**The Contribution of Fibronectin ED-A Expression to Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis**

Keisuke Ina, Hirokazu Kitamura, Shuji Tatsukawa and Yoshihisa Fujikura *Oita University Japan* 

#### **1. Introduction**

108 Diabetic Nephropathy

[63] Wendt T, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, et al. RAGE drives the development

[64] Yonekura H, Yamamoto Y, Sakurai S, *et al.* Novel splice variants of the receptor for

[65] Harashima A, Yamamoto Y, Cheng C, *et al*. Identification of mouse orthologue of

[66] Cuccurullo C, Lezzi A, Fazia ML, *et al*. Suppression of RAGE as a basis of simvastatin-

[67] Santilli F, Bucciarelli L, Noto D, *et al.* Decreased plasma soluble RAGE in patients with hypercholesterolemia: effects of statins. *Free Radic Biol Med* 2007; 43(9): 1255-1262.

diabetic nephropathy. *Am J Pathol* 2003; 162: 1123-1137.

function and expression. *Biochem J* 2006; 396(1): 109-115.

*J* 2003; 370(3): 1097-1109.

2006; 26(12): 2716-2723.

of glomeruloslerosis and implicates podocyte activation in the pathogenesis of

advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury. *Biochem* 

endogenous secretory receptor for advanced glycation end-products: structure,

dependent plaque stabilization in type 2 diabetes. *Arterioscler Thromb Vasc Biol* 

The number of cases in which hemodialysis therapy for diabetic nephropathy is required has been increasing. Currently, the goal of treatment for renal fibrosis is not only to prevent the development and progression of the disease, but also to promote its remission and regression.

It is well known that glomerulosclerosis and tubulointerstitial fibrosis occur during the early stages of diabetic nephropathy. It has been demonstrated that the development of tubulointerstitial lesions is more closely correlated with a progressive decline in renal function compared to glomerular lesions (Bohle et al., 1991). Tubulointerstitial fibrosis ultimately leads to renal failure as a result of renal atrophy. The myofibroblasts emerging in tubulointerstitial fibrosis tissue have been indicated to play a crucial role in the development and progression of fibrosis (Simonson, 2007). They overproduce extracellular matrix (ECM) molecules, including type I collagen and fibronectin, and repress ECM degradation through the production of tissue inhibitor of metalloproteinase-1 (TIMP-1) (Edwards et al., 1987) and plasminogen activator inhibitor-1 (PAI-1) in response to transforming growth factor-β1 (TGF-β1), which is increased in DN (Laiho et al., 1987), followed by ECM accumulation (i.e. fibrosis). Furthermore, the myofibroblasts may induce fibrosis tissue contraction via increased cell contraction promoted by αsmooth muscle actin (α-SMA) expression, leading to renal atrophy and failure. Myofibroblasts are considered to be derived from various kinds of cells, e.g. fibroblasts (Strutz and Zeisberg, 2006), epithelial cells (Iwano et al., 2002), endothelial cells (Zeisberg et al., 2008), pericytes (Humphreys et al., 2010), and bone marrow-originated cells (Keeley et al., 2010), via stimulation by TGF-β1.

Although α-SMA expression, which is the most pronounced characteristic of myofibroblasts, has been considered to cause tissue contraction, the detailed mechanism has not yet been determined. Myofiboblasts were first described in wound-healing granulation tissue

The Contribution of Fibronectin ED-A Expression to

Fig. 1. The molecular structure of fibronectin ED-A and ED-B.

approximate intron positions determined in the murine gene.

kits were obtained from Cusabio (Wuhan, China).

**2. Materials and methods** 

**2.1 Materials** 

**2.2 Cell culture** 

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 111

The fibronectin subunit is composed of three different repeating sequences: type I (rectangles), type II (ovals) and type III (circles). Sets of repeats constitute binding domains for fibrin, fibronectin (FN), collagen, cells and heparin as described. Sequence variations can occur in all three points of the fibronectin subunit: extra domains A and B (ED-A and ED-B, respectively) and the IIICS (Wierzbicka-Patynowski & Schwarzbauer, 2003). Allows indicate

Porcine type I collagen solution was purchased from Nitta Gelatin (Osaka, Japan), recombinant human TGF-β1 was purchased from R and D Systems (MN, USA), and the mouse monoclonal antibodies against α-SMA and ED-A were obtained from Sigma-Aldrich (MO, USA) and Abcam (Cambridge, UK), respectively. The fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody against these monoclonal antibodies was purchased from Caltag Laboratories (CA, USA). Rat type I collagen ELISA

Normal rat kidney fibroblasts (NRK 49F cells) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). The cells were maintained in Dulbecco's minimal essential medium (D-MEM) (Nissui Pharmaceutical, Tokyo, Japan) containing 100 IU ml-1 penicillin, 100 μg ml-1 streptomycin, and 10% fetal bovine serum (FBS) (JRH Biosciences, KS) at 37°C in a humidified, 5% CO2 atmosphere. FBS had been heat-inactivated at 56°C for 30 min prior to use. Cells from passage 3 to passage 8 were used in the experiments described below.

(Gabbiani et al., 1971), where they were referred to as "modified fibroblasts" on the basis of their ultrastructural identification in transmission electron microscopic studies. These cells include a fibrillar system corresponding to stress fibers, nuclear indentation implying cell contraction, and cell-to-ECM and cell-to-cell junctions. Thus, modified fibroblasts (myofibroblasts) were described as an intermediate type of cell between fibroblasts and smooth muscle cells due to their ability to produce ECM and contract. Later investigators have noted that α-SMA expression in the stress fibers was the major characteristic of myofibroblasts.

At present, cells with α-SMA-positive stress fibers in the interstitium, except for vascular components, are recognized to be myofibroblasts. In vertebrate tissues, six actin isoforms have been identified. Four actin isoforms, including the α-skeletal, α-cardiac, α-vascular and γ-enteric isoforms, are tissue-restricted and involved in cell contraction. The other two actin isoforms, the β-cytoplasmic and γ-cytoplasmic isoforms, are ubiquitously expressed. These have been considered to constitute noncontractile cytoskeletons. The α-vascular actin is the SMA isoform, referred to as "α-SMA". During myofibroblast transdifferentiation, the actin isoform transitions, at least in part, from the cytosolic actin to α-SMA. It has been proposed that myofibroblasts have the ability to contract due to their expression of α-SMA.

Fibronectin, a 440 kDa dimeric glycoprotein, is one of the ECM molecules that exerts various functions, including adhesion, migration and differentiation. The fibronectin subunit is composed of three internally homologous repeats and contains binding sites for cell surface receptors and for other ECM components (Magnusson & Mosher, 1998; Peterson et al., 1983). Fibronectin polymorphisms arise from the alternative splicing of mRNA at three regions, ED-A, ED-B and IIICS (Norton & Hynes, 1987) (Fig. 1). The two former sequences are either omitted or included, while the latter one varies in length. Fibronectins are present in an insoluble form in the ECM, and in a soluble form in circulation, namely as cellular fibronectin and plasma fibronectin. Only cellular fibronectin contains ED-A and ED-B (Vartio et al., 1987). The fibronectin ED-A and fibronectin ED-B isoforms are predominantly expressed in embryonic tissue, and are seldom found in normal adult tissues. However, fibronectin ED-A has been demonstrated to be re-expressed in a variety of reactive and pathological processes, such as wound healing (Ffrench-Constant et al., 1989), arterial internal thickening (Glukhova et al., 1989) and liver fibrosis (Jamagin et al., 1994). *In vitro*, TGF-β1 has been shown to increase fibronectin production, and it regulates the splicing pattern of fibronectin pre-messenger RNA, followed by expression of the ED-A domain (Balza et al., 1988; Borsi et al., 1990). Moreover, it has been suggested that the fibronectin ED-A isoform modulates various cells to generate myofibroblasts which express α-SMA (Bochaton-Piallat et al., 2000; Ffrench-Constant et al., 1989; Jamagin et al., 1994). Gabbiani and coworkers demonstrated that the fibronectin ED-A isoform plays a crucial role in TGFβ1-induced myofibroblast transdifferentiation in an *in vitro* study using human subcutaneous fibroblasts (Serini et al., 1998).

In the present study, we examined whether TGF-β1 induces the expression of the ED-A domain in fibronectin, and whether this, in turn, leads to α-SMA expression in renal fibroblasts by using an *in vitro* model of fibrosis due to diabetic nephropathy. Furthermore, whether the fibronectin ED-A isoform stimulates the contraction of the collagen gel through the expression of α-SMA and the accumulation of type I collagen was examined.

Fig. 1. The molecular structure of fibronectin ED-A and ED-B.

The fibronectin subunit is composed of three different repeating sequences: type I (rectangles), type II (ovals) and type III (circles). Sets of repeats constitute binding domains for fibrin, fibronectin (FN), collagen, cells and heparin as described. Sequence variations can occur in all three points of the fibronectin subunit: extra domains A and B (ED-A and ED-B, respectively) and the IIICS (Wierzbicka-Patynowski & Schwarzbauer, 2003). Allows indicate approximate intron positions determined in the murine gene.

#### **2. Materials and methods**

#### **2.1 Materials**

110 Diabetic Nephropathy

(Gabbiani et al., 1971), where they were referred to as "modified fibroblasts" on the basis of their ultrastructural identification in transmission electron microscopic studies. These cells include a fibrillar system corresponding to stress fibers, nuclear indentation implying cell contraction, and cell-to-ECM and cell-to-cell junctions. Thus, modified fibroblasts (myofibroblasts) were described as an intermediate type of cell between fibroblasts and smooth muscle cells due to their ability to produce ECM and contract. Later investigators have noted that α-SMA expression in the stress fibers was the major characteristic of

At present, cells with α-SMA-positive stress fibers in the interstitium, except for vascular components, are recognized to be myofibroblasts. In vertebrate tissues, six actin isoforms have been identified. Four actin isoforms, including the α-skeletal, α-cardiac, α-vascular and γ-enteric isoforms, are tissue-restricted and involved in cell contraction. The other two actin isoforms, the β-cytoplasmic and γ-cytoplasmic isoforms, are ubiquitously expressed. These have been considered to constitute noncontractile cytoskeletons. The α-vascular actin is the SMA isoform, referred to as "α-SMA". During myofibroblast transdifferentiation, the actin isoform transitions, at least in part, from the cytosolic actin to α-SMA. It has been proposed

Fibronectin, a 440 kDa dimeric glycoprotein, is one of the ECM molecules that exerts various functions, including adhesion, migration and differentiation. The fibronectin subunit is composed of three internally homologous repeats and contains binding sites for cell surface receptors and for other ECM components (Magnusson & Mosher, 1998; Peterson et al., 1983). Fibronectin polymorphisms arise from the alternative splicing of mRNA at three regions, ED-A, ED-B and IIICS (Norton & Hynes, 1987) (Fig. 1). The two former sequences are either omitted or included, while the latter one varies in length. Fibronectins are present in an insoluble form in the ECM, and in a soluble form in circulation, namely as cellular fibronectin and plasma fibronectin. Only cellular fibronectin contains ED-A and ED-B (Vartio et al., 1987). The fibronectin ED-A and fibronectin ED-B isoforms are predominantly expressed in embryonic tissue, and are seldom found in normal adult tissues. However, fibronectin ED-A has been demonstrated to be re-expressed in a variety of reactive and pathological processes, such as wound healing (Ffrench-Constant et al., 1989), arterial internal thickening (Glukhova et al., 1989) and liver fibrosis (Jamagin et al., 1994). *In vitro*, TGF-β1 has been shown to increase fibronectin production, and it regulates the splicing pattern of fibronectin pre-messenger RNA, followed by expression of the ED-A domain (Balza et al., 1988; Borsi et al., 1990). Moreover, it has been suggested that the fibronectin ED-A isoform modulates various cells to generate myofibroblasts which express α-SMA (Bochaton-Piallat et al., 2000; Ffrench-Constant et al., 1989; Jamagin et al., 1994). Gabbiani and coworkers demonstrated that the fibronectin ED-A isoform plays a crucial role in TGFβ1-induced myofibroblast transdifferentiation in an *in vitro* study using human

In the present study, we examined whether TGF-β1 induces the expression of the ED-A domain in fibronectin, and whether this, in turn, leads to α-SMA expression in renal fibroblasts by using an *in vitro* model of fibrosis due to diabetic nephropathy. Furthermore, whether the fibronectin ED-A isoform stimulates the contraction of the collagen gel through

the expression of α-SMA and the accumulation of type I collagen was examined.

that myofibroblasts have the ability to contract due to their expression of α-SMA.

subcutaneous fibroblasts (Serini et al., 1998).

myofibroblasts.

Porcine type I collagen solution was purchased from Nitta Gelatin (Osaka, Japan), recombinant human TGF-β1 was purchased from R and D Systems (MN, USA), and the mouse monoclonal antibodies against α-SMA and ED-A were obtained from Sigma-Aldrich (MO, USA) and Abcam (Cambridge, UK), respectively. The fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody against these monoclonal antibodies was purchased from Caltag Laboratories (CA, USA). Rat type I collagen ELISA kits were obtained from Cusabio (Wuhan, China).

#### **2.2 Cell culture**

Normal rat kidney fibroblasts (NRK 49F cells) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). The cells were maintained in Dulbecco's minimal essential medium (D-MEM) (Nissui Pharmaceutical, Tokyo, Japan) containing 100 IU ml-1 penicillin, 100 μg ml-1 streptomycin, and 10% fetal bovine serum (FBS) (JRH Biosciences, KS) at 37°C in a humidified, 5% CO2 atmosphere. FBS had been heat-inactivated at 56°C for 30 min prior to use. Cells from passage 3 to passage 8 were used in the experiments described below.

The Contribution of Fibronectin ED-A Expression to

**supernatants of the cultures** 

**2.6 Statistical analysis** 

at P<0.05.

**3. Results** 

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 113

NRK49F cell suspension (5000 cells per 5μl) was placed in a very small area near the margin of the gel, and incubated for 1h at 37°C. Then, preincubation was done as above. After rinsing, the gels were cultured in the control medium and the medium with 5ng ml-1 TGF-β<sup>1</sup> for 48h at 37°C following gel detachment. After 48h, the shapes of the gels were observed.

The supernatants from monolayer cultures were added to the wells of 96 well plate, which were pre-coated with a mouse monoclonal antibody against rat type I collagen, and were incubated for 2 h at 37°C. After removing the liquid from each well, a biotin-conjugated anti-mouse IgG secondary antibody solution was added, and cells were incubated for 1 h at 37°C. After rinsing, each well was exposed to the horseradish peroxidase-avidin solution for 1 h at 37°C. After washing, the TMB substrate was added, and cells were incubated for 20 min at 37°C. Then, the stop solution was added to each well, while the optical density of each well was determined using a microplate reader, the Multiskan GO (Thermo Fisher Scientific, Yokohama, Japan), at 450 nm. The value of the optical density was converted to

The experimental data are presented as the means ±SD. The statistical analyses were performed using Student's *t-*test. Differences were considered to be statistically significant

ED-A expression was induced in a dot or short linear pattern on the cell surface by TGF-β1 by 24 h after initiation of culture (the 1D・TGF-β1 group), and was further increased after the 48 h culture (the 2D・TGF-β1 group) (Figs. 2c, 2d). On the other hand, the expression of intracellular α-SMA was not observed 24 h after the initiation of culture (the 1D・TGF-β<sup>1</sup> group), but was induced in stress fiber formations after 48 h in culture (the 2D・TGF-β1 group) (Figs. 2g, 2h). The expression of ED-A and α-SMA was not observed in the cells cultured in the

**2.5 Enzyme-linked immunosolvent assay for type I collagen accumulation in the** 

the type I collagen concentration in the supernatant of the monolayer culture.

medium without TGF-β1 (the 1D and 2D control groups) (Figs. 2a, 2b, 2e, 2f).

anti-ED-A antibody group (36%), and the 2D IgG group (95%) (Fig. 3B).

