evidence that RAAS plays a role in the pathogenesis and progress of diabetic nephropathy by proving that enalapril decreases glomerular capillary hypertension, structural glomerular damage, and proteinuria in diabetic rats. Later studies have confirmed that angiotensin II plays the key role in the functional and structural changes linking proteinuria with the development of diabetic nephropathy. Along with pleiotropic effects, angiotensin II has effects on the structure of glomerular filtration membrane, inducing the remodeling of the podocytic cytoskeleton and causing their apoptosis, which contributes to easier ultrafiltration of plasma proteins [93]. Renoprotective effect of ACEI and ARB has been confirmed in a metaanalysis showing that ACEI and ARB decrease albuminuria in patients with DM more effectively than antihypertensive medications whose mechanism of action excludes RAAS


#### **Strict Blood Pressure Control**

Strict blood pressure control is important in the prevention of progress of diabetic nephropathy and other complications in patients with type 2 DM. The optimum lower range of systolic blood pressure is not clearly defined. According to the UKPDS study, a reduction in systolic blood pressure by 10 mm Hg decreases the risk of development of diabetic complications by 12%; the risk is the lowest where systolic blood pressure values are below 120 mm Hg [85]. The Irbesartan Diabetic Nephropathy Trial showed that decreasing systolic blood pressure to the lower limit value of 120 mm Hg reduces the risk of cardiovascular mortality and heart failure (but not of myocardial infarction) and the risk of double increase in serum creatinine or progress to terminal kidney failure [86].

According to the current Guidelines on Arterial Hypertension Treatment [87], the target blood pressure in patients with DM should be <130/80 mm Hg. Antihypertensive therapy may be started even when blood pressure values are in the upper normal range.

#### **Inhibition of Renin-Angiotensin-Aldosterone System**

Angiotensin II is the most effective factor of renin-angiotensin-aldosterone system (RAAS), resulting from a range of proteolytic reactions that begin with the conversion of angiotensinogen to angiotensin I through the catalytic action of renin (Figure 7).

RAAS is directly associated with blood pressure regulation, body fluid volume, and vascular response to injury and inflammation. Inappropriate activation of this system increases the blood pressure and has anti-inflammatory, prothrombotic, and proatherogenic effects, which in the long run lead to irreversible damage of target organs. Although aldosterone, renin, and end-products of angiotensin degradation are also involved in this process, majority of the RAAS effects on target organs are mediated by angiotensin II, which is present in the bloodstream and tissues. Angiotensin II, which is produced in the heart, brain, and kidneys through alternative pathways by kinase and endopeptidase activity, is more effective than angiotensin II produced in the bloodstream [88]. Angiotensin II binds to AT1 i AT2 receptors. AT1 receptor activation is responsible for vasoconstriction, release of aldosterone, vascular remodeling, oxidative stress, and has anti-inflammatory, proatherogenic, and prothrombotic effects [89]. The activation of AT2 receptors leads not

recurrence of nephropathy in kidney transplant [84].

confirmed by semiquantitative method (test strip). e. It reduces mesangial cell number and mesangial matrix.

in serum creatinine or progress to terminal kidney failure [86].

**Inhibition of Renin-Angiotensin-Aldosterone System** 

g. The progress of tubular atrophy is slowed down.

**Strict Blood Pressure Control** 

c. It stabilizes or decreases the elimination of proteins in patients with pronounced proteinuria. This effect is not apparent in patients who are not relatively normogycemic during two years. Furthermore, re-established normoglycemia after combined kidney and pancreas transplantation in patients with type 1 DM has preventive effects on

d. It slows down the progress of kidney disease in case of already developed proteinuria

f. In some patients, the thickness of glomerular and tubular basement membranes and

Strict blood pressure control is important in the prevention of progress of diabetic nephropathy and other complications in patients with type 2 DM. The optimum lower range of systolic blood pressure is not clearly defined. According to the UKPDS study, a reduction in systolic blood pressure by 10 mm Hg decreases the risk of development of diabetic complications by 12%; the risk is the lowest where systolic blood pressure values are below 120 mm Hg [85]. The Irbesartan Diabetic Nephropathy Trial showed that decreasing systolic blood pressure to the lower limit value of 120 mm Hg reduces the risk of cardiovascular mortality and heart failure (but not of myocardial infarction) and the risk of double increase

According to the current Guidelines on Arterial Hypertension Treatment [87], the target blood pressure in patients with DM should be <130/80 mm Hg. Antihypertensive therapy

Angiotensin II is the most effective factor of renin-angiotensin-aldosterone system (RAAS), resulting from a range of proteolytic reactions that begin with the conversion of

