**3. The involvement of kidneys in glucose homeostasis**

The plasma glucose concentration is determined by the amount of glucose synthesized, and the one removed from the circulation and metabolized. This concentration must be maintained within a relatively narrow range despite the wide daily fluctuations in glucose ingestion and glucose demands in various tissues [4]. Other substrates such as free fatty acids (FFAs), glycerol, lactate and ketone bodies have greater daily fluctuations. This can be explained by the need of the body to protect himself against hyper- and hypoglycaemia. Hyperglycaemia is associated with both chronic effects (such as nephropathy, retinopathy, neuropathy and premature atherosclerosis) and also acute complications (including diabetic ketoacidosis and hyperosmolar hyperglycaemic state that are associated with higher morbidity and mortality). Hypoglycaemia is also harmful because it can cause neurological events (including coma, seizures), cardiac arrhythmias and death [4].

The regulation of endogenous production of glucose is determined by hormonal and neural factors [15]. In the acute phase, glucoregulatory mechanisms involve insulin, glucagon and catecholamines and they can effect changes in plasma glucose levels in a matter of minutes. Insulin is able to suppress glucose release in both the kidney and liver by direct enzyme activation ⁄ deactivation and by reducing the availability of gluconeogenic substrates. Gluca‐ gon has no effect on the kidneys, but it stimulates glycogenolysis and gluconeogenesis in the liver [16]. Catecholamines also have multiple acute actions. They can stimulate renal glucose release and glucagon secretion and inhibit insulin secretion [4].

The kidneys are involved in maintaining glucose homeostasis through three different mech‐ anisms: gluconeogenesis; glucose uptake from the blood for its own energy requests and reabsorption into the general circulation of glucose from glomerular filtrate in order to preserve energy [4].

#### **3.1. Renal gluconeogenesis**

From the point of view of glucose utilization, the kidney is considered as 2 separate organs; the renal medulla is characterized mainly by glucose utilization and the renal cortex is responsible for glucose release. The separation of these activities represents the consequence of differences in the distribution of numerous enzymes along the nephron. The cells in the renal medulla can use only glucose for their needs (like the brain) and they have enzymes capable of glucose-phosphorylation and glycolysis. They can therefore phosphorylate important amounts of glucose and accumulate glycogen but, because these cells do not have glucose-6-phosphatase or any other gluconeogenic enzymes, they are unable to release glucose into the bloodstream. Moreover, the cells in the renal cortex have gluconeogenic enzymes and they can produce and release glucose into the circulation. However these cells cannot synthe‐ size glycogen because they have little phosphorylating capacity [6].

After a 16-h overnight fast, approximately 10 µmol ⁄ (kg /min) of glucose is released into the circulation [17]. Almost 50% of this is the result of glycogenolysis from the liver stocks and the other half is produced by liver and kidney gluconeogenesis. The renal cortex (like the liver) contains gluconeogenic enzymes and it can synthesize glucose-6-phosphate from precursors (lactate, glutamine, glycerol and alanine). Because it contains glucose-6-phosphatase, it is able to release glucose into the blood stream [18] and the human liver and kidneys are the only organs that can perform gluconeogenesis. Therefore, after an overnight fast, the liver produces 75–80% of glucose released into the circulation and the remaining 20–25% is derived from the kidneys [4].

Several studies have indicated that human kidneys and liver provide approximately the same amounts of glucose through gluconeogenesis in postabsorptive period. If the duration of fasting is increased, the glycogen stores are depleted and gluconeogenesis produces all the glucose released into circulation.

An important aspect is that kidney and liver use different gluconeogenic precursors and several hormones have different effects on their release of glucose. Lactate represents the predominant gluconeogenic precursor in both organs, but regarding the aminoacids, the kidney prefers to use glutamine, whereas the liver preferentially uses alanine [19]. Insulin can suppress glucose release in both organs with almost comparable efficacy [20], whereas glucagon stimulates hepatic glucose release only [21]. Catecholamines normally have a direct effect only on renal glucose release [22], but their effect on both hepatic and renal glucose release may be indirect by increasing the quantity of gluconeogenic substrates available and by suppressing insulin secretion. Other hormones, such as growth hormone, cortisol and thyroid hormones can stimulate hepatic glucose release over a great period of time [15]. Their effects on the kidneys regarding glucose release in humans are not completely deciphered.

In the postprandial state the situation changes significantly. Postprandial glucose levels in the plasma are determined by insulin and glucagon levels. After glucose ingestion, plasma glucose levels reach the peak in 60–90 minutes and they return to post-absorptive levels in almost 3– 4 h. The plasma insulin increases four times and the plasma glucagon levels decrease by 50% [15]. Meyer et al. indicated that endogenous glucose release is reduced by almost 60% and hepatic glycogenolysis drops to zero in the 4- to 6-h period after meal ingestion [23].This is happening because this period determines the refilling of hepatic glycogen stores and inhibition of endogenous glucose release is able to limit postprandial hyperglycaemia. There is also a reduction in hepatic gluconeogenesis by 82% and glucose molecules generated through hepatic gluconeogenesis are also directed into hepatic glycogen, not only released in the circulation.