**3.3 The characteristics of TGF-β1-induced gel shrinkage** 

**3.2 The effects of the anti-ED-A antibody on TGF-β1- induced α-SMA expression** 

The TGF-β1- induced α-SMA expression was markedly decreased by adding an anti-ED-A antibody to the medium (Fig. 3A). The rates of α-SMA-positive cells in stress fiber formations were as follows: the 2D control group (0%), the 2D TGF-β1 group (97%), the 2D

When the cells were ubiquitously poured on the gel, TGF-β1-induced gel shrinkage occurred in the shape of a circle after culture for 48h (Fig. 4Aa). The diameters of gels were 99.4±1.0%

**3.1 TGF-β1- induced the expression of ED-A and α-SMA** 

Monolayer cultures of 2 X 104 cells ml-1 were grown on two-well Labtek Chamber Slides (AS ONE, Osaka, Japan). The cell cultures were divided into 4 groups: a control group, cultured with 0.5% FBS; a TGF-β1 group, cultured with 0.5% FBS + 5 ng ml-1 TGF-β1; an anti-ED-A antibody group, cultured with 0.5% FBS + 5 ng ml-1 TGF-β1 + 10 μg ml-1 anti-ED-A antibody; an IgG group, cultured with 0.5%FBS + 5ng ml-1 TGF-β1 + 10 μg ml-1 mouse IgG (negative control, R &D Systems, Inc., MN, USA). Furthermore, each group was sub-divided into two groups: the 1D (1 day) group, which was cultured for 24h after the initiation of culture; the 2D (2 day) group, which was cultured for 24h after refeeding the cells the same medium 24 h after culture initiation. The cells and the supernatants of all groups were subjected to immunofluorescence studies for fibronectin ED-A or α-SMA, and to an ELISA for type I collagen, respectively.

#### **2.3 Immunofluorescence microscopy for fibronectin ED-A and α-SMA**

The cells from monolayer cultures were fixed in 4% paraformaldehyde in 0.1 M phosphatebuffered saline (pH 7.4) for 10 min. After rinsing, the cells were permeabilized with 0.5% Triton X-100 for 20 min in order to identify α-SMA in the cells, this process was omitted in studies for detecting fibronectin ED-A. After washing, the cells were incubated with the primary antibody against α-SMA or fibronectin ED-A for 2 h at room temperature. After being rinsed, they were incubated with FITC-conjugated goat anti-mouse IgG secondary antibody for 1 h at room temperature. After staining, the cells were rinsed, observed and photographed using an Olympus BX 60 microscope equipped with epifluorescence optics. The assessment of the percentage of cells with α-SMA-positive stress fibers was performed as follows: when there were one or more stress fibers with α-SMA-positive staining in a cell, the cell was considered to be α-SMA-positive and was referred to as a "myofibroblast". The percentage of myofibroblasts was represented by counting the α-SMA-positive cells among 100 to 500 cells.

#### **2.4 Gel contraction assay**

A total of 7 ml of a collagen solution (3 mg/ml stock) was gently mixed with 2 ml of 5-foldconcentrated D-MEM on ice. The mixture was adjusted to pH 7.4 with 1 ml of 200 mM HEPES plus 2.2% NaHCO3 in distilled water. This collagen mixture (0.3ml) was added to each well of a 24 well plate (Becton-Dickinson Labware, NJ). Then, the collagen gel was formed by the incubation of the mixture for 30 min at 37°C. After gelatinization, an NRK 49F cell suspension (2 X104 cells ml-1) was poured on the gel in each well. Preincubation of the cell-populated collagen lattice was performed in D-MEM + 10% FBS for 24 h. Next, the gels were rinsed with serum-free D-MEM three times for 30 min. Then, the gel cultures were divided into 4 groups similar to the immunofluorescence studies: a control group, cultured in 0.5% FBS; a TGF-β1 group, cultured in 0.5% FBS + 5 ng ml-1 TGF-β1; an anti-ED-A antibody group, cultured in 0.5% FBS +5 ng ml-1 TGF-β1+ 10 μg ml-1 anti-ED-A antibody and an IgG group, cultured in 0.5%FBS + 5ng ml-1 TGF-β1 + 10 μg ml-1 mouse IgG. The gels were then detached from the lateral wall and bottom of each well with a microspatula and cultured for 48 h. After 48 h in culture, the diameter of each gel was measured with a ruler to assess the gel contraction after treatment with the reagents. In addition, to confirm that the shrinkage of the cell-populated collagen lattice occurred due to gel contraction, rather than gel digestion, another study was performed. For this study, after gelatinization, the NRK49F cell suspension (5000 cells per 5μl) was placed in a very small area near the margin of the gel, and incubated for 1h at 37°C. Then, preincubation was done as above. After rinsing, the gels were cultured in the control medium and the medium with 5ng ml-1 TGF-β<sup>1</sup> for 48h at 37°C following gel detachment. After 48h, the shapes of the gels were observed.

#### **2.5 Enzyme-linked immunosolvent assay for type I collagen accumulation in the supernatants of the cultures**

The supernatants from monolayer cultures were added to the wells of 96 well plate, which were pre-coated with a mouse monoclonal antibody against rat type I collagen, and were incubated for 2 h at 37°C. After removing the liquid from each well, a biotin-conjugated anti-mouse IgG secondary antibody solution was added, and cells were incubated for 1 h at 37°C. After rinsing, each well was exposed to the horseradish peroxidase-avidin solution for 1 h at 37°C. After washing, the TMB substrate was added, and cells were incubated for 20 min at 37°C. Then, the stop solution was added to each well, while the optical density of each well was determined using a microplate reader, the Multiskan GO (Thermo Fisher Scientific, Yokohama, Japan), at 450 nm. The value of the optical density was converted to the type I collagen concentration in the supernatant of the monolayer culture.

#### **2.6 Statistical analysis**

The experimental data are presented as the means ±SD. The statistical analyses were performed using Student's *t-*test. Differences were considered to be statistically significant at P<0.05.

#### **3. Results**

112 Diabetic Nephropathy

Monolayer cultures of 2 X 104 cells ml-1 were grown on two-well Labtek Chamber Slides (AS ONE, Osaka, Japan). The cell cultures were divided into 4 groups: a control group, cultured with 0.5% FBS; a TGF-β1 group, cultured with 0.5% FBS + 5 ng ml-1 TGF-β1; an anti-ED-A antibody group, cultured with 0.5% FBS + 5 ng ml-1 TGF-β1 + 10 μg ml-1 anti-ED-A antibody; an IgG group, cultured with 0.5%FBS + 5ng ml-1 TGF-β1 + 10 μg ml-1 mouse IgG (negative control, R &D Systems, Inc., MN, USA). Furthermore, each group was sub-divided into two groups: the 1D (1 day) group, which was cultured for 24h after the initiation of culture; the 2D (2 day) group, which was cultured for 24h after refeeding the cells the same medium 24 h after culture initiation. The cells and the supernatants of all groups were subjected to immunofluorescence studies for fibronectin ED-A or α-SMA, and to an ELISA for type I

The cells from monolayer cultures were fixed in 4% paraformaldehyde in 0.1 M phosphatebuffered saline (pH 7.4) for 10 min. After rinsing, the cells were permeabilized with 0.5% Triton X-100 for 20 min in order to identify α-SMA in the cells, this process was omitted in studies for detecting fibronectin ED-A. After washing, the cells were incubated with the primary antibody against α-SMA or fibronectin ED-A for 2 h at room temperature. After being rinsed, they were incubated with FITC-conjugated goat anti-mouse IgG secondary antibody for 1 h at room temperature. After staining, the cells were rinsed, observed and photographed using an Olympus BX 60 microscope equipped with epifluorescence optics. The assessment of the percentage of cells with α-SMA-positive stress fibers was performed as follows: when there were one or more stress fibers with α-SMA-positive staining in a cell, the cell was considered to be α-SMA-positive and was referred to as a "myofibroblast". The percentage of myofibroblasts was represented by counting the α-SMA-positive cells among

A total of 7 ml of a collagen solution (3 mg/ml stock) was gently mixed with 2 ml of 5-foldconcentrated D-MEM on ice. The mixture was adjusted to pH 7.4 with 1 ml of 200 mM HEPES plus 2.2% NaHCO3 in distilled water. This collagen mixture (0.3ml) was added to each well of a 24 well plate (Becton-Dickinson Labware, NJ). Then, the collagen gel was formed by the incubation of the mixture for 30 min at 37°C. After gelatinization, an NRK 49F cell suspension (2 X104 cells ml-1) was poured on the gel in each well. Preincubation of the cell-populated collagen lattice was performed in D-MEM + 10% FBS for 24 h. Next, the gels were rinsed with serum-free D-MEM three times for 30 min. Then, the gel cultures were divided into 4 groups similar to the immunofluorescence studies: a control group, cultured in 0.5% FBS; a TGF-β1 group, cultured in 0.5% FBS + 5 ng ml-1 TGF-β1; an anti-ED-A antibody group, cultured in 0.5% FBS +5 ng ml-1 TGF-β1+ 10 μg ml-1 anti-ED-A antibody and an IgG group, cultured in 0.5%FBS + 5ng ml-1 TGF-β1 + 10 μg ml-1 mouse IgG. The gels were then detached from the lateral wall and bottom of each well with a microspatula and cultured for 48 h. After 48 h in culture, the diameter of each gel was measured with a ruler to assess the gel contraction after treatment with the reagents. In addition, to confirm that the shrinkage of the cell-populated collagen lattice occurred due to gel contraction, rather than gel digestion, another study was performed. For this study, after gelatinization, the

**2.3 Immunofluorescence microscopy for fibronectin ED-A and α-SMA** 

collagen, respectively.

100 to 500 cells.

**2.4 Gel contraction assay** 

#### **3.1 TGF-β1- induced the expression of ED-A and α-SMA**

ED-A expression was induced in a dot or short linear pattern on the cell surface by TGF-β1 by 24 h after initiation of culture (the 1D・TGF-β1 group), and was further increased after the 48 h culture (the 2D・TGF-β1 group) (Figs. 2c, 2d). On the other hand, the expression of intracellular α-SMA was not observed 24 h after the initiation of culture (the 1D・TGF-β<sup>1</sup> group), but was induced in stress fiber formations after 48 h in culture (the 2D・TGF-β1 group) (Figs. 2g, 2h). The expression of ED-A and α-SMA was not observed in the cells cultured in the medium without TGF-β1 (the 1D and 2D control groups) (Figs. 2a, 2b, 2e, 2f).

#### **3.2 The effects of the anti-ED-A antibody on TGF-β1- induced α-SMA expression**

The TGF-β1- induced α-SMA expression was markedly decreased by adding an anti-ED-A antibody to the medium (Fig. 3A). The rates of α-SMA-positive cells in stress fiber formations were as follows: the 2D control group (0%), the 2D TGF-β1 group (97%), the 2D anti-ED-A antibody group (36%), and the 2D IgG group (95%) (Fig. 3B).

#### **3.3 The characteristics of TGF-β1-induced gel shrinkage**

When the cells were ubiquitously poured on the gel, TGF-β1-induced gel shrinkage occurred in the shape of a circle after culture for 48h (Fig. 4Aa). The diameters of gels were 99.4±1.0%

The Contribution of Fibronectin ED-A Expression to

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 115

in the control medium group and 86.4±1.7% in the TGF-β1 group (p<0.001) (Fig. 4Ab). On the other hand, when the cells were placed in a small region close to the margin of the gel, the gel exhibited indentation toward the cell mass in response to TGF-β1 (Fig. 4B). If gel shrinkage occurs due to gel digestion, the gel would reveal the features of digestion, such as the dissolution of the gel around the cell mass, and would form a thin layer or disappear. However, because the present study did not show these features, gel digestion was not considered to be responsible for the observed gel shrinkage. The observed indentation was

Gel contraction did not occur in the control medium throughout the experimental period. In addition, no gel contraction was observed in any of the 1D groups. In the 2D groups, TGF-β<sup>1</sup> remarkably evoked gel contraction. The treatment with the anti-ED-A antibody was shown to repress TGF-β1-induced gel contraction. The diameters of the gels in the four groups were as follows: the 2D control group (100%), the 2D TGF-β1 group (84.0±1.9%), the 2D anti-ED-A antibody group (89.5±1.9%, p<0.05 vs the 2D TGF-β1 group), and the 2D IgG group

thought to be caused by retraction of the gel resulting from cell contraction.

(83.7±1.6%, p<0.05 vs the 2D anti-ED-A antibody group) (Fig. 5).

**accumulation in the supernatants of the cell cultures** 

**4. Discussion** 

**3.4 The effects of the anti-ED-A antibody on TGF-β1-induced gel contraction** 

**3.5 The effects of the anti-ED-A antibody on TGF-β1-stimulated type I collagen** 

There were no significant differences in the accumulation of type I collagen in the cell supernatants between the three 1D groups (the control group, 1420±200; the TGF-β1 group, 1470±100; the anti-ED-A antibody group, 1510±160 ; the IgG group, 1513±26 pg ml-1). In the 2D groups, the accumulation of type I collagen in the supernatant of the TGF-β1 group was increased. The increased accumulation of collagen was repressed by addition of the anti-ED-A antibody to the medium with TGF-β1 (Fig. 5). The concentrations of the supernatants in the four 2D groups (control, TGF-β1, anti-ED-A antibody, IgG) were 1980±220, 2730±110, 2490±18, 2570±3 pg ml-1, respectively (p<0.001, the control group vs the TGF-β1 group; p<0.05, the TGF-β1 group and the IgG group vs the anti-ED-A antibody group) (Fig. 6).

Myofibroblasts expressing α-SMA have been shown to emerge in fibrotic diseases (Tomasek et al., 2002). In general, α-SMA is considered to be profoundly involved in the progression of fibrosis, but the detailed significance of α-SMA expression remains unresolved. For example, it is unclear whether myofibroblasts are really essential for fibrosis. In contrast to general considerations, there have been some reports indicating that myofibroblasts are not necessary for the progression of fibrosis. For example, Takeji et al. suggested that α-SMA expression provided benefits for fibrosis (Takeji et al., 2006). It was demonstrated that defects in α-SMA enhanced the progression of fibrosis by performing unilateral ureteral obstruction in a renal interstitial fibrosis model in an α-SMA knockout mouse. Furthermore, transfection of the α-SMA gene into α-SMA-/- myofibroblasts from these mice suppressed fibrosis. These results indicated that α-SMA expression suppresses, rather than accelerates, fibrosis. The α-SMA-/- myofibroblasts that were obtained possessed the ability to migrate, proliferate, and overproduced type I collagen. These cells did not express α-SMA, but did start to overexpress skeletal muscle α-actin, smooth muscle γ-actin, and related molecules.

Fig. 2. Immunofluorescent staining for ED-A and α-SMA induced by TGF-β1. a, c, e, g, ID groups; b, d, f, h, 2D groups; a, b, c, d, Immunostaining for ED-A; e, f, g, h, Immunostaining for α-SMA; a, b, e, f, control groups; c, d, g, h, TGF-β1 groups. a, No immunostaining. b, No immunostaining. c, Immunostaining for ED-A is shown by the dot or short linear pattern on the cell surface. d, Immunostaining for ED-A was increased. e, No immunostaining. f, No immunostaining, g, No immunostaining. h, Immunostaining for α-SMA is seen in the stress fiber formation in the cells. Bar, 10 μm.

Fig. 2. Immunofluorescent staining for ED-A and α-SMA induced by TGF-β1.

SMA is seen in the stress fiber formation in the cells. Bar, 10 μm.

a, c, e, g, ID groups; b, d, f, h, 2D groups; a, b, c, d, Immunostaining for ED-A; e, f, g, h, Immunostaining for α-SMA; a, b, e, f, control groups; c, d, g, h, TGF-β1 groups. a, No immunostaining. b, No immunostaining. c, Immunostaining for ED-A is shown by the dot or short linear pattern on the cell surface. d, Immunostaining for ED-A was increased. e, No immunostaining. f, No immunostaining, g, No immunostaining. h, Immunostaining for αin the control medium group and 86.4±1.7% in the TGF-β1 group (p<0.001) (Fig. 4Ab). On the other hand, when the cells were placed in a small region close to the margin of the gel, the gel exhibited indentation toward the cell mass in response to TGF-β1 (Fig. 4B). If gel shrinkage occurs due to gel digestion, the gel would reveal the features of digestion, such as the dissolution of the gel around the cell mass, and would form a thin layer or disappear. However, because the present study did not show these features, gel digestion was not considered to be responsible for the observed gel shrinkage. The observed indentation was thought to be caused by retraction of the gel resulting from cell contraction.