RAAS is directly associated with blood pressure regulation, body fluid volume, and vascular response to injury and inflammation. Inappropriate activation of this system increases the blood pressure and has anti-inflammatory, prothrombotic, and proatherogenic effects, which in the long run lead to irreversible damage of target organs. Although aldosterone, renin, and end-products of angiotensin degradation are also involved in this process, majority of the RAAS effects on target organs are mediated by angiotensin II, which is present in the bloodstream and tissues. Angiotensin II, which is produced in the heart, brain, and kidneys through alternative pathways by kinase and endopeptidase activity, is more effective than angiotensin II produced in the bloodstream [88]. Angiotensin II binds to AT1 i AT2 receptors. AT1 receptor activation is responsible for vasoconstriction, release of aldosterone, vascular remodeling, oxidative stress, and has anti-inflammatory, proatherogenic, and prothrombotic effects [89]. The activation of AT2 receptors leads not

may be started even when blood pressure values are in the upper normal range.

angiotensinogen to angiotensin I through the catalytic action of renin (Figure 7).

mesangial cell number become normal and glomerular nodules disappear.

**Figure 7.** The RAAS and examples of RAAS inhibitors that are available for oral treatment. ACE, angiotensin-converting enzyme; Ang I, angiotensin I; Ang II, angiotensin II; ARB, angiotensin-IIreceptor blocker; AT 1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; RAAS, reninangiotensin-aldosterone system.

only to vasodilatation, growth inhibition, and antiatherogenic effects, but also to heart hypertrophy and poorer revascularization after the obstruction of coronary or peripheral artery [90]. In 1977, Ondetti et al. [91] started a new era in the research of pathophysiological role of the RAAS in kidney disease by developing the first ACEI (captopril) for the treatment of renovascular hypertension. In 1986, Zatz et al. [92] provided evidence that RAAS plays a role in the pathogenesis and progress of diabetic nephropathy by proving that enalapril decreases glomerular capillary hypertension, structural glomerular damage, and proteinuria in diabetic rats. Later studies have confirmed that angiotensin II plays the key role in the functional and structural changes linking proteinuria with the development of diabetic nephropathy. Along with pleiotropic effects, angiotensin II has effects on the structure of glomerular filtration membrane, inducing the remodeling of the podocytic cytoskeleton and causing their apoptosis, which contributes to easier ultrafiltration of plasma proteins [93]. Renoprotective effect of ACEI and ARB has been confirmed in a metaanalysis showing that ACEI and ARB decrease albuminuria in patients with DM more effectively than antihypertensive medications whose mechanism of action excludes RAAS [94]. Early treatment with ACEI may prevent microalbuminuria, which is the early sign of glomerular damage and marker of cardiovascular risk in patients with DM. Delayed treatment with ACEI or ARB in patients with type 2 DM, diabetic nephropathy, and proteinuria is not effective enough. Increasing ACEI and ARB dosages above the recommended values for the treatment of hypertension or their combination is very effective in reducing albuminuria [95]. Aldosteron receptor antagonists and renin inhibitors also decrease albuminuria in patients with DM, but large randomized trial are needed to determine their possible advantage over ACEI and ARB either as monotherapy or combined therapy [96].

Diabetic Nephropathy 89

**New Treatment Strategies** 

proteinuria [108].

**10. Conclusion** 

diabetic nephropathy.

**Author details** 

*Rijeka, Croatia* 

Božidar Vujičić and Sanjin Rački

Tamara Turk and Željka Crnčević-Orlić

options are being investigated.

randomized clinical trials are still not available.

between the treatment and control groups [110].

and cardiovascular mortality in patients with DM.

Current treatment has not always been effective in all patients. Therefore, new treatment

High doses of thiamine and its derivative benfotiamine (S-benzoylthiamine Omonophosphate) were shown to slow down the development of microalbuminuria in animal models, most likely by decreasing the activation of PKC, protein glycation, and oxidative stress [105]. In experimental animals treated with ALT-711, which metabolizes AGEs, a decrease in blood pressure and kidney damage was observed [106]. PKC-beta inhibitor (ruboxistaurin) normalizes GFR, reduces or decreases albuminuria, and improves kidney function in experimental animals [107]. Pimagedin (second generation AGE inhibitor) reduces albuminuria and GFR decrease in patients with type 1 DM and

Smaller clinical trials have produced contradictory results, while the results of large

In an experimental model of induced glomerulosclerosis, modified heparin glycosaminoglycan prevented albuminuria, accumulation of extracellular matrix proteins, and increased expression of TGF-beta [109]. Although animal models held promise, the administration of sulodexid in a large multicentric SUN-Micro-Trial did not achieve the primary outcome, i.e., there were no significant differences in the reduction of albuminuria

In the last several years, we have witnessed an enormous progress made not only in our understanding of the risk factors and mechanism of the development of diabetic nephropathy, but also in the treatment possibilities aimed at preventing the progression of