#### **3.4 The effects of the anti-ED-A antibody on TGF-β1-induced gel contraction**

Gel contraction did not occur in the control medium throughout the experimental period. In addition, no gel contraction was observed in any of the 1D groups. In the 2D groups, TGF-β<sup>1</sup> remarkably evoked gel contraction. The treatment with the anti-ED-A antibody was shown to repress TGF-β1-induced gel contraction. The diameters of the gels in the four groups were as follows: the 2D control group (100%), the 2D TGF-β1 group (84.0±1.9%), the 2D anti-ED-A antibody group (89.5±1.9%, p<0.05 vs the 2D TGF-β1 group), and the 2D IgG group (83.7±1.6%, p<0.05 vs the 2D anti-ED-A antibody group) (Fig. 5).

#### **3.5 The effects of the anti-ED-A antibody on TGF-β1-stimulated type I collagen accumulation in the supernatants of the cell cultures**

There were no significant differences in the accumulation of type I collagen in the cell supernatants between the three 1D groups (the control group, 1420±200; the TGF-β1 group, 1470±100; the anti-ED-A antibody group, 1510±160 ; the IgG group, 1513±26 pg ml-1). In the 2D groups, the accumulation of type I collagen in the supernatant of the TGF-β1 group was increased. The increased accumulation of collagen was repressed by addition of the anti-ED-A antibody to the medium with TGF-β1 (Fig. 5). The concentrations of the supernatants in the four 2D groups (control, TGF-β1, anti-ED-A antibody, IgG) were 1980±220, 2730±110, 2490±18, 2570±3 pg ml-1, respectively (p<0.001, the control group vs the TGF-β1 group; p<0.05, the TGF-β1 group and the IgG group vs the anti-ED-A antibody group) (Fig. 6).

#### **4. Discussion**

Myofibroblasts expressing α-SMA have been shown to emerge in fibrotic diseases (Tomasek et al., 2002). In general, α-SMA is considered to be profoundly involved in the progression of fibrosis, but the detailed significance of α-SMA expression remains unresolved. For example, it is unclear whether myofibroblasts are really essential for fibrosis. In contrast to general considerations, there have been some reports indicating that myofibroblasts are not necessary for the progression of fibrosis. For example, Takeji et al. suggested that α-SMA expression provided benefits for fibrosis (Takeji et al., 2006). It was demonstrated that defects in α-SMA enhanced the progression of fibrosis by performing unilateral ureteral obstruction in a renal interstitial fibrosis model in an α-SMA knockout mouse. Furthermore, transfection of the α-SMA gene into α-SMA-/- myofibroblasts from these mice suppressed fibrosis. These results indicated that α-SMA expression suppresses, rather than accelerates, fibrosis. The α-SMA-/- myofibroblasts that were obtained possessed the ability to migrate, proliferate, and overproduced type I collagen. These cells did not express α-SMA, but did start to overexpress skeletal muscle α-actin, smooth muscle γ-actin, and related molecules.

The Contribution of Fibronectin ED-A Expression to

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 117

Fig. 4. Morphological alterations of gels in response to TGF-β1 according to differences in the

A. Ubiquitous pouring of cells. a. The shape of the gel b. the diameters of the gels. B. Spot pouring of cells. Aa. The change in shape induced by TGF-β1 is indicated by the circle. Ab. TGF-β1 significantly induced gel shrinkage. The data represent the diameters of the gels compared to the well. B. The gel was deformed, as shown by the indentation torward the

pouring methods.

cell mass (arrows).

These cells also overproduced type I collagen compared to conventional myofibroblasts expressing α-SMA. Because the cells which were established by transfection of the α-SMA gene into the α-SMA-/- cells failed to produce type I collagen, these cells might resemble smooth muscle cells. It is likely that myofibroblasts are an intermediate type of cells between α-SMA-/- cells and smooth muscle cells, given their ability to express α-SMA and produce type I collagen. Taken together, their report does not refute the generally accepted concept

Fig. 3. Immunofluorescent staining for α-SMA was influenced by the anti-ED-A antibody Aa, the 2D TGF-β1 group; b, the 2D anti-ED-A antibody group; c, the 2D IgG group; B, Counts of cells immunostained for α-SMA in the stress fiber formation. Aa and Ac, Immunostaining for α-SMA was shown in the stress fiber formation. Ab, Cells expressing α-SMA in the stress fiber formation were markedy decreased in number. Immunostaining for ED-A was seen in response to addition of the anti-ED-A antibody to the medium. B. Cells with α-SMA expression induced by TGF-β1 were decreased in number in the anti-ED-A antibody group. Bar, 10 μm.

These cells also overproduced type I collagen compared to conventional myofibroblasts expressing α-SMA. Because the cells which were established by transfection of the α-SMA gene into the α-SMA-/- cells failed to produce type I collagen, these cells might resemble smooth muscle cells. It is likely that myofibroblasts are an intermediate type of cells between α-SMA-/- cells and smooth muscle cells, given their ability to express α-SMA and produce type I collagen. Taken together, their report does not refute the generally accepted concept

Fig. 3. Immunofluorescent staining for α-SMA was influenced by the anti-ED-A antibody Aa, the 2D TGF-β1 group; b, the 2D anti-ED-A antibody group; c, the 2D IgG group; B, Counts of cells immunostained for α-SMA in the stress fiber formation. Aa and Ac,

Immunostaining for α-SMA was shown in the stress fiber formation. Ab, Cells expressing α-SMA in the stress fiber formation were markedy decreased in number. Immunostaining for ED-A was seen in response to addition of the anti-ED-A antibody to the medium. B. Cells with α-SMA expression induced by TGF-β1 were decreased in number in the anti-ED-

A antibody group. Bar, 10 μm.

Fig. 4. Morphological alterations of gels in response to TGF-β1 according to differences in the pouring methods.

A. Ubiquitous pouring of cells. a. The shape of the gel b. the diameters of the gels. B. Spot pouring of cells. Aa. The change in shape induced by TGF-β1 is indicated by the circle. Ab. TGF-β1 significantly induced gel shrinkage. The data represent the diameters of the gels compared to the well. B. The gel was deformed, as shown by the indentation torward the cell mass (arrows).

The Contribution of Fibronectin ED-A Expression to

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 119

Fig. 6. Type I collagen accumulated in the supernatants of the monolayer cultures.

antibody.

The TGF-β1 and anti-ED-A antibodies do not influence the type I collagen accumulation in 1D groups. TGF-β1 increased the type I collagen accumulation in the 2D TGF-β1 and IgG groups. The TGF-β1- accelerated collagen accumulation was repressed by the anti-ED-A

Fig. 5. Type I collagen gel contraction.

No gel contraction occurred in any of the 1D groups. TGF-β1 was shown to evoke gel contraction in the 2D TGF-β1 and IgG groups. TGF-β1 -induced gel contraction was attenuated by adding the anti-ED-A antibody. The data represent the diameters of the gels compared to the well.

Fig. 5. Type I collagen gel contraction.

compared to the well.

No gel contraction occurred in any of the 1D groups. TGF-β1 was shown to evoke gel contraction in the 2D TGF-β1 and IgG groups. TGF-β1 -induced gel contraction was

attenuated by adding the anti-ED-A antibody. The data represent the diameters of the gels

Fig. 6. Type I collagen accumulated in the supernatants of the monolayer cultures. The TGF-β1 and anti-ED-A antibodies do not influence the type I collagen accumulation in 1D groups. TGF-β1 increased the type I collagen accumulation in the 2D TGF-β1 and IgG groups. The TGF-β1- accelerated collagen accumulation was repressed by the anti-ED-A antibody.

The Contribution of Fibronectin ED-A Expression to

Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 121

Fig. 7. The hypothesis: about the contribution of fibronectin ED-A expression to α-SMA

We hypothesize the mechanism ad follows: TGF-β1 induces ED-A domain expression through the pathway involving Smad 3 in the fibronectin molecule. The fibronectin ED-A is anchored to the cell via a combination of the cell-binding domain and the integrin on the cell. Subsequently, the ED-A domain binds to the receptor (the Toll-like receptor) and stimulates it. Furthermore, the signaling transfers to one of the TGF-β1-activated pathways, which may be the p38 MAPK pathway. Finally, the ED-A-stimulated pathway induces α-SMA expression and type I collagen accumulation, which involves the increased synthesis of collagen and decreased degradation of collagen via the production of proteinase

expression and type I collagen accumulation.

inhibitors, TIMPs and PAI-1.

that α-SMA contributes to the progression of fibrosis, but do add an additional role for α-SMA producing cells. There was another report (Leemans et al., 2009) that suggested that renal fibrogenesis persists despite decreasing numbers of α-SMA+ myofibroblasts. This also does not contradict the general consensus about the role of myofibroblasts, because it can be considered that after myofibroblasts induce the accumulation of collagen fibrils by forming rigid fibrils which are resistant to proteolysis by proteinases, they undergo apoptosis. Subsequently, the α-SMA fibroblasts might add collagen fibers to these rigid fibers. In this case, myofibroblasts would make up the nucleus of the accumulated collagen fibrils.

In the present study, TGF-β1 induced the production of fibronectin ED-A, which was anchored to kidney fibroblasts, and subsequently induced α-SMA expression in the stress fiber formations in cells. Stress fiber formation is involved in cell contraction, which results in gel contraction via the consecutive binding of α-SMA, focal adhesion, integrins, and type I collagen. In addition, the α-SMA expression was strongly inhibited by exposing the cells to an anti-ED-A antibody. Therefore, the TGF-β1 -induced expression of α-SMA was considered to be, at least to a large extent, dependent on ED-A expression. This is compatible with the fact that the fibronectin ED-A isoform contributes to the transdifferentiation of a variety of cells into myofibroblasts with α-SMA (Bochaton-Piallat et al., 2000; Ffrench-Constant et al., 1989; Jamagin et al., 1994).

It was shown in the renal fibroblasts that the intracellular signaling pathways of TGF-β1 induced fibronectin ED-A expression involves Smad 3 (Bondi et al., 2010). The pathways leading to α-SMA expression and myofibroblast transdifferentiation differed between reports. Our previous report suggested that the pathway of TGF-β1-induced α-SMA expression involved p38 MAPK (Ina et al., 2011). This finding is consistent with a study by Miura et al. (Miura et al., 2006), in which retinal pigment epithelial cells were used. On the other hand, Masszi et al. (Masszi et al., 2003) have found that the activation of the Rho kinase pathway was involved in the α-SMA expression in renal tubular epithelial cells. The contrast between these findings might be due to the differences in the cells used. How ED-A expression activated the p38 MAPK pathway in the present study remains to be determined. However, ED-A has been identified as an endogenous ligand for Toll-like receptor-4 (Gondokaryono et al., 2007; Lefebvre et al., 2011; Ricupero et al., 2001) which is well known to be expressed in inflammatory cells such as macrophages, mast cells, and leukocytes, and is associated with innate immunity. Recently, it was demonstrated that Toll-like receptor 4 is also expressed in renal interstitial fibroblasts (Sorensen et al., 2011). Furthermore, the activation of Toll-like receptor 4 was reported to promote renal tubulointerstitial fibrosis (Campbell et al., 2011; Sorensen et al., 2011). It is therefore possible that ED-A binds to Tolllike receptor 4 and activates it, leading to stimulation of the related signaling pathways, eventually promoting the signaling by TGF-β1 and stimulating α-SMA expression.

Because the immunostaining for fibronectin ED-A was positive in both conventional immunocytochemistry and in that without the first antibody (the anti-ED-A antibody) of the cells exposed to the antibody in culture, the epitope appears to be present in a region other than the receptor-binding site. In this case, the binding of the antibody to the epitope might cause alterations in the conformation of the receptor binding site, and inhibit the binding of ED-A to the receptor. In the current study, since contraction of the NRK49F cell-populated collagen lattice was suppressed by the adding anti-ED-A antibody, the ED-A expression was considered to be involved in TGF-β1-induced gel contraction.

that α-SMA contributes to the progression of fibrosis, but do add an additional role for α-SMA producing cells. There was another report (Leemans et al., 2009) that suggested that renal fibrogenesis persists despite decreasing numbers of α-SMA+ myofibroblasts. This also does not contradict the general consensus about the role of myofibroblasts, because it can be considered that after myofibroblasts induce the accumulation of collagen fibrils by forming rigid fibrils which are resistant to proteolysis by proteinases, they undergo apoptosis.

case, myofibroblasts would make up the nucleus of the accumulated collagen fibrils.

al., 2000; Ffrench-Constant et al., 1989; Jamagin et al., 1994).

In the present study, TGF-β1 induced the production of fibronectin ED-A, which was anchored to kidney fibroblasts, and subsequently induced α-SMA expression in the stress fiber formations in cells. Stress fiber formation is involved in cell contraction, which results in gel contraction via the consecutive binding of α-SMA, focal adhesion, integrins, and type I collagen. In addition, the α-SMA expression was strongly inhibited by exposing the cells to an anti-ED-A antibody. Therefore, the TGF-β1 -induced expression of α-SMA was considered to be, at least to a large extent, dependent on ED-A expression. This is compatible with the fact that the fibronectin ED-A isoform contributes to the transdifferentiation of a variety of cells into myofibroblasts with α-SMA (Bochaton-Piallat et

It was shown in the renal fibroblasts that the intracellular signaling pathways of TGF-β1 induced fibronectin ED-A expression involves Smad 3 (Bondi et al., 2010). The pathways leading to α-SMA expression and myofibroblast transdifferentiation differed between reports. Our previous report suggested that the pathway of TGF-β1-induced α-SMA expression involved p38 MAPK (Ina et al., 2011). This finding is consistent with a study by Miura et al. (Miura et al., 2006), in which retinal pigment epithelial cells were used. On the other hand, Masszi et al. (Masszi et al., 2003) have found that the activation of the Rho kinase pathway was involved in the α-SMA expression in renal tubular epithelial cells. The contrast between these findings might be due to the differences in the cells used. How ED-A expression activated the p38 MAPK pathway in the present study remains to be determined. However, ED-A has been identified as an endogenous ligand for Toll-like receptor-4 (Gondokaryono et al., 2007; Lefebvre et al., 2011; Ricupero et al., 2001) which is well known to be expressed in inflammatory cells such as macrophages, mast cells, and leukocytes, and is associated with innate immunity. Recently, it was demonstrated that Toll-like receptor 4 is also expressed in renal interstitial fibroblasts (Sorensen et al., 2011). Furthermore, the activation of Toll-like receptor 4 was reported to promote renal tubulointerstitial fibrosis (Campbell et al., 2011; Sorensen et al., 2011). It is therefore possible that ED-A binds to Tolllike receptor 4 and activates it, leading to stimulation of the related signaling pathways,

eventually promoting the signaling by TGF-β1 and stimulating α-SMA expression.

considered to be involved in TGF-β1-induced gel contraction.

Because the immunostaining for fibronectin ED-A was positive in both conventional immunocytochemistry and in that without the first antibody (the anti-ED-A antibody) of the cells exposed to the antibody in culture, the epitope appears to be present in a region other than the receptor-binding site. In this case, the binding of the antibody to the epitope might cause alterations in the conformation of the receptor binding site, and inhibit the binding of ED-A to the receptor. In the current study, since contraction of the NRK49F cell-populated collagen lattice was suppressed by the adding anti-ED-A antibody, the ED-A expression was

fibroblasts might add collagen fibers to these rigid fibers. In this

Subsequently, the α-SMA-

We hypothesize the mechanism ad follows: TGF-β1 induces ED-A domain expression through the pathway involving Smad 3 in the fibronectin molecule. The fibronectin ED-A is anchored to the cell via a combination of the cell-binding domain and the integrin on the cell. Subsequently, the ED-A domain binds to the receptor (the Toll-like receptor) and stimulates it. Furthermore, the signaling transfers to one of the TGF-β1-activated pathways, which may be the p38 MAPK pathway. Finally, the ED-A-stimulated pathway induces α-SMA expression and type I collagen accumulation, which involves the increased synthesis of collagen and decreased degradation of collagen via the production of proteinase inhibitors, TIMPs and PAI-1.

The Contribution of Fibronectin ED-A Expression to

diabetic nephropathy.

**6. Acknowledgement** 

511, 1474-1733

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Myofibroblast Transdifferentiation in Diabetic Renal Fibrosis 123

inhibition of ED-A expression or activity would likely be minimal. Taken together, our findings and those of previous studies suggest that ED-A may be a target for treatment of

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The previous study demonstrated that TGF-β1 caused the contraction of the fibroblast (without α-SMA expression)-populated collagen lattice in culture medium with 10% FBS. The TGF-β1-induced gel contraction was accelerated by expressing α-SMA, which was induced by a low concentration of FBS and the addition of TGF-β1 (Ina et al., 2011). Therefore, TGF-β1 was suggested to exert distinct functions on α-SMA expression and gel contraction. In addition, it was shown that TGF-β1-induced gel contraction occurred via the p38 MAPK pathway and the Rho kinase pathway (Ina et al., 2011). Taken together, since the p38 MARK pathway is involved in both α-SMA expression and gel contraction, it is likely that ED-A expression might stimulate the p38 MAPK pathway activated by TGF-β1, followed by myofibroblast transdifferentiation and gel contraction.

These findings still have not clarified what gel contraction means in the fibrosis of diabetic nephropathy. We believe that it corresponds to the renal atrophy following fibrosis. However, it is also possible that gel contraction might reflect the shrinkage of collagen fibrils, allowing collagen molecules to become nearer to each other, followed by increased crosslinking, which leads to the formation of rigid fibrils. These are resistant to proteolysis by proteinases, thus leading to the accumulation of fibrils, and therefore, fibrosis.

In the present study, we also demonstrated that accumulation of type I collagen was, at least in part, dependent on ED-A expression. To our knowledge, this is the first report that ED-A mediates TGF-β1-stimulated type I collagen accumulation, while there was a previous report that ED-A stimulated type I collagen mRNA expression (Serini et al., 1998). There are both Smad-dependent and independent pathways, including the p38 MAPK pathway, via which TGF-β1 can stimulate the production of ECM containing type I collagen (Eickelberg, 2001). The observed α-SMA expression, gel contraction, and type I collagen accumulation are, at least in part, dependent on ED-A expression. The α-SMA expression was the most dependent of these events on ED-A. We therefore hypothesized that α-SMA expression is, to a large extent, dependent on ED-A expression via the p38 MAPK pathway. On the other hand, gel contraction and type I collagen accumulation were, to a lesser extent, dependent on ED-A expression, because they are comparatively mediated by pathways other than the p38 MAPK pathway. ED-A binds to Toll-like receptor-4 and activates it, followed by signaling via diverse pathways in inflammatory cells (Akira & Takeda, 2004). The p38 MAPK pathway is also included in these pathways. Thus, it is most likely that the α-SMA expression, gel contraction, and type I collagen accumulation induced by ED-A expression are mediated by activating the p38 MAPK pathway (Fig. 6). In other words, ED-A appears to enhance the activity of the p38 MARK pathway stimulated by TGF-β1.

#### **5. Conclusion**

The TGF-β1-induced α-SMA expression, followed by gel contraction and stimulated type I collagen accumulation, were, at least in part, dependent on fibronectin ED-A expression. These findings suggest that tubulointerstitial fibrosis associated with diabetic nephropathy and fibrotic tissue contraction leading to renal atrophy are partly mediated by TGF-β1-induced fibronectin ED-A expression. In addition, since the fibronectin ED-A isoform is seldom found in normal adult subjects, the negative effects induced by inhibition of ED-A expression or activity would likely be minimal. Taken together, our findings and those of previous studies suggest that ED-A may be a target for treatment of diabetic nephropathy.

#### **6. Acknowledgement**

The authors thank Ms. Yukari Goto for her excellent secretarial assistance.

#### **7. References**

122 Diabetic Nephropathy

The previous study demonstrated that TGF-β1 caused the contraction of the fibroblast (without α-SMA expression)-populated collagen lattice in culture medium with 10% FBS. The TGF-β1-induced gel contraction was accelerated by expressing α-SMA, which was induced by a low concentration of FBS and the addition of TGF-β1 (Ina et al., 2011). Therefore, TGF-β1 was suggested to exert distinct functions on α-SMA expression and gel contraction. In addition, it was shown that TGF-β1-induced gel contraction occurred via the p38 MAPK pathway and the Rho kinase pathway (Ina et al., 2011). Taken together, since the p38 MARK pathway is involved in both α-SMA expression and gel contraction, it is likely that ED-A expression might stimulate the p38 MAPK pathway activated by TGF-β1,

These findings still have not clarified what gel contraction means in the fibrosis of diabetic nephropathy. We believe that it corresponds to the renal atrophy following fibrosis. However, it is also possible that gel contraction might reflect the shrinkage of collagen fibrils, allowing collagen molecules to become nearer to each other, followed by increased crosslinking, which leads to the formation of rigid fibrils. These are resistant to proteolysis

In the present study, we also demonstrated that accumulation of type I collagen was, at least in part, dependent on ED-A expression. To our knowledge, this is the first report that ED-A mediates TGF-β1-stimulated type I collagen accumulation, while there was a previous report that ED-A stimulated type I collagen mRNA expression (Serini et al., 1998). There are both Smad-dependent and independent pathways, including the p38 MAPK pathway, via which TGF-β1 can stimulate the production of ECM containing type I collagen (Eickelberg, 2001). The observed α-SMA expression, gel contraction, and type I collagen accumulation are, at least in part, dependent on ED-A expression. The α-SMA expression was the most dependent of these events on ED-A. We therefore hypothesized that α-SMA expression is, to a large extent, dependent on ED-A expression via the p38 MAPK pathway. On the other hand, gel contraction and type I collagen accumulation were, to a lesser extent, dependent on ED-A expression, because they are comparatively mediated by pathways other than the p38 MAPK pathway. ED-A binds to Toll-like receptor-4 and activates it, followed by signaling via diverse pathways in inflammatory cells (Akira & Takeda, 2004). The p38 MAPK pathway is also included in these pathways. Thus, it is most likely that the α-SMA expression, gel contraction, and type I collagen accumulation induced by ED-A expression are mediated by activating the p38 MAPK pathway (Fig. 6). In other words, ED-A appears to enhance the activity of the p38 MARK

The TGF-β1-induced α-SMA expression, followed by gel contraction and stimulated type I collagen accumulation, were, at least in part, dependent on fibronectin ED-A expression. These findings suggest that tubulointerstitial fibrosis associated with diabetic nephropathy and fibrotic tissue contraction leading to renal atrophy are partly mediated by TGF-β1-induced fibronectin ED-A expression. In addition, since the fibronectin ED-A isoform is seldom found in normal adult subjects, the negative effects induced by

by proteinases, thus leading to the accumulation of fibrils, and therefore, fibrosis.

followed by myofibroblast transdifferentiation and gel contraction.

pathway stimulated by TGF-β1.

**5. Conclusion** 


The Contribution of Fibronectin ED-A Expression to

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Sörensen, I., Susnik, N., Inhester, T., Degen, J.L., Melk, A., Haller, H., & Schmitt, R (2011).

Strutz, F., & Zeisberg, M. (2006). Renal fibroblasts and myofibroblasts in chronic kidney

Takeji, M., Moriyama, T., Oseto, S., Kawada, N., Hori, M., Imai, E., & Miwa, T. (2006).

Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C., & Brown, R.A. (2002). Myofibroblasts

Vartio, T., Laitinen, L., Narvanen, O., Cutolo, M., Thornell, L.E., Zardi, L., &Virtanen, I.

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(1987). Differential expression of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. *Journal of Cell Science,* Vol. 88,


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**8** 

*Spain* 

**Immunoinflammation in Diabetic Nephropathy:** 

Virginia Lopez-Parra, Beñat Mallavia, Jesus Egido and Carmen Gomez-Guerrero

*Renal and Vascular Inflammation, Nephrology Department, IIS-Fundacion Jimenez Diaz, Autonoma University, Madrid* 

**Molecular Mechanisms and Therapeutic Options** 

The prevalence of diabetes mellitus, predominantly of type 2, has dramatically increased worldwide (Ritz et al., 2011). Diabetic nephropathy (DN) affects approximately one third of people with type 1 or type 2 diabetes mellitus (Reutens et al., 2011). In developed countries, the proportion of patients with diabetic kidney disease has stabilized despite increased use of glucose-lowering medications and renin-angiotensin-aldosterone system (RAAS)

DN typically develops after many years of diabetes, and is usually manifested clinically by gradually worsening albuminuria, followed by a decline in glomerular filtration rate, which over years or decades leads to end-stage renal disease. DN is characterized by specific renal morphological and functional alterations. Features of early diabetic renal changes are glomerular hyperfiltration, glomerular and renal hypertrophy, increased urinary albumin excretion, increased basement membrane thickness, and mesangial expansion with the accumulation of extracellular matrix proteins such as collagens, fibronectin, and laminin. Pathologic alterations of the tubulointerstitium such as fibrosis and tubular atrophy are also closely linked to the deterioration of renal function in patients with diabetes of both types 1

Despite the modern therapies like antidiabetic, antihypertensive, and antioxidant drugs available to treat DN, most of patients continue to show progressive renal damage. It suggests that the key pathogenic mechanisms involved in the induction and progression of DN are still remaining active and unmodified by the present therapies (Balakumar et al., 2009). Many studies have attempted to elucidate the molecular signaling mechanisms that lead to DN so that effective therapies and preventive strategies might be developed. Through these efforts the general understanding of the pathogenic signaling factors that lead to progressive DN has expanded considerably during the past decade (Balakumar et

In recent years, extensive research has elucidated several pathways involved in the development and progression of diabetic kidney disease beyond the relevant role of high blood glucose (Schrijvers et al., 2004). Our knowledge of the pathophysiological processes in

**1. Introduction** 

inhibitors (de Boer et al., 2011).

and 2 (Wolf, 2000; Schrijvers et al., 2004).

al., 2009; Brosius et al., 2010).

Zeisberg, E.M., Potenta, S.E., Sugimono, H., Zeisberg, M., & Kalluri, R. (2008). Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. *The Journal of American Society of Nephrology,* Vol. 19, pp. 2282-2287, 1046-6673

## **Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options**

Virginia Lopez-Parra, Beñat Mallavia, Jesus Egido and Carmen Gomez-Guerrero *Renal and Vascular Inflammation, Nephrology Department, IIS-Fundacion Jimenez Diaz, Autonoma University, Madrid Spain* 

#### **1. Introduction**

126 Diabetic Nephropathy

Zeisberg, E.M., Potenta, S.E., Sugimono, H., Zeisberg, M., & Kalluri, R. (2008). Fibroblasts in

*American Society of Nephrology,* Vol. 19, pp. 2282-2287, 1046-6673

kidney fibrosis emerge via endothelial-to-mesenchymal transition. *The Journal of* 

The prevalence of diabetes mellitus, predominantly of type 2, has dramatically increased worldwide (Ritz et al., 2011). Diabetic nephropathy (DN) affects approximately one third of people with type 1 or type 2 diabetes mellitus (Reutens et al., 2011). In developed countries, the proportion of patients with diabetic kidney disease has stabilized despite increased use of glucose-lowering medications and renin-angiotensin-aldosterone system (RAAS) inhibitors (de Boer et al., 2011).

DN typically develops after many years of diabetes, and is usually manifested clinically by gradually worsening albuminuria, followed by a decline in glomerular filtration rate, which over years or decades leads to end-stage renal disease. DN is characterized by specific renal morphological and functional alterations. Features of early diabetic renal changes are glomerular hyperfiltration, glomerular and renal hypertrophy, increased urinary albumin excretion, increased basement membrane thickness, and mesangial expansion with the accumulation of extracellular matrix proteins such as collagens, fibronectin, and laminin. Pathologic alterations of the tubulointerstitium such as fibrosis and tubular atrophy are also closely linked to the deterioration of renal function in patients with diabetes of both types 1 and 2 (Wolf, 2000; Schrijvers et al., 2004).

Despite the modern therapies like antidiabetic, antihypertensive, and antioxidant drugs available to treat DN, most of patients continue to show progressive renal damage. It suggests that the key pathogenic mechanisms involved in the induction and progression of DN are still remaining active and unmodified by the present therapies (Balakumar et al., 2009). Many studies have attempted to elucidate the molecular signaling mechanisms that lead to DN so that effective therapies and preventive strategies might be developed. Through these efforts the general understanding of the pathogenic signaling factors that lead to progressive DN has expanded considerably during the past decade (Balakumar et al., 2009; Brosius et al., 2010).

In recent years, extensive research has elucidated several pathways involved in the development and progression of diabetic kidney disease beyond the relevant role of high blood glucose (Schrijvers et al., 2004). Our knowledge of the pathophysiological processes in

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 129

proinflammatory cytokines, such as interleukins (ILs), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ), all of which exacerbate inflammation and tissue injury *in vivo*  (Mantovani et al., 2004). Alternatively activated M2 macrophages represent the other end of the spectrum and they participate in the resolution of inflammation through the secretion of antiinflammatory factors such as IL-10 and transforming growth factor-β (TGF-β), as well as by inhibiting the production of proinflammatory cytokines, chemokines, and superoxide anion (Martinez et al., 2008; Ricardo et al., 2008). Recent studies demonstrated a reduction in the severity of glomerular inflammation with adoptive transfer of cytokine-programmed M2 macrophages (Ricardo et al., 2008). Furthermore, the antiinflammatory effects of statins (Fujita et al., 2010) and the angiotensin II type 1 receptor blocker olmesartan (Aki et al., 2010) in experimental glomerulonephritis are mediated through downregulation of M1 macrophage infiltration as well as augmentation of antiinflammatory M2 macrophages and cytokines. Based on this, strategies to control the dynamic balance of macrophage polarization could have therapeutic interests in DN, and future studies will determine the

T lymphocytes are known to play a significant role in renal injury induced by non-immune insults including ischaemia or toxins (e.g. adriamycin) (Lim et al., 2010). In patients with type 1 diabetes, the presence of nephropathy and proteinuria has been associated with increased activated peripheral blood T cells and also infiltration of T cells into the kidney (Xiao et al., 2009; Moriya et al., 2004; Ichinose et al., 2007), thus suggesting that lymphocyte activation may play a role in early DN. Activated T cells can cause injury directly through cytotoxic effects and indirectly by recruiting and activating macrophages. In addition, kidney autoantigens may develop during chronic diabetic renal injury and, if this occurs, B cells could present these antigens to T cells to promote their activation. Furthermore, diabetic patients have increased levels of serum immunoglobulins, which include antibodies (Abs) against proteins modified by glycoxidation or lipoxidation. These circulating Abs can form immune complexes (Atchley et al., 2002), which may deposit in glomeruli and promote activation of complement or macrophages via receptor interactions. Elements of the diabetic milieu can directly or indirectly activate T cells in diabetic kidneys. CD4+ T cells express the receptor for advanced glycation end products (AGEs) and can respond to AGEs by producing IFN-γ, which could exacerbate inflammation in the diabetic kidney. In addition, hyperglycemia induces macrophage production of IL-12, which can also stimulate CD4 cell production of IFN-γ (Wen et al., 2006; Lim et al., 2010). T lymphocyte-directed immunotherapies with anti-CD3 and anti-CD4 monoclonal Abs also induce disease remission in non-obese diabetic mice (Mehta et al., 2010). Some of them are currently in Phase III clinical trials for prevention of type 1 diabetes (Miller & St, 2011), although their

The infiltration of leukocytes into sites of inflammation is mediated by sequential binding to specific cell adhesion molecules and chemokine and cytokine release that together promote rolling, arrest, firm adhesion, transmigration, and activation (Hogg & Berlin, 1995). A large array of cell adhesion molecules, chemokines, and cytokines have been shown to be important in leukocyte accumulation and renal injury in models of non-diabetic and diabetic kidney damage, and some of these mediators are also found elevated in renal biopsies from diabetic patients (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Shikata

place of these novel approaches in diabetic patients.

renoprotective effects are not documented.

**2.2 Inflammatory mediators** 

DN has notably improved on a genetic and molecular level. Thus, the classic view of metabolic and hemodynamic alterations as the main causes of renal injury in diabetes has been transformed significantly, with clear evidence indicating that these traditional factors are only a partial aspect of a much more complex picture. One of the most important changes is related to the participation of immune-mediated inflammatory processes in the pathophysiology of diabetes mellitus and its complications (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Shikata & Makino, 2001; Chow et al., 2004).

Although DN is traditionally considered a non-immune disease, accumulating evidence now indicates that immunologic and inflammatory mechanisms play a significant role in its development and progression. DN also includes a variety of inflammatory responses induced by hyperglycemic conditions. Furthermore, microinflammation is a common major mechanism for the progression of DN. This process is mediated by elements of the immune system, including lymphocytes and monocytes/macrophages, as well as cytokines, growth factors, chemokines, adhesion molecules, enzymes, and nuclear factors. In this review we summarized cell processes, mediators and intracellular pathways participating in the immune and inflammatory response during the development of diabetic renal damage.