Early detection of this chronic DM complication along with the treatment of main risk factors (hyperglycemia, hypertension, and dyslipidemia) and use of renoprotective drugs (ACEI and ARB) may decrease the progression of this kidney disease. The treatment of increased blood pressure is a priority. All listed measures lead to a decrease in the overall

*Department of Nephrology and Dialysis, Clinical Hospital Centre Rijeka, Rijeka, Croatia* 

*Department of Endocrinology, Diabetes and Metabolic Diseases, Clinical Hospital Centre Rijeka,* 

#### **Dyslipidemia**

Dyslipidemia occurs in all patients with DM, and its occurrence increases with the development of diabetic nephropathy. Aggressive plasma lipid reduction is an important therapeutic intervention, because patients with DM have an increased risk of coronary disease. In addition, dyslipidemia contributes to the development of diabetic nephropathy. Treating dyslipidemia with statins slows down the progression of diabetic nephropathy [97]. In addition to statins, fenofibrate also decreases the progression of albuminuria in patients with DM [98]. In addition to anti-inflammatory effect, it decreases the production of collagen type 1 in mesangial cells via nuclear peroxisome proliferator-activated receptors (PPAR) alpha [99]. Intensive glycemic control, blood pressure control by RAAS inhibitors, and decreasing serum lipid concentration is an optimal therapeutic approach in patients with DM and diabetic nephropathy (including the microalbuminuria stage).

#### **The Role of Other Factors**

Transforming growth factor beta (TGF-beta) has effects on cell hypertrophy and increased collagen synthesis. Inhibition of TGF-beta in experimental DM model prevented the development and progression of diabetic nephropathy [100]. Experimental studies have shown that non-dihydropyridine calcium channel blocker (diltiazem) slows down the progression of most morphological changes in diabetic nephropathy [101]. On the other hand, diltiazem monotherapy leads to the increased tubulointerstitial fibrosis and global, but not segmental, glomerulosclerosis. This negative effect of diltiazem can be corrected by ACEI therapy.

Peroxisome proliferator-activated receptors (PPAR) play a significant role in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, inflammation, and blood pressure control; however, they also seem to play a significant role in the development of diabetic nephropathy in type 2 DM patients [102]. In an experimental animal model of diabetic nephropathy, PPAR gamma agonists, such as tiazolidinedones (oral hypoglicemic agents), were shown to reduce fibrosis, mesangial proliferation, and inflammation [103]. In addition, these agents reduce albuminuria in different stages of diabetic nephropathy and decrease blood pressure [104]. Their possible renoprotective effects still need to be confirmed in randomized clinical trials including a large number of patients.

#### **New Treatment Strategies**

88 Pathophysiology and Complications of Diabetes Mellitus

therapy [96].

**Dyslipidemia** 

**The Role of Other Factors** 

ACEI therapy.

[94]. Early treatment with ACEI may prevent microalbuminuria, which is the early sign of glomerular damage and marker of cardiovascular risk in patients with DM. Delayed treatment with ACEI or ARB in patients with type 2 DM, diabetic nephropathy, and proteinuria is not effective enough. Increasing ACEI and ARB dosages above the recommended values for the treatment of hypertension or their combination is very effective in reducing albuminuria [95]. Aldosteron receptor antagonists and renin inhibitors also decrease albuminuria in patients with DM, but large randomized trial are needed to determine their possible advantage over ACEI and ARB either as monotherapy or combined

Dyslipidemia occurs in all patients with DM, and its occurrence increases with the development of diabetic nephropathy. Aggressive plasma lipid reduction is an important therapeutic intervention, because patients with DM have an increased risk of coronary disease. In addition, dyslipidemia contributes to the development of diabetic nephropathy. Treating dyslipidemia with statins slows down the progression of diabetic nephropathy [97]. In addition to statins, fenofibrate also decreases the progression of albuminuria in patients with DM [98]. In addition to anti-inflammatory effect, it decreases the production of collagen type 1 in mesangial cells via nuclear peroxisome proliferator-activated receptors (PPAR) alpha [99]. Intensive glycemic control, blood pressure control by RAAS inhibitors, and decreasing serum lipid concentration is an optimal therapeutic approach in patients

Transforming growth factor beta (TGF-beta) has effects on cell hypertrophy and increased collagen synthesis. Inhibition of TGF-beta in experimental DM model prevented the development and progression of diabetic nephropathy [100]. Experimental studies have shown that non-dihydropyridine calcium channel blocker (diltiazem) slows down the progression of most morphological changes in diabetic nephropathy [101]. On the other hand, diltiazem monotherapy leads to the increased tubulointerstitial fibrosis and global, but not segmental, glomerulosclerosis. This negative effect of diltiazem can be corrected by

Peroxisome proliferator-activated receptors (PPAR) play a significant role in the regulation of adipogenesis, lipid metabolism, insulin sensitivity, inflammation, and blood pressure control; however, they also seem to play a significant role in the development of diabetic nephropathy in type 2 DM patients [102]. In an experimental animal model of diabetic nephropathy, PPAR gamma agonists, such as tiazolidinedones (oral hypoglicemic agents), were shown to reduce fibrosis, mesangial proliferation, and inflammation [103]. In addition, these agents reduce albuminuria in different stages of diabetic nephropathy and decrease blood pressure [104]. Their possible renoprotective effects still need to be confirmed in

with DM and diabetic nephropathy (including the microalbuminuria stage).

randomized clinical trials including a large number of patients.