#### **2. Mechanisms of immune cell infiltration in the diabetic kidney**

#### **2.1 Immune cells**

Yet, the molecular and cellular mechanisms of intrarenal inflammation in DN remain poorly characterized. Macrophages are the major inflammatory cells found in diabetic kidneys and their accumulation is a recognized feature in renal biopsies from diabetic patients (Xiao et al., 2009). In different experimental models of DN, renal macrophage accumulation correlates with the severity of glomerular and tubulointerstitial injury (Chow et al., 2004; Chow et al., 2005). However, it remains to be established whether macrophages are a major effector cell of diabetic renal damage, or merely recruited as a response to injury. Previous studies in diabetic animals reported the protective effect of mycophenolate or irradiation via reduction of renal macrophage infiltration (Wu et al., 2006). However, these treatments have additional effects on the kidney and immune system, and cannot be used for determining the long-term effects of macrophages on the progression of DN.

Macrophages mediate immunopathology and tissue remodeling in both non-renal and renal diseases, and blocking macrophage recruitment prevents the progression of many types of kidney disease models (Ricardo et al., 2008). There are at least two subtypes of resident macrophages in tissues: the M1 macrophages, classically activated by Th1 stimuli, that express proinflammatory cytokines and enhances tissue inflammatory response; and the M2 macrophages, alternatively activated by Th2 stimuli, that express antiinflammatory cytokines, and participate in the promotion of tissue repair, remodeling and vasculogenesis (Gordon, 2003). Obviously, modulating the macrophage phenotype is as important as reducing their overall number to prevent glomerular damage. The relative abundance of M1 and M2 macrophages in the injured kidney changes dynamically by recruitment of polarized monocytes or through the effects of local cytokines on macrophages. Different chemokines are able to recruit circulating monocytes to extravascular compartments such as the glomerulus. Classically activated M1 macrophages represent one end of the spectrum as they produce upon stimulation high amounts of reactive oxygen species (ROS), and proinflammatory cytokines, such as interleukins (ILs), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ), all of which exacerbate inflammation and tissue injury *in vivo*  (Mantovani et al., 2004). Alternatively activated M2 macrophages represent the other end of the spectrum and they participate in the resolution of inflammation through the secretion of antiinflammatory factors such as IL-10 and transforming growth factor-β (TGF-β), as well as by inhibiting the production of proinflammatory cytokines, chemokines, and superoxide anion (Martinez et al., 2008; Ricardo et al., 2008). Recent studies demonstrated a reduction in the severity of glomerular inflammation with adoptive transfer of cytokine-programmed M2 macrophages (Ricardo et al., 2008). Furthermore, the antiinflammatory effects of statins (Fujita et al., 2010) and the angiotensin II type 1 receptor blocker olmesartan (Aki et al., 2010) in experimental glomerulonephritis are mediated through downregulation of M1 macrophage infiltration as well as augmentation of antiinflammatory M2 macrophages and cytokines. Based on this, strategies to control the dynamic balance of macrophage polarization could have therapeutic interests in DN, and future studies will determine the place of these novel approaches in diabetic patients.

T lymphocytes are known to play a significant role in renal injury induced by non-immune insults including ischaemia or toxins (e.g. adriamycin) (Lim et al., 2010). In patients with type 1 diabetes, the presence of nephropathy and proteinuria has been associated with increased activated peripheral blood T cells and also infiltration of T cells into the kidney (Xiao et al., 2009; Moriya et al., 2004; Ichinose et al., 2007), thus suggesting that lymphocyte activation may play a role in early DN. Activated T cells can cause injury directly through cytotoxic effects and indirectly by recruiting and activating macrophages. In addition, kidney autoantigens may develop during chronic diabetic renal injury and, if this occurs, B cells could present these antigens to T cells to promote their activation. Furthermore, diabetic patients have increased levels of serum immunoglobulins, which include antibodies (Abs) against proteins modified by glycoxidation or lipoxidation. These circulating Abs can form immune complexes (Atchley et al., 2002), which may deposit in glomeruli and promote activation of complement or macrophages via receptor interactions. Elements of the diabetic milieu can directly or indirectly activate T cells in diabetic kidneys. CD4+ T cells express the receptor for advanced glycation end products (AGEs) and can respond to AGEs by producing IFN-γ, which could exacerbate inflammation in the diabetic kidney. In addition, hyperglycemia induces macrophage production of IL-12, which can also stimulate CD4 cell production of IFN-γ (Wen et al., 2006; Lim et al., 2010). T lymphocyte-directed immunotherapies with anti-CD3 and anti-CD4 monoclonal Abs also induce disease remission in non-obese diabetic mice (Mehta et al., 2010). Some of them are currently in Phase III clinical trials for prevention of type 1 diabetes (Miller & St, 2011), although their renoprotective effects are not documented.

#### **2.2 Inflammatory mediators**

128 Diabetic Nephropathy

DN has notably improved on a genetic and molecular level. Thus, the classic view of metabolic and hemodynamic alterations as the main causes of renal injury in diabetes has been transformed significantly, with clear evidence indicating that these traditional factors are only a partial aspect of a much more complex picture. One of the most important changes is related to the participation of immune-mediated inflammatory processes in the pathophysiology of diabetes mellitus and its complications (Navarro-Gonzalez & Mora-

Although DN is traditionally considered a non-immune disease, accumulating evidence now indicates that immunologic and inflammatory mechanisms play a significant role in its development and progression. DN also includes a variety of inflammatory responses induced by hyperglycemic conditions. Furthermore, microinflammation is a common major mechanism for the progression of DN. This process is mediated by elements of the immune system, including lymphocytes and monocytes/macrophages, as well as cytokines, growth factors, chemokines, adhesion molecules, enzymes, and nuclear factors. In this review we summarized cell processes, mediators and intracellular pathways participating in the immune and inflammatory response during the development of diabetic renal damage.

Yet, the molecular and cellular mechanisms of intrarenal inflammation in DN remain poorly characterized. Macrophages are the major inflammatory cells found in diabetic kidneys and their accumulation is a recognized feature in renal biopsies from diabetic patients (Xiao et al., 2009). In different experimental models of DN, renal macrophage accumulation correlates with the severity of glomerular and tubulointerstitial injury (Chow et al., 2004; Chow et al., 2005). However, it remains to be established whether macrophages are a major effector cell of diabetic renal damage, or merely recruited as a response to injury. Previous studies in diabetic animals reported the protective effect of mycophenolate or irradiation via reduction of renal macrophage infiltration (Wu et al., 2006). However, these treatments have additional effects on the kidney and immune system, and cannot be used for determining

Macrophages mediate immunopathology and tissue remodeling in both non-renal and renal diseases, and blocking macrophage recruitment prevents the progression of many types of kidney disease models (Ricardo et al., 2008). There are at least two subtypes of resident macrophages in tissues: the M1 macrophages, classically activated by Th1 stimuli, that express proinflammatory cytokines and enhances tissue inflammatory response; and the M2 macrophages, alternatively activated by Th2 stimuli, that express antiinflammatory cytokines, and participate in the promotion of tissue repair, remodeling and vasculogenesis (Gordon, 2003). Obviously, modulating the macrophage phenotype is as important as reducing their overall number to prevent glomerular damage. The relative abundance of M1 and M2 macrophages in the injured kidney changes dynamically by recruitment of polarized monocytes or through the effects of local cytokines on macrophages. Different chemokines are able to recruit circulating monocytes to extravascular compartments such as the glomerulus. Classically activated M1 macrophages represent one end of the spectrum as they produce upon stimulation high amounts of reactive oxygen species (ROS), and

Fernandez, 2008; Galkina & Ley, 2006; Shikata & Makino, 2001; Chow et al., 2004).

**2. Mechanisms of immune cell infiltration in the diabetic kidney** 

the long-term effects of macrophages on the progression of DN.

**2.1 Immune cells** 

The infiltration of leukocytes into sites of inflammation is mediated by sequential binding to specific cell adhesion molecules and chemokine and cytokine release that together promote rolling, arrest, firm adhesion, transmigration, and activation (Hogg & Berlin, 1995). A large array of cell adhesion molecules, chemokines, and cytokines have been shown to be important in leukocyte accumulation and renal injury in models of non-diabetic and diabetic kidney damage, and some of these mediators are also found elevated in renal biopsies from diabetic patients (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Shikata

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 131

small-molecule CCR2 antagonist, reduced glomerular macrophage content, glomerulosclerosis, and albuminuria in diabetic mice, and also improved glomerular filtration rate (Sayyed et al., 2011; Camilla et al., 2011). Furthermore, the renoprotective effect of several compounds, including pioglitazone (Hu et al., 2010), clarithromycin (Tone et al., 2011), and exenatide (Wu et al., 2011) has been related to the local reduction of MCP-1 activity within the kidney. Furthermore, the indazolic derivative bindarit (AF-2838) is a potent antiinflammatory agent that inhibits chemokine synthesis, particularly MCP-1. Phase II trials in rheumatoid arthritis and lupus nephritis have shown that bindarit significantly reduced urinary MCP-1 and albumin excretion rate. A clinical trial aimed to reduce albuminuria and renal disease progression with bindarit added onto RAAS blockade therapy is ongoing in type 2 diabetic

There is growing support for the notion that circulating proinflammatory cytokines, such as ILs and TNF-α, are strongly associated with the risk of developing diabetic complications (Shikata & Makino, 2001). ILs comprise a large group of cytokines secreted by leukocytes and other body cells that can be classified as proinflammatory and antiinflammatory. In particular, the proinflammatory IL-1 increases the expression of chemotactic factors and adhesion molecules, enhances vascular endothelial permeability, and stimulates the proliferation of mesangial cells and matrix synthesis (Rivero et al., 2009). Renal IL-1 expression is found increased in diabetic animals and correlates with albuminuria and macrophage content (Hasegawa et al., 1991; Sassy-Prigent et al., 2000; Navarro et al., 2006). Specific blockade of IL-1 activity by the IL-1 receptor antagonist anakinra reduced the release of inflammatory cytokines and chemokines in pancreatic islet from diabetic rats, and also decreased hyperglycemia and improved insulin sensitivity (Ehses et al., 2009). In type 2 diabetic patients, anakinra improved glycemia and beta-cell secretory function and reduced markers of systemic inflammation (Larsen et al., 2007). Further studies are needed to

IL-6 is a pleiotropic cytokine secreted by renal cells in response to a diabetic milieu (Min et al., 2009; Tang et al., 2010a) that stimulates mesangial cell proliferation, affects extracellular matrix dynamics in renal cells, and enhances endothelial permeability (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Shikata & Makino, 2001; Chow et al., 2004). Serum IL-6 levels are significantly increased in patients with type 2 DN compared to levels observed in diabetic patients without nephropathy (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Dalla et al., 2005), and studies in renal biopsies revealed a significant association between the severity of diabetic glomerulopathy and the expression levels of IL-6 in glomerular cells (Suzuki et al., 1995), thus suggesting a role for IL-6 in the pathogenesis of DN. There are no direct data of treatment against elevated IL-6 levels in DN, however there are indirect evidences. In a recent study in patients with incipient and established DN, the treatment with pentoxyfylline, a methylxanthine derivate and nonselective phosphodiesterase inhibitor, caused a decrease in the urinary albumin excretion, and this renoprotective effect was attributable in part to reduced levels of IL-6 among other proinflammatory mediators

TNF-α is a pleiotropic cytokine produced mainly by monocytes/macrophages that is involved in systemic inflammation (Sugimoto et al., 1999). TNF-α exerts cytotoxic effects on renal cells (McCarthy et al., 1998; Min et al., 2009), and it has been shown to participate in

patients with micro- or macroalbuminuria (Cortinovis et al., 2008).

demonstrate the biological effects of this compound on diabetic kidneys.

(Hasegawa et al., 1991; Sassy-Prigent et al., 2000; Navarro et al., 2006).

**2.2.3 Cytokines: IL-1, IL-6, and TNF-α** 

& Makino, 2001; Chow et al., 2004). The most representative members for each family are discussed below.

#### **2.2.1 Adhesion molecules: ICAM-1**

Intercellular adhesion molecule (ICAM)-1 is a 90-kD cell surface glycoprotein of the Ig superfamily involved in the firm attachment of leukocytes to endothelium (Staunton et al., 1988), which interacts with lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) that are present on most leukocytes. ICAM-1 expression is upregulated and associated with leukocyte infiltration and disease progression in experimental models of type 1 and type 2 DN (Sugimoto et al., 1997; Coimbra et al., 2000) and also in diabetic patients (Rubio-Guerra et al., 2009). ICAM-1 is induced by factors common to both types of diabetes, such as hyperglycemia, AGEs, hyperfiltration, and oxidative stress, but it can also be increased by additional elements such as hyperlipidemia, hyperinsulinemia, and elevated levels of circulating TNF-α (Chow et al., 2006). Previous evidence from genetically deficient mice demonstrated that ICAM-1 is a critical mediator of macrophage accumulation in diabetic kidneys both in early and late stages of diabetes (Okada et al., 2003; Chow et al., 2005), while treatment with anti-ICAM-1 monoclonal Ab prevents mononuclear cell infiltration into diabetic glomeruli (Sugimoto et al., 1997). Furthermore, the reduced ICAM-1 overexpression is one of the renoprotective effecs of taurine treatment in streptozotocin-induced diabetic rats (Wang et al., 2008).

#### **2.2.2 Chemokines: MCP-1**

Monocyte chemotactic protein-1 (MCP-1) is a small cytokine belonging to the CC chemokine family that is also known as chemokine (C-C motif) ligand 2 (CCL2). MCP-1 recruits monocytes, T cells, macrophages and dendritic cells to sites of tissue injury, infection, and inflammation, and is gaining interest as a mediator of DN. MCP-1 is induced by high glucose concentrations, AGEs and cytokines in cultured renal cells (Chow et al., 2006), and its expression increases progressively in diabetic kidneys from different animal models. In diabetic patients, MCP-1 urinary levels correlate with albuminuria, therefore being considered a marker of renal function decline (Sayyed et al., 2011; Camilla et al., 2011).

MCP-1 is a potent chemokine involved in the accumulation and function of macrophages (Chow et al., 2006), thus playing a role in the inflammatory phase of DN. Renal cells like podocytes and mesangial cells are also able to produce MCP-1 in response to metabolic mediators, such as high glucose, and AGEs, and also by activation of RAAS and TGF-β (Yamagishi & Matsui, 2010). Renal cells are susceptible to paracrine and autocrine activation by MCP-1, through the interaction with CCR2, the main receptor of MCP-1 (Sayyed et al., 2011; Camilla et al., 2011). In fact, MCP-1/CCR2 system is involved in podocyte cytoskeleton reorganization and motility, and also in mesangial expression of fibronectin and type IV collagen (Lee et al., 2009; Park et al., 2008). Diabetic mice with gene deficiency in MCP-1 are protected from glomerular macrophage infiltration, renal injury, and development of albuminuria (Chow et al., 2006).

As upregulation of kidney MCP-1 is a feature of human diabetic renal injury associated with macrophage recruitment and disease progression, neutralizing MCP-1 activity should be viewed as an important therapeutic goal in the treatment of DN. Preclinical studies have demonstrated that blockade of MCP-1/CCR2 system with RO5234444, an orally active small-molecule CCR2 antagonist, reduced glomerular macrophage content, glomerulosclerosis, and albuminuria in diabetic mice, and also improved glomerular filtration rate (Sayyed et al., 2011; Camilla et al., 2011). Furthermore, the renoprotective effect of several compounds, including pioglitazone (Hu et al., 2010), clarithromycin (Tone et al., 2011), and exenatide (Wu et al., 2011) has been related to the local reduction of MCP-1 activity within the kidney. Furthermore, the indazolic derivative bindarit (AF-2838) is a potent antiinflammatory agent that inhibits chemokine synthesis, particularly MCP-1. Phase II trials in rheumatoid arthritis and lupus nephritis have shown that bindarit significantly reduced urinary MCP-1 and albumin excretion rate. A clinical trial aimed to reduce albuminuria and renal disease progression with bindarit added onto RAAS blockade therapy is ongoing in type 2 diabetic patients with micro- or macroalbuminuria (Cortinovis et al., 2008).

#### **2.2.3 Cytokines: IL-1, IL-6, and TNF-α**

130 Diabetic Nephropathy

& Makino, 2001; Chow et al., 2004). The most representative members for each family are

Intercellular adhesion molecule (ICAM)-1 is a 90-kD cell surface glycoprotein of the Ig superfamily involved in the firm attachment of leukocytes to endothelium (Staunton et al., 1988), which interacts with lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) that are present on most leukocytes. ICAM-1 expression is upregulated and associated with leukocyte infiltration and disease progression in experimental models of type 1 and type 2 DN (Sugimoto et al., 1997; Coimbra et al., 2000) and also in diabetic patients (Rubio-Guerra et al., 2009). ICAM-1 is induced by factors common to both types of diabetes, such as hyperglycemia, AGEs, hyperfiltration, and oxidative stress, but it can also be increased by additional elements such as hyperlipidemia, hyperinsulinemia, and elevated levels of circulating TNF-α (Chow et al., 2006). Previous evidence from genetically deficient mice demonstrated that ICAM-1 is a critical mediator of macrophage accumulation in diabetic kidneys both in early and late stages of diabetes (Okada et al., 2003; Chow et al., 2005), while treatment with anti-ICAM-1 monoclonal Ab prevents mononuclear cell infiltration into diabetic glomeruli (Sugimoto et al., 1997). Furthermore, the reduced ICAM-1 overexpression is one of the renoprotective effecs of

taurine treatment in streptozotocin-induced diabetic rats (Wang et al., 2008).

Monocyte chemotactic protein-1 (MCP-1) is a small cytokine belonging to the CC chemokine family that is also known as chemokine (C-C motif) ligand 2 (CCL2). MCP-1 recruits monocytes, T cells, macrophages and dendritic cells to sites of tissue injury, infection, and inflammation, and is gaining interest as a mediator of DN. MCP-1 is induced by high glucose concentrations, AGEs and cytokines in cultured renal cells (Chow et al., 2006), and its expression increases progressively in diabetic kidneys from different animal models. In diabetic patients, MCP-1 urinary levels correlate with albuminuria, therefore being considered a marker of renal function decline (Sayyed et al., 2011; Camilla et al., 2011).

MCP-1 is a potent chemokine involved in the accumulation and function of macrophages (Chow et al., 2006), thus playing a role in the inflammatory phase of DN. Renal cells like podocytes and mesangial cells are also able to produce MCP-1 in response to metabolic mediators, such as high glucose, and AGEs, and also by activation of RAAS and TGF-β (Yamagishi & Matsui, 2010). Renal cells are susceptible to paracrine and autocrine activation by MCP-1, through the interaction with CCR2, the main receptor of MCP-1 (Sayyed et al., 2011; Camilla et al., 2011). In fact, MCP-1/CCR2 system is involved in podocyte cytoskeleton reorganization and motility, and also in mesangial expression of fibronectin and type IV collagen (Lee et al., 2009; Park et al., 2008). Diabetic mice with gene deficiency in MCP-1 are protected from glomerular macrophage infiltration, renal injury, and

As upregulation of kidney MCP-1 is a feature of human diabetic renal injury associated with macrophage recruitment and disease progression, neutralizing MCP-1 activity should be viewed as an important therapeutic goal in the treatment of DN. Preclinical studies have demonstrated that blockade of MCP-1/CCR2 system with RO5234444, an orally active

discussed below.

**2.2.1 Adhesion molecules: ICAM-1** 

**2.2.2 Chemokines: MCP-1** 

development of albuminuria (Chow et al., 2006).

There is growing support for the notion that circulating proinflammatory cytokines, such as ILs and TNF-α, are strongly associated with the risk of developing diabetic complications (Shikata & Makino, 2001). ILs comprise a large group of cytokines secreted by leukocytes and other body cells that can be classified as proinflammatory and antiinflammatory. In particular, the proinflammatory IL-1 increases the expression of chemotactic factors and adhesion molecules, enhances vascular endothelial permeability, and stimulates the proliferation of mesangial cells and matrix synthesis (Rivero et al., 2009). Renal IL-1 expression is found increased in diabetic animals and correlates with albuminuria and macrophage content (Hasegawa et al., 1991; Sassy-Prigent et al., 2000; Navarro et al., 2006). Specific blockade of IL-1 activity by the IL-1 receptor antagonist anakinra reduced the release of inflammatory cytokines and chemokines in pancreatic islet from diabetic rats, and also decreased hyperglycemia and improved insulin sensitivity (Ehses et al., 2009). In type 2 diabetic patients, anakinra improved glycemia and beta-cell secretory function and reduced markers of systemic inflammation (Larsen et al., 2007). Further studies are needed to demonstrate the biological effects of this compound on diabetic kidneys.

IL-6 is a pleiotropic cytokine secreted by renal cells in response to a diabetic milieu (Min et al., 2009; Tang et al., 2010a) that stimulates mesangial cell proliferation, affects extracellular matrix dynamics in renal cells, and enhances endothelial permeability (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Shikata & Makino, 2001; Chow et al., 2004). Serum IL-6 levels are significantly increased in patients with type 2 DN compared to levels observed in diabetic patients without nephropathy (Navarro-Gonzalez & Mora-Fernandez, 2008; Galkina & Ley, 2006; Dalla et al., 2005), and studies in renal biopsies revealed a significant association between the severity of diabetic glomerulopathy and the expression levels of IL-6 in glomerular cells (Suzuki et al., 1995), thus suggesting a role for IL-6 in the pathogenesis of DN. There are no direct data of treatment against elevated IL-6 levels in DN, however there are indirect evidences. In a recent study in patients with incipient and established DN, the treatment with pentoxyfylline, a methylxanthine derivate and nonselective phosphodiesterase inhibitor, caused a decrease in the urinary albumin excretion, and this renoprotective effect was attributable in part to reduced levels of IL-6 among other proinflammatory mediators (Hasegawa et al., 1991; Sassy-Prigent et al., 2000; Navarro et al., 2006).

TNF-α is a pleiotropic cytokine produced mainly by monocytes/macrophages that is involved in systemic inflammation (Sugimoto et al., 1999). TNF-α exerts cytotoxic effects on renal cells (McCarthy et al., 1998; Min et al., 2009), and it has been shown to participate in

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 133

receptors in the kidney by various mechanisms, and angiotensin converting enzyme (ACE) inhibition reduces serum and urinary levels of TGF-β (Wolf, 2006). In diabetic patients, treatment with the ACE inhibitor perindopril reduced the intrarenal TGF-β expression and activity (Langham et al., 2006). Furthermore, the antifibrotic agent N-acetyl-serylaspartyl-lysyl-proline, which reduced TGF-β-induced extracellular matrix production and prevented renal fibrosis and albuminuria in diabetic db/db mice, conferred an additional renoprotective effect when combined with the angiotensin II receptor antagonist losartan

A range of novel compounds has been recently examined to inhibit TGF-β and TGF-βdependent pathways in diabetes. Several blocking Abs against TGF-β effectively reduce mesangial matrix accumulation and glomerulosclerosis in diabetic mouse models (Ziyadeh et al., 2000; Goh et al., 2008), and particularly the TGF-AY1 Ab is in clinical development for the treatment of chronic kidney disease, with focus on DN (Cortinovis et al., 2008). In addition, the soluble human TGF-β type II receptor (sT β RII-Fc), a high-affinity TGF-β1 binding molecule, has been proposed as a potential new agent for the treatment of fibrosis

A potential therapeutic approach is the use of micro RNA (miRNA)-based strategies. The miRNAs are short noncoding nucleotides that regulate target messenger RNAs at the posttranscriptional level and are involved in many biological processes (Lorenzen et al., 2011). Recent studies have identified miRNA-mediated circuits controlling auto-upregulation of TGF-β1 and amplification of TGF-β1 signaling that accelerate chronic fibrotic kidney diseases including DN (Kato et al., 2011). In particular, miRNA-92c and miRNA-192 are induced in renal cells by high glucose and TGF-β, and mediate cell apoptosis and extracellular matrix accumulation (Kato et al., 2011; Long et al., 2011). Renal expression of these miRNAs increased in type 1 and type 2 diabetic animals, and *in vivo* knockdown prevented progression of DN. Their widespread and distinct expression patterns under normal and disease states make miRNAs attractive molecular therapeutic targets for human diseases. In fact, different miRNA modulators (such as antagomirs and locked nucleic acid antimiRs) have been developed for specific targeting of miRNAs and respective downstream gene networks (Lorenzen et al., 2011). The therapeutic potential of miRNA-

Several reports have described an increased expression of CTGF in diabetic kidneys, that is therefore being considered a marker and a mediator of disease. Synthesis of CTGF is stimulated by TGF-β, hyperglycemia and AGEs, as well as CTGF itself. CTGF induces mesangial cell hypertrophy and cytoskeletal disassembling, upregulates cell production of fibronectin and collagens, and is also involved in epithelial-to-mesenchymal transition of

CTGF is also an important downstream mediator of the profibrotic activity of TGF-β. But in contrast to TGF-β, CTGF is not centrally involved in the modulation of inflammation or immune reactions (Goldschmeding et al., 2000) and thus this profibrotic factor may be a more attractive target for new renoprotective therapies. In fact, decreased CTGF expression in the kidney has been suggested as the mechanism involved in the inhibition of diabetic renal damage by different agents including the AGE inhibitors aminoguanidine (Twigg &

(Sugaru et al., 2006).

and albuminuria in DN (Russo et al., 2007).

based treatment in DN requires further study.

tubular cells (Twigg, 2010; Connolly et al., 2003).

**3.1.2 CTGF** 

renal damage development in experimental models of renal disease including lupus nephritis, glomerulonephritis, nephropathy, hypertension, and diabetes (McCarthy et al., 1998; Elmarakby & Sullivan, 2010). A role for TNF-α in DN is supported by the finding that urinary albumin excretion significantly correlates with renal TNF-α levels and urinary TNFα excretion in streptozotocin-induced diabetic rats (Navarro et al., 2005). TNF-α also contributes to sodium retention and renal hypertrophy, which are early characteristic signs of streptozotocin-induced DN (DiPetrillo et al., 2003). Renal TNF-α expression, particularly in the glomerulus and tubulointerestitium, is increased in streptozotocin diabetic rat kidneys, and serum TNF-α is increased in type 2 diabetic patients (Navarro et al., 2005). Therefore, TNF-α plays an important role in the incidence and progression of DN and renal TNF-α levels correlate with markers of DN.

Strategies to inhibit TNF-α have been successfully used in experimental diabetes. DiPetrillo et al. (DiPetrillo et al., 2003) reported that treatment of diabetic rats with the anti-TNF-α agent TNFR:Fc, a soluble TNF-α receptor fusion protein, reduced urinary TNF-α excretion and prevented sodium retention and renal hypertrophy. Similarly, TNF-α inhibition with infliximab, a chimeric monoclonal Ab directed against TNF-α, significantly reduced both albuminuria and urinary TNF-α in streptozotocin-induced diabetic rats (Moriwaki et al., 2007). Unfortunately, no other parameters such as structural changes or hemodynamics were studied. A recent retrospective study evaluated the effects of anti–TNF-α agents on control of type 2 diabetes in patients with rheumatoid arthritis and Crohn's disease. Anti-TNF treatment improved glucose tolerance and control, although future prospective studies are needed to solidify these results (Gupta-Ganguli et al., 2011).

#### **3. Molecules involved in the progression of diabetic renal disease**

#### **3.1 Growth factors: TGF-β, CTGF, and VEGF**

Hyperglycemia stimulates resident and non-resident renal cells to produce cytokines and growth factors that contribute to the development of renal injury. In particular, the expression of the profibrotic factor TGF-β is increased in both type 1 and type 2 diabetes (Cortinovis et al., 2008). Other growth factors are also implicated, including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), among others acting through several intracellular signaling pathways comprising protein kinases and transcription factors (Goh et al., 2008). Recently, specific Abs against different growth factors that might be useful for the treatment of chronic nephropathies, including DN, have been developed (Cortinovis et al., 2008).

#### **3.1.1 TGF-β**

TGF-β is a well-known profibrogenic factor which controls synthesis and degradation of extracellular matrix proteins by stimulating transcription of extracellular matrix genes in renal cells and reducing collagenase production, eventually inhibiting matrix turnover. Furthermore TGF-β is involved in tubuloglomerular sclerosis and podocyte apoptosis in diabetes. TGF-β gene and protein levels are significantly increased in glomeruli and tubulointerstitium of type 1 and 2 diabetic patients and animals (Yamagishi & Matsui, 2010; Goh et al., 2008). Factors that regulate TGF-β expression in renal cells include hyperglycemia, AGEs, endothelin, lipids and products of oxidative stress. TGF-β is also modulated by the RAAS (Goh et al., 2008). In fact, angiotensin II stimulates TGF-β and its receptors in the kidney by various mechanisms, and angiotensin converting enzyme (ACE) inhibition reduces serum and urinary levels of TGF-β (Wolf, 2006). In diabetic patients, treatment with the ACE inhibitor perindopril reduced the intrarenal TGF-β expression and activity (Langham et al., 2006). Furthermore, the antifibrotic agent N-acetyl-serylaspartyl-lysyl-proline, which reduced TGF-β-induced extracellular matrix production and prevented renal fibrosis and albuminuria in diabetic db/db mice, conferred an additional renoprotective effect when combined with the angiotensin II receptor antagonist losartan (Sugaru et al., 2006).

A range of novel compounds has been recently examined to inhibit TGF-β and TGF-βdependent pathways in diabetes. Several blocking Abs against TGF-β effectively reduce mesangial matrix accumulation and glomerulosclerosis in diabetic mouse models (Ziyadeh et al., 2000; Goh et al., 2008), and particularly the TGF-AY1 Ab is in clinical development for the treatment of chronic kidney disease, with focus on DN (Cortinovis et al., 2008). In addition, the soluble human TGF-β type II receptor (sT β RII-Fc), a high-affinity TGF-β1 binding molecule, has been proposed as a potential new agent for the treatment of fibrosis and albuminuria in DN (Russo et al., 2007).

A potential therapeutic approach is the use of micro RNA (miRNA)-based strategies. The miRNAs are short noncoding nucleotides that regulate target messenger RNAs at the posttranscriptional level and are involved in many biological processes (Lorenzen et al., 2011). Recent studies have identified miRNA-mediated circuits controlling auto-upregulation of TGF-β1 and amplification of TGF-β1 signaling that accelerate chronic fibrotic kidney diseases including DN (Kato et al., 2011). In particular, miRNA-92c and miRNA-192 are induced in renal cells by high glucose and TGF-β, and mediate cell apoptosis and extracellular matrix accumulation (Kato et al., 2011; Long et al., 2011). Renal expression of these miRNAs increased in type 1 and type 2 diabetic animals, and *in vivo* knockdown prevented progression of DN. Their widespread and distinct expression patterns under normal and disease states make miRNAs attractive molecular therapeutic targets for human diseases. In fact, different miRNA modulators (such as antagomirs and locked nucleic acid antimiRs) have been developed for specific targeting of miRNAs and respective downstream gene networks (Lorenzen et al., 2011). The therapeutic potential of miRNAbased treatment in DN requires further study.

#### **3.1.2 CTGF**

132 Diabetic Nephropathy

renal damage development in experimental models of renal disease including lupus nephritis, glomerulonephritis, nephropathy, hypertension, and diabetes (McCarthy et al., 1998; Elmarakby & Sullivan, 2010). A role for TNF-α in DN is supported by the finding that urinary albumin excretion significantly correlates with renal TNF-α levels and urinary TNFα excretion in streptozotocin-induced diabetic rats (Navarro et al., 2005). TNF-α also contributes to sodium retention and renal hypertrophy, which are early characteristic signs of streptozotocin-induced DN (DiPetrillo et al., 2003). Renal TNF-α expression, particularly in the glomerulus and tubulointerestitium, is increased in streptozotocin diabetic rat kidneys, and serum TNF-α is increased in type 2 diabetic patients (Navarro et al., 2005). Therefore, TNF-α plays an important role in the incidence and progression of DN and renal

Strategies to inhibit TNF-α have been successfully used in experimental diabetes. DiPetrillo et al. (DiPetrillo et al., 2003) reported that treatment of diabetic rats with the anti-TNF-α agent TNFR:Fc, a soluble TNF-α receptor fusion protein, reduced urinary TNF-α excretion and prevented sodium retention and renal hypertrophy. Similarly, TNF-α inhibition with infliximab, a chimeric monoclonal Ab directed against TNF-α, significantly reduced both albuminuria and urinary TNF-α in streptozotocin-induced diabetic rats (Moriwaki et al., 2007). Unfortunately, no other parameters such as structural changes or hemodynamics were studied. A recent retrospective study evaluated the effects of anti–TNF-α agents on control of type 2 diabetes in patients with rheumatoid arthritis and Crohn's disease. Anti-TNF treatment improved glucose tolerance and control, although future prospective studies

TNF-α levels correlate with markers of DN.

are needed to solidify these results (Gupta-Ganguli et al., 2011).