Current treatment has not always been effective in all patients. Therefore, new treatment options are being investigated.

High doses of thiamine and its derivative benfotiamine (S-benzoylthiamine Omonophosphate) were shown to slow down the development of microalbuminuria in animal models, most likely by decreasing the activation of PKC, protein glycation, and oxidative stress [105]. In experimental animals treated with ALT-711, which metabolizes AGEs, a decrease in blood pressure and kidney damage was observed [106]. PKC-beta inhibitor (ruboxistaurin) normalizes GFR, reduces or decreases albuminuria, and improves kidney function in experimental animals [107]. Pimagedin (second generation AGE inhibitor) reduces albuminuria and GFR decrease in patients with type 1 DM and proteinuria [108].

Smaller clinical trials have produced contradictory results, while the results of large randomized clinical trials are still not available.

In an experimental model of induced glomerulosclerosis, modified heparin glycosaminoglycan prevented albuminuria, accumulation of extracellular matrix proteins, and increased expression of TGF-beta [109]. Although animal models held promise, the administration of sulodexid in a large multicentric SUN-Micro-Trial did not achieve the primary outcome, i.e., there were no significant differences in the reduction of albuminuria between the treatment and control groups [110].

## **10. Conclusion**

In the last several years, we have witnessed an enormous progress made not only in our understanding of the risk factors and mechanism of the development of diabetic nephropathy, but also in the treatment possibilities aimed at preventing the progression of diabetic nephropathy.

Early detection of this chronic DM complication along with the treatment of main risk factors (hyperglycemia, hypertension, and dyslipidemia) and use of renoprotective drugs (ACEI and ARB) may decrease the progression of this kidney disease. The treatment of increased blood pressure is a priority. All listed measures lead to a decrease in the overall and cardiovascular mortality in patients with DM.

## **Author details**

Božidar Vujičić and Sanjin Rački *Department of Nephrology and Dialysis, Clinical Hospital Centre Rijeka, Rijeka, Croatia* 

Tamara Turk and Željka Crnčević-Orlić *Department of Endocrinology, Diabetes and Metabolic Diseases, Clinical Hospital Centre Rijeka, Rijeka, Croatia* 

#### Gordana Đorđević

*Department of Pathology and Pathologic Anatomy, Faculty of Medicine, University of Rijeka, Rijeka, Croatia* 

Diabetic Nephropathy 91

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**Chapter 5** 

© 2012 Malathi and Shanthi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Malathi and Shanthi, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Wavelet Image Fusion Approach for** 

**Classification of Ultrasound Placenta** 

G. Malathi and V. Shanthi

http://dx.doi.org/10.5772/53530

assist the experts in their diagnosis.

obstetrics and gynecology domain.

**1. Introduction** 

Additional information is available at the end of the chapter

**Complicated by Gestational Diabetes Mellitus** 

The steady increase in population correspondingly increases the number of diseases people are prone to. The early diagnosis of a disease is of paramount importance, which is a major challenge faced by the medical experts. Health information, especially, clinical information increases on a daily basis and is extremely variable and is also complicate to assess. As a result, there is a demand for finding the criteria that can be used to evaluate the quality of hidden information. One of the most important problems of medical diagnosis, in general, is the subjectivity of the specialist. All these factors have resulted in the use of computers to

Computer assisted information retrieval may assist to support quality decision making and avoid human error. Although human decision-making is often optimal, it is poor when huge amounts of data are involved for classification. Computer Aided Diagnosis (CAD) is a fast growing research field that has set a new horizon in the medical domain. It has increased the quality of current medical imaging technologies by bringing in new developments in medical imaging technology. CAD has already been successfully implemented for a number of medical problems which includes cancer, fractures etc. Even though CAD software's were developed for uncovering many diseases like microcalcification in mammograms, chest, colon, brain, liver, skeletal and vascular systems, is lacking application to ultrasound

The human placenta is a fetus's lifeline during gestation, providing nutrients and antibodies, while eliminating waste products via the mother's blood supply. The placenta is an integral part of the child's development, but is generally disposed of, after delivery. The relatively new field of placenta analysis within the field of prenatal pathology investigates