**3.1 Growth factors: TGF-β, CTGF, and VEGF** 

developed (Cortinovis et al., 2008).

**3.1.1 TGF-β** 

**3. Molecules involved in the progression of diabetic renal disease** 

Hyperglycemia stimulates resident and non-resident renal cells to produce cytokines and growth factors that contribute to the development of renal injury. In particular, the expression of the profibrotic factor TGF-β is increased in both type 1 and type 2 diabetes (Cortinovis et al., 2008). Other growth factors are also implicated, including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), among others acting through several intracellular signaling pathways comprising protein kinases and transcription factors (Goh et al., 2008). Recently, specific Abs against different growth factors that might be useful for the treatment of chronic nephropathies, including DN, have been

TGF-β is a well-known profibrogenic factor which controls synthesis and degradation of extracellular matrix proteins by stimulating transcription of extracellular matrix genes in renal cells and reducing collagenase production, eventually inhibiting matrix turnover. Furthermore TGF-β is involved in tubuloglomerular sclerosis and podocyte apoptosis in diabetes. TGF-β gene and protein levels are significantly increased in glomeruli and tubulointerstitium of type 1 and 2 diabetic patients and animals (Yamagishi & Matsui, 2010; Goh et al., 2008). Factors that regulate TGF-β expression in renal cells include hyperglycemia, AGEs, endothelin, lipids and products of oxidative stress. TGF-β is also modulated by the RAAS (Goh et al., 2008). In fact, angiotensin II stimulates TGF-β and its Several reports have described an increased expression of CTGF in diabetic kidneys, that is therefore being considered a marker and a mediator of disease. Synthesis of CTGF is stimulated by TGF-β, hyperglycemia and AGEs, as well as CTGF itself. CTGF induces mesangial cell hypertrophy and cytoskeletal disassembling, upregulates cell production of fibronectin and collagens, and is also involved in epithelial-to-mesenchymal transition of tubular cells (Twigg, 2010; Connolly et al., 2003).

CTGF is also an important downstream mediator of the profibrotic activity of TGF-β. But in contrast to TGF-β, CTGF is not centrally involved in the modulation of inflammation or immune reactions (Goldschmeding et al., 2000) and thus this profibrotic factor may be a more attractive target for new renoprotective therapies. In fact, decreased CTGF expression in the kidney has been suggested as the mechanism involved in the inhibition of diabetic renal damage by different agents including the AGE inhibitors aminoguanidine (Twigg &

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 135

Ha, 2011; Elmarakby & Sullivan, 2010). Among many pathways, nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase and mitochondrial dysfunction have been recognized as two major sources of ROS generation in diabetic kidneys, and NADPH oxidase-derived ROS has been shown to facilitate renal mitochondrial superoxide

Inhibition of ROS production and their activity has been demonstrated to be effective in preventing the development and progression of experimental diabetes. Different approaches including oral administration of resveratrol (Palsamy & Subramanian, 2011) and dietary antioxidant supplementation with N-acetylcysteine, vitamins C and E (Park et al., 2011) and curcuminoids (Sharma et al., 2006) have been shown to reduce oxidative stress and renal inflammation in diabetic animals. Clinical observations have also revealed a positive effect on oxidative stress in diabetic patients (Gupta et al., 2011). Alternative strategies based on upregulation of antioxidant proteins, such as superoxide dismutase, heme oxygenase-1, and catalase, have also been proven to diminish high glucose-induced ROS in cell cultures and animal models (Li et al., 2011). A recent study in diabetic animals reported the renoprotective effects of luteolin (Wang et al., 2011), a plant-derived flavonoid with antiinflammatory and antitumorigenic properties (Lopez-Lazaro, 2009). The improvement of renal function in luteolin treated animals was associated with changes in superoxide dismutase activity, malondialdehyde content and expression of heme oxygenase-1 expression (Wang et al., 2011). New strategies targeting NF-E2-related factor 2, the transcription factor that controls antioxidant protein expression, mitochondrial dysfunction, and NADPH oxidase might provide a potential approach for the prevention and treatment of DN (Noh & Ha, 2011). Presently, designing new antioxidant therapies focus on effective, cell compartment-specific agents that would improve renoprotection in combination with current therapies (Forbes et al., 2008). Reports on their efficacy in clinical trials for

inflammatory-associated pathologies, including diabetes, have yet to be published.

Intense investigation revealed that numerous inflammatory signaling pathways such as phospholipases, protein kinase cascades and transcription factors, are implicated in the pathogenesis of DN, from early phase to the progression and final complications. Among them, nuclear factor-κB (NF-κB) and janus kinase/signal transducers and activators of transcription (JAK/STAT) have a relevant role in the control of immunoinflammatory responses in the diabetic kidney. Mechanisms of action and therapeutic opportunities are

The transcription factor NF-κB is induced by various cell stress-associated stimuli including growth factors, vasoactive agents, cytokines, and oxidative stress. NF-κB in turn controls the regulation of numerous genes activated during inflammation, such as cytokines, chemokines, growth factors, cellular ligands, and adhesion molecules (Karin & Greten, 2005). The activation and nuclear translocation of NF-κB has been demonstrated in diabetic kidneys from human and rodents (Mezzano et al., 2004; Liu et al., 2010), and also in proximal tubular cells in the urinary sediment of patients with type 2 diabetes (Brosius et al., 2010). Furthermore, a study integrating gene-expression profiling in human renal biopsies

**4. Intracellular pathways activated in diabetic kidney** 

discussed.

**4.1 NF-κB** 

production in hyperglycemia (Tojo et al., 2007).

Cooper, 2004) and XLF-III-43 (Li et al., 2010), the aldosterone receptor blocker spironolactone (Han et al., 2006), and the flavonoid compound astilbin (Li et al., 2009). More specific therapies include FG-3019, a humanized monoclonal Ab that neutralizes the effects of CTGF in diabetic animals (Cortinovis et al., 2008). FG-3019 is currently under development for idiopathic pulmonary fibrosis, pancreatic cancer and diabetes. In an openlabel, dose-escalation Phase Ib trial in type 1 or 2 diabetic patients with incipient nephropathy, FG-3019 effectively decreased albuminuria (Adler et al., 2010), although further validation in a prospective, randomized, blinded study is required.

#### **3.1.3 VEGF**

VEGF is a potent inducer of vasopermeability and angiogenesis that plays a major pathophysiological role in DN, despite VEGF exhibiting protective roles in non-diabetic renal disease. Serum levels of VEGF correlate with albuminuria and increase with DN stage in patients with type 1 and 2 diabetes (Hovind et al., 2000). Several experimental model studies have demonstrated that VEGF may contribute to some of the hemodynamic changes in DN, including hyperfiltration and albuminuria. Expression of VEGF and its receptors are modulated by high glucose, endothelin 1, AGEs, angiotensin II, stretch and TGF-β, and their renal expression is increased in diabetic kidneys (Chen & Ziyadeh, 2008; Cooper et al., 1999). Furthermore, VEGF is a trophic factor for glomerular endothelial cells, affects podocyte function, and is also involved in macrophage influx during early DN (Chen & Ziyadeh, 2008).

Since VEGF levels are elevated in patients and animal models of DN, a number of studies have examined the effect of inhibition of VEGF receptor binding or activation. Antagonism of VEGF using a variety of different strategies has been reported to improve the outcome in experimental nephropathies of various origins, including DN. Neutralizing VEGF Abs ameliorate early and long-term renal changes in diabetic animals (De Vriese et al., 2001; Schrijvers et al., 2006), while treatment with the pan-VEGF receptor tyrosine kinase inhibitor SU5416 ameliorates diabetic albuminuria in mice (Sung et al., 2006). Several clinical studies have evaluated the beneficial effect of these anti-VEGF agents in the treatment of diabetic retinopathy, but more studies are needed to determine the viability of such strategy in diabetic renal disease.

#### **3.2 Oxidative stress**

Oxidative stress defined as an excessive production of ROS surpassing existing antioxidative defense mechanisms plays a critical role in the pathogenesis of diabetes, and more importantly in the development of diabetic complications, including DN. Free radicals are capable of disturb physiological cell function both directly, by damaging cellular macromolecules such as DNA, proteins, and lipids, and indirectly through the stimulation of multiple pathways, such as protein kinases, polyol and hexosamine pathways and AGEs formation. In addition, low antioxidant bioavailability promotes cellular oxidative stress leading to additional cellular damage (Forbes et al., 2008; Elmarakby & Sullivan, 2010; Noh & Ha, 2011).

Overproduction of ROS in the diabetic milieu is both a direct consequence of hyperglycemia and an indirect consequence through AGEs or mediators of glucotoxicity such as cytokines and growth factors (Noh & Ha, 2011; Forbes et al., 2008). The effects of ROS in renal cells comprise mesangial cell proliferation, expression of growth factors, extracellular matrix accumulation, RAAS activation and induction of epithelial-mesenchymal transition (Noh & Ha, 2011; Elmarakby & Sullivan, 2010). Among many pathways, nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase and mitochondrial dysfunction have been recognized as two major sources of ROS generation in diabetic kidneys, and NADPH oxidase-derived ROS has been shown to facilitate renal mitochondrial superoxide production in hyperglycemia (Tojo et al., 2007).

Inhibition of ROS production and their activity has been demonstrated to be effective in preventing the development and progression of experimental diabetes. Different approaches including oral administration of resveratrol (Palsamy & Subramanian, 2011) and dietary antioxidant supplementation with N-acetylcysteine, vitamins C and E (Park et al., 2011) and curcuminoids (Sharma et al., 2006) have been shown to reduce oxidative stress and renal inflammation in diabetic animals. Clinical observations have also revealed a positive effect on oxidative stress in diabetic patients (Gupta et al., 2011). Alternative strategies based on upregulation of antioxidant proteins, such as superoxide dismutase, heme oxygenase-1, and catalase, have also been proven to diminish high glucose-induced ROS in cell cultures and animal models (Li et al., 2011). A recent study in diabetic animals reported the renoprotective effects of luteolin (Wang et al., 2011), a plant-derived flavonoid with antiinflammatory and antitumorigenic properties (Lopez-Lazaro, 2009). The improvement of renal function in luteolin treated animals was associated with changes in superoxide dismutase activity, malondialdehyde content and expression of heme oxygenase-1 expression (Wang et al., 2011). New strategies targeting NF-E2-related factor 2, the transcription factor that controls antioxidant protein expression, mitochondrial dysfunction, and NADPH oxidase might provide a potential approach for the prevention and treatment of DN (Noh & Ha, 2011). Presently, designing new antioxidant therapies focus on effective, cell compartment-specific agents that would improve renoprotection in combination with current therapies (Forbes et al., 2008). Reports on their efficacy in clinical trials for inflammatory-associated pathologies, including diabetes, have yet to be published.

#### **4. Intracellular pathways activated in diabetic kidney**

Intense investigation revealed that numerous inflammatory signaling pathways such as phospholipases, protein kinase cascades and transcription factors, are implicated in the pathogenesis of DN, from early phase to the progression and final complications. Among them, nuclear factor-κB (NF-κB) and janus kinase/signal transducers and activators of transcription (JAK/STAT) have a relevant role in the control of immunoinflammatory responses in the diabetic kidney. Mechanisms of action and therapeutic opportunities are discussed.

#### **4.1 NF-κB**

134 Diabetic Nephropathy

Cooper, 2004) and XLF-III-43 (Li et al., 2010), the aldosterone receptor blocker spironolactone (Han et al., 2006), and the flavonoid compound astilbin (Li et al., 2009). More specific therapies include FG-3019, a humanized monoclonal Ab that neutralizes the effects of CTGF in diabetic animals (Cortinovis et al., 2008). FG-3019 is currently under development for idiopathic pulmonary fibrosis, pancreatic cancer and diabetes. In an openlabel, dose-escalation Phase Ib trial in type 1 or 2 diabetic patients with incipient nephropathy, FG-3019 effectively decreased albuminuria (Adler et al., 2010), although

VEGF is a potent inducer of vasopermeability and angiogenesis that plays a major pathophysiological role in DN, despite VEGF exhibiting protective roles in non-diabetic renal disease. Serum levels of VEGF correlate with albuminuria and increase with DN stage in patients with type 1 and 2 diabetes (Hovind et al., 2000). Several experimental model studies have demonstrated that VEGF may contribute to some of the hemodynamic changes in DN, including hyperfiltration and albuminuria. Expression of VEGF and its receptors are modulated by high glucose, endothelin 1, AGEs, angiotensin II, stretch and TGF-β, and their renal expression is increased in diabetic kidneys (Chen & Ziyadeh, 2008; Cooper et al., 1999). Furthermore, VEGF is a trophic factor for glomerular endothelial cells, affects podocyte function, and is also involved in macrophage influx during early DN (Chen & Ziyadeh, 2008). Since VEGF levels are elevated in patients and animal models of DN, a number of studies have examined the effect of inhibition of VEGF receptor binding or activation. Antagonism of VEGF using a variety of different strategies has been reported to improve the outcome in experimental nephropathies of various origins, including DN. Neutralizing VEGF Abs ameliorate early and long-term renal changes in diabetic animals (De Vriese et al., 2001; Schrijvers et al., 2006), while treatment with the pan-VEGF receptor tyrosine kinase inhibitor SU5416 ameliorates diabetic albuminuria in mice (Sung et al., 2006). Several clinical studies have evaluated the beneficial effect of these anti-VEGF agents in the treatment of diabetic retinopathy, but more studies are needed to determine the viability of such strategy in

Oxidative stress defined as an excessive production of ROS surpassing existing antioxidative defense mechanisms plays a critical role in the pathogenesis of diabetes, and more importantly in the development of diabetic complications, including DN. Free radicals are capable of disturb physiological cell function both directly, by damaging cellular macromolecules such as DNA, proteins, and lipids, and indirectly through the stimulation of multiple pathways, such as protein kinases, polyol and hexosamine pathways and AGEs formation. In addition, low antioxidant bioavailability promotes cellular oxidative stress leading to additional cellular

Overproduction of ROS in the diabetic milieu is both a direct consequence of hyperglycemia and an indirect consequence through AGEs or mediators of glucotoxicity such as cytokines and growth factors (Noh & Ha, 2011; Forbes et al., 2008). The effects of ROS in renal cells comprise mesangial cell proliferation, expression of growth factors, extracellular matrix accumulation, RAAS activation and induction of epithelial-mesenchymal transition (Noh &

damage (Forbes et al., 2008; Elmarakby & Sullivan, 2010; Noh & Ha, 2011).

further validation in a prospective, randomized, blinded study is required.

**3.1.3 VEGF** 

diabetic renal disease.

**3.2 Oxidative stress** 

The transcription factor NF-κB is induced by various cell stress-associated stimuli including growth factors, vasoactive agents, cytokines, and oxidative stress. NF-κB in turn controls the regulation of numerous genes activated during inflammation, such as cytokines, chemokines, growth factors, cellular ligands, and adhesion molecules (Karin & Greten, 2005). The activation and nuclear translocation of NF-κB has been demonstrated in diabetic kidneys from human and rodents (Mezzano et al., 2004; Liu et al., 2010), and also in proximal tubular cells in the urinary sediment of patients with type 2 diabetes (Brosius et al., 2010). Furthermore, a study integrating gene-expression profiling in human renal biopsies

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 137

2008; Amiri et al., 2002; de Prati et al., 2005; Lu et al., 2009). JAK/STAT also mediates the mitogenic and fibrotic actions of cytokines and angiotensin II in the kidney (Marrero et al., 2006), suggesting that modulation of this pathway may prevent diabetic renal disease. Importantly, studies in experimental models of diabetes suggest that the renal protective effects of current drugs like captopril, statins and rosiglitazone (Banes et al., 2004; Shi et al., 2007; Tang et al., 2010b) could be partially attributed to a modulation in JAK/STAT phosphorylation. More selective therapies, such as inhibitors of JAK2 and JAK3, and STAT1 antisense oligonucleotides have been proven to counteract the harmful effects of JAK/STAT activation in cultured renal cells and in experimental models of DN (Wang et al., 2002; Shi et

The JAK/STAT pathway is controlled through different mechanisms: 1) receptor internalization; 2) protein tyrosine phosphatases; 3) protein inhibitors of activated STAT; and 4) suppressors of cytokine signaling (SOCS). In particular, SOCS family of intracellular proteins has emerged as a potential target to modulate the magnitude and duration of JAK/STAT signaling (Yoshimura et al., 2007). SOCS are induced by many pathologic stimuli (*e.g.*, cytokines, angiotensin II, chemokines, insulin, immunoglobulins, and lipoproteins) thus indicating their involvement in many biologic processes (Ortiz-Munoz et al., 2009; Yoshimura et al., 2007; Hernandez-Vargas et al., 2005; Gomez-Guerrero et al., 2004). Our group has recently reported an increased expression of SOCS proteins in renal samples from patients with progressive DN and diabetic animals (Ortiz-Munoz et al., 2010). *In vitro*, SOCS induction prevented the expression of STAT-dependent genes including adhesion molecules, chemokines, and cytokines. *In vivo* gene therapy with SOCS-expressing adenovirus reduced JAK/STAT activation and ameliorated the early renal changes in diabetic rats. Further research into inducers of SOCS expression or SOCS mimetics could have therapeutic value to prevent or retard the progression of diabetic complications.

The current knowledge of the cellular and molecular processes involved in the initiation and progression of diabetic renal injury continues to expand (Figure 1). Inflammation is now recognized as an important player in the pathogenesis of DN, and a number of studies have been designed to address whether blockade or modulation of specific inflammatory molecules can be beneficial for this disease. Furthermore, the increased understanding of the functionality of signal transduction pathways will lead to identification of therapeutic targets able to specifically downregulate proinflammatory responses and mediators, potentially even harnessing some of the sophisticated regulatory systems designed to normally limit the inflammatory response. Nevertheless, because of multiple molecular links between inflammation, immune response and diabetes complications, it seems unlikely that suppressing one single specific effector molecule could be sufficient to produce clinically relevant benefit. Effective treatment and/or prevention of diabetic renal disease will therefore require an integrated approach combining multiple strategies to target the underlying inflammatory processes. In addition, as diabetes complications require chronic treatment, long-term antiinflammatory therapies could potentially have other side effects despite improving renal function. Future research will provide answers to these uncertainties regarding multiple and long-term interventional therapies based on modulation of immunoinflammatory responses. In fact, progress towards therapeutics

al., 2007; de Prati et al., 2005).

**5. Conclusion** 

with promoter modeling has identified the specific set of target genes, especially chemokines, containing a specific NF-κB promoter module (NFKB\_IRFF\_01) with a NF-κB binding site, that were activated in progressive DN (Schmid et al., 2006). These findings emphasize the NF-κB proinflammatory pathway as potentially a major upstream target for developing new renoprotective agents in diabetes. There is a raising number of reported inhibitors of NF-κB pathway with potential benefits for future therapies in humans. Inhibitors can be divided into basic categories according to the step at which NF-κB is blocked: 1) upstream of IκB kinase complex; 2) IκB phosphorylation/degradation; 3) nuclear translocation; 4) DNA binding; and 5) gene transactivation (Gomez-Guerrero et al., 2011). Some of these compounds are in clinical development against various inflammatory diseases, but studies in diabetic patients are scarce.

It has been suggested that the preventive effects of thiazolidinedione, a ligand for peroxisome proliferator-activated receptor-γ, are mediated by its antiinflammatory actions, including inhibition of NF-κB. Thiazolidinedione caused a reduction in intranuclear NF-κB binding activity in type 2 diabetic patients with obesity (Aljada et al., 2001), and ameliorated renal injury in experimental diabetic rats through NF-κB inhibition (Ohga et al., 2007). Other studies demonstrated that 1,25-dihydroxyvitamin D3 suppresses hyperglycemia-induced gene expression by blocking NF-κB activation in mesangial cells (Zhang et al., 2007). Statins and fenofibrate also exhibit a downregulating effect on NF-κB pathway in kidneys from diabetic rats (Usui et al., 2003; Chen et al., 2008). In the same way, a recent report demonstrates that the ameliorative effects of the plant alkaloid berberine on renal dysfunction in diabetic rats is associated with its inhibitory function on NF-κB signal pathway in the kidney (Liu et al., 2010).

NF-κB is modulated by upstream enzymes like poly(ADP-ribose) polymerase (PARP). Increased PARP activity has been shown to participate in the pathogenesis of diabetic complications. Pharmacological inhibition of PARP by two different inhibitors (PJ-34 and INO-1001) decreased kidney hypertrophy in type 1 diabetic mice (Drel et al., 2011) and this effect was associated with a decrease in NF-κB p50 nuclear translocation (Goh et al., 2008). This provides rationale reasons for development and further studies of PARP inhibitors as promising approaches to DN. In fact, PARP inhibitors are currently being tested in clinical trials for cancer.

#### **4.2 JAK/STAT**

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an essential intracellular mechanism of cytokines and other stimuli that regulates gene expression and cellular activation, proliferation, and differentiation. Members of the JAK/STAT pathway have been claimed as new molecular targets of antiinflammatory treatment in acute and chronic inflammatory diseases (de Prati et al., 2005; Marrero et al., 2006), and their activation is involved in the development of the diabetes complications. A recent report using a transcriptomic approach documented enhanced expression of a large number of JAK/STAT mRNAs and JAK2 protein in glomerular and tubulointerstitial regions from patients with both early and progressive DN (Berthier et al., 2009).

JAK/STAT pathway, especially the JAK2/STAT1/STAT3-dependent axis, contributes to high glucose mediated renal cell responses, including enhanced expression of genes involved in leukocyte infiltration, cell growth, and fibrosis (Brosius et al., 2010; Brosius, III, 2008; Amiri et al., 2002; de Prati et al., 2005; Lu et al., 2009). JAK/STAT also mediates the mitogenic and fibrotic actions of cytokines and angiotensin II in the kidney (Marrero et al., 2006), suggesting that modulation of this pathway may prevent diabetic renal disease. Importantly, studies in experimental models of diabetes suggest that the renal protective effects of current drugs like captopril, statins and rosiglitazone (Banes et al., 2004; Shi et al., 2007; Tang et al., 2010b) could be partially attributed to a modulation in JAK/STAT phosphorylation. More selective therapies, such as inhibitors of JAK2 and JAK3, and STAT1 antisense oligonucleotides have been proven to counteract the harmful effects of JAK/STAT activation in cultured renal cells and in experimental models of DN (Wang et al., 2002; Shi et al., 2007; de Prati et al., 2005).

The JAK/STAT pathway is controlled through different mechanisms: 1) receptor internalization; 2) protein tyrosine phosphatases; 3) protein inhibitors of activated STAT; and 4) suppressors of cytokine signaling (SOCS). In particular, SOCS family of intracellular proteins has emerged as a potential target to modulate the magnitude and duration of JAK/STAT signaling (Yoshimura et al., 2007). SOCS are induced by many pathologic stimuli (*e.g.*, cytokines, angiotensin II, chemokines, insulin, immunoglobulins, and lipoproteins) thus indicating their involvement in many biologic processes (Ortiz-Munoz et al., 2009; Yoshimura et al., 2007; Hernandez-Vargas et al., 2005; Gomez-Guerrero et al., 2004). Our group has recently reported an increased expression of SOCS proteins in renal samples from patients with progressive DN and diabetic animals (Ortiz-Munoz et al., 2010). *In vitro*, SOCS induction prevented the expression of STAT-dependent genes including adhesion molecules, chemokines, and cytokines. *In vivo* gene therapy with SOCS-expressing adenovirus reduced JAK/STAT activation and ameliorated the early renal changes in diabetic rats. Further research into inducers of SOCS expression or SOCS mimetics could have therapeutic value to prevent or retard the progression of diabetic complications.

#### **5. Conclusion**

136 Diabetic Nephropathy

with promoter modeling has identified the specific set of target genes, especially chemokines, containing a specific NF-κB promoter module (NFKB\_IRFF\_01) with a NF-κB binding site, that were activated in progressive DN (Schmid et al., 2006). These findings emphasize the NF-κB proinflammatory pathway as potentially a major upstream target for developing new renoprotective agents in diabetes. There is a raising number of reported inhibitors of NF-κB pathway with potential benefits for future therapies in humans. Inhibitors can be divided into basic categories according to the step at which NF-κB is blocked: 1) upstream of IκB kinase complex; 2) IκB phosphorylation/degradation; 3) nuclear translocation; 4) DNA binding; and 5) gene transactivation (Gomez-Guerrero et al., 2011). Some of these compounds are in clinical development against various inflammatory

It has been suggested that the preventive effects of thiazolidinedione, a ligand for peroxisome proliferator-activated receptor-γ, are mediated by its antiinflammatory actions, including inhibition of NF-κB. Thiazolidinedione caused a reduction in intranuclear NF-κB binding activity in type 2 diabetic patients with obesity (Aljada et al., 2001), and ameliorated renal injury in experimental diabetic rats through NF-κB inhibition (Ohga et al., 2007). Other studies demonstrated that 1,25-dihydroxyvitamin D3 suppresses hyperglycemia-induced gene expression by blocking NF-κB activation in mesangial cells (Zhang et al., 2007). Statins and fenofibrate also exhibit a downregulating effect on NF-κB pathway in kidneys from diabetic rats (Usui et al., 2003; Chen et al., 2008). In the same way, a recent report demonstrates that the ameliorative effects of the plant alkaloid berberine on renal dysfunction in diabetic rats is associated with its inhibitory function on NF-κB signal

NF-κB is modulated by upstream enzymes like poly(ADP-ribose) polymerase (PARP). Increased PARP activity has been shown to participate in the pathogenesis of diabetic complications. Pharmacological inhibition of PARP by two different inhibitors (PJ-34 and INO-1001) decreased kidney hypertrophy in type 1 diabetic mice (Drel et al., 2011) and this effect was associated with a decrease in NF-κB p50 nuclear translocation (Goh et al., 2008). This provides rationale reasons for development and further studies of PARP inhibitors as promising approaches to DN. In fact, PARP inhibitors are currently being tested in clinical

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is an essential intracellular mechanism of cytokines and other stimuli that regulates gene expression and cellular activation, proliferation, and differentiation. Members of the JAK/STAT pathway have been claimed as new molecular targets of antiinflammatory treatment in acute and chronic inflammatory diseases (de Prati et al., 2005; Marrero et al., 2006), and their activation is involved in the development of the diabetes complications. A recent report using a transcriptomic approach documented enhanced expression of a large number of JAK/STAT mRNAs and JAK2 protein in glomerular and tubulointerstitial

JAK/STAT pathway, especially the JAK2/STAT1/STAT3-dependent axis, contributes to high glucose mediated renal cell responses, including enhanced expression of genes involved in leukocyte infiltration, cell growth, and fibrosis (Brosius et al., 2010; Brosius, III,

regions from patients with both early and progressive DN (Berthier et al., 2009).

diseases, but studies in diabetic patients are scarce.

pathway in the kidney (Liu et al., 2010).

trials for cancer.

**4.2 JAK/STAT** 

The current knowledge of the cellular and molecular processes involved in the initiation and progression of diabetic renal injury continues to expand (Figure 1). Inflammation is now recognized as an important player in the pathogenesis of DN, and a number of studies have been designed to address whether blockade or modulation of specific inflammatory molecules can be beneficial for this disease. Furthermore, the increased understanding of the functionality of signal transduction pathways will lead to identification of therapeutic targets able to specifically downregulate proinflammatory responses and mediators, potentially even harnessing some of the sophisticated regulatory systems designed to normally limit the inflammatory response. Nevertheless, because of multiple molecular links between inflammation, immune response and diabetes complications, it seems unlikely that suppressing one single specific effector molecule could be sufficient to produce clinically relevant benefit. Effective treatment and/or prevention of diabetic renal disease will therefore require an integrated approach combining multiple strategies to target the underlying inflammatory processes. In addition, as diabetes complications require chronic treatment, long-term antiinflammatory therapies could potentially have other side effects despite improving renal function. Future research will provide answers to these uncertainties regarding multiple and long-term interventional therapies based on modulation of immunoinflammatory responses. In fact, progress towards therapeutics

Immunoinflammation in Diabetic Nephropathy: Molecular Mechanisms and Therapeutic Options 139

The authors have been granted by Ministry of Science (SAF2007/63648 and SAF2009/11794), Ministry of Health (Instituto de Salud Carlos III, Red RECAVA RD06/0014/0035), Ramon

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Fig. 1. Immunoinflammatory mechanisms in DN. Elements of the diabetic milieu (high glucose, AGEs, angiotensin, and oxidative stress) induce the expression of chemokines and adhesion molecules by renal cells, which favours leukocyte infiltration into the kidney. Further exposure of kidney macrophages and resident cells to diabetic milieu promotes cell activation, with the subsequent release of proinflammatory cytokines (e.g. IL-1, IL-6, TNFα), ROS, and profibrotic growth factors (e.g. TGF-β). The initial inflammatory response is self-amplified then causing renal injury and cell death. Furthermore, the fibrotic response induces proliferation and extracellular matrix accumulation in mesangial and tubular cells. Diabetic renal injury then progresses to glomerulosclerosis and tubulointerstitial fibrosis.

#### **6. Acknowledgment**

The authors have been granted by Ministry of Science (SAF2007/63648 and SAF2009/11794), Ministry of Health (Instituto de Salud Carlos III, Red RECAVA RD06/0014/0035), Ramon Areces Foundation, and Comunidad de Madrid (S2006/GEN-0247).

#### **7. References**

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designed to target specific cytokines, chemokines, growth factors and even transcription factors is already well underway. Besides the assessment of pharmacological safety, bioavailability, and efficacy *in vivo*, more clinical studies will further support the potential of

Fig. 1. Immunoinflammatory mechanisms in DN. Elements of the diabetic milieu (high glucose, AGEs, angiotensin, and oxidative stress) induce the expression of chemokines and adhesion molecules by renal cells, which favours leukocyte infiltration into the kidney. Further exposure of kidney macrophages and resident cells to diabetic milieu promotes cell activation, with the subsequent release of proinflammatory cytokines (e.g. IL-1, IL-6, TNFα), ROS, and profibrotic growth factors (e.g. TGF-β). The initial inflammatory response is self-amplified then causing renal injury and cell death. Furthermore, the fibrotic response induces proliferation and extracellular matrix accumulation in mesangial and tubular cells. Diabetic renal injury then progresses to glomerulosclerosis and tubulointerstitial fibrosis.

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**9** 

*Pakistan* 

**Study of Diabetic Hypertensive Nephropathy in** 

*University of the Punjab, Lahore, Pakistan and FPGMI, Shaikh Zayed Hospital Lahore* 

Diabetes mellitus is a metabolic disorder that is characterized by high blood sugar and it is because of either problem in insulin secretion (type 1-β cell destruction) or either because of combination of insulin resistance or improper insulin secretion to compensate (type 2).

Despite ingestion of dietary carbohydrates, serum glucose level remain relatively steady throughout the day. This requires the concerted actions of several different tissues. Pancreatic β-cells, for example, secrete insulin in response to the elevation in glucose that occur after eating. Insulin promotes glucose disposal in adipose tissue and muscle, and also prevents the liver from producing more glucose by suppressing glycogenolysis and gluconeogenesis. In the fasting state, low insulin levels combined with elevated counterregulatory hormones such as glucagon, adrenaline and corticosteroids promote hepatic glucose production. Recently, evidence has emerged that the brain coordinates many of these effects as well, through direct and indirect glucose sensing and neural outputs to

Diabetes results from the dysregulation of multiple glucoregulatory hormones that normally acto to maintain glucose homeostasis. For example any defect in insulin production lead to improper regulation of glucose in the blood and result in diabetes. Similarly in patients with type1 and type 2 diabetes, post prandial glucagon secretion is abnormally elevated. This inappropriate secretion of glucagon leads to excess hepatic glucose production and is

Deficient insulin action results from inadequate insulin secretion or decrease the tissue response.Insulin resistance is define as lack of sufficient insulin receptors, in target tissue, defect in intracellular transport of glucose and or an altered insulin secondary pattern that is dys-synchronization between pancreatic β cell release of insulin and the body's insulin

important contributor to postprandial hyperglycemia in patients with diabetes.

**1. Introduction** 

(Riaz, S. 2009)

peripheral organs.

**3. Insulin resistance** 

requirement.( Defronzo, R.A., 1992)

**2. Mechanism of action** 

**the Local Population of Pakistan** 

Samreen Riaz and Saadia Shahzad Alam