Renal gluconeogenesis can increase by approximately twofold and it can represent ~60% of endogenous glucose production in the postprandial state [24]. This mechanism is believed to facilitate the repletion of glycogen stocks in the liver.

A new concept of hepatorenal glucose reciprocity emerged from the differences observed in regulation and interchange between renal and hepatic glucose release [24]. This concept refers to the facts that a pathological or physiological reduction in glucose release by kidney or liver determines a compensatory increase in glucose release of the other one (liver or kidney) in order to avoid hypoglycaemia. This situation occurs in the anhepatic phase during liver transplantation, prolonged fasting, meal ingestion, acidosis and insulin overdoses in diabetes mellitus [24].

### **3.2. Glycogenolysis**

The kidneys are involved in maintaining glucose homeostasis through three different mech‐ anisms: gluconeogenesis; glucose uptake from the blood for its own energy requests and reabsorption into the general circulation of glucose from glomerular filtrate in order to

From the point of view of glucose utilization, the kidney is considered as 2 separate organs; the renal medulla is characterized mainly by glucose utilization and the renal cortex is responsible for glucose release. The separation of these activities represents the consequence of differences in the distribution of numerous enzymes along the nephron. The cells in the renal medulla can use only glucose for their needs (like the brain) and they have enzymes capable of glucose-phosphorylation and glycolysis. They can therefore phosphorylate important amounts of glucose and accumulate glycogen but, because these cells do not have glucose-6-phosphatase or any other gluconeogenic enzymes, they are unable to release glucose into the bloodstream. Moreover, the cells in the renal cortex have gluconeogenic enzymes and they can produce and release glucose into the circulation. However these cells cannot synthe‐

After a 16-h overnight fast, approximately 10 µmol ⁄ (kg /min) of glucose is released into the circulation [17]. Almost 50% of this is the result of glycogenolysis from the liver stocks and the other half is produced by liver and kidney gluconeogenesis. The renal cortex (like the liver) contains gluconeogenic enzymes and it can synthesize glucose-6-phosphate from precursors (lactate, glutamine, glycerol and alanine). Because it contains glucose-6-phosphatase, it is able to release glucose into the blood stream [18] and the human liver and kidneys are the only organs that can perform gluconeogenesis. Therefore, after an overnight fast, the liver produces 75–80% of glucose released into the circulation and the remaining 20–25% is derived from the

Several studies have indicated that human kidneys and liver provide approximately the same amounts of glucose through gluconeogenesis in postabsorptive period. If the duration of fasting is increased, the glycogen stores are depleted and gluconeogenesis produces all the

An important aspect is that kidney and liver use different gluconeogenic precursors and several hormones have different effects on their release of glucose. Lactate represents the predominant gluconeogenic precursor in both organs, but regarding the aminoacids, the kidney prefers to use glutamine, whereas the liver preferentially uses alanine [19]. Insulin can suppress glucose release in both organs with almost comparable efficacy [20], whereas glucagon stimulates hepatic glucose release only [21]. Catecholamines normally have a direct effect only on renal glucose release [22], but their effect on both hepatic and renal glucose release may be indirect by increasing the quantity of gluconeogenic substrates available and by suppressing insulin secretion. Other hormones, such as growth hormone, cortisol and thyroid hormones can stimulate hepatic glucose release over a great period of time [15]. Their effects on the kidneys regarding glucose release in humans are not completely deciphered.

size glycogen because they have little phosphorylating capacity [6].

preserve energy [4].

6 Treatment of Type 2 Diabetes

kidneys [4].

glucose released into circulation.

**3.1. Renal gluconeogenesis**

Glycogenolysis is the breakdown of glycogen to glucose-6-phosphate and a hydrolysis reaction (using glucose-6-phosphatase) in order to free glucose. The liver is the only organ that contains glucose-6-phosphatase. So, the cleavage of hepatic glycogen releases glucose, while the cleavage of glycogen from other sources can release only lactate. Lactate, that is generated via glycolysis, is often absorbed by other organs and helps regenerating glucose [6].

#### **3.3. Glucose reabsorption**

Apart from the important role in gluconeogenesis and the role of renal cortex in glucose uptake, the kidneys contribute to glucose homeostasis by filtering and reabsorbing glucose. In normal conditions, the kidneys can reabsorb as much glucose as possible, the result being a virtually glucose free urine. Approximately 180 grams of glucose are filtered by the glomeruli from plasma, daily but all of this quantity is reabsorbed through glucose transporters that are present in cell membranes located in the proximal tubules [24].

These glucose transporters have a limited capacity of reabsorption. If this capacity is exceeded, glucose usually appears in the urine. The tubular maximum for glucose (TmG), the term used for the maximum capacity, can vary from 260 to 350 mg/min/1.73 m2 in healthy subjects. It corresponds to blood glucose levels of 180-200 mg/dL [24]. When the blood glucose is very high and the TmG is reached, the transporters cannot reabsorb all the glucose and glucosuria occurs (Figure 2). Nevertheless, there can be slight differences between the nephrons and the inaccurate nature of biological systems may potentially lead to the development of glucosuria when blood glucose is below TmG. Glucosuria may occur at lower plasma glucose levels in certain conditions of hyperfiltration (eg. pregnancy), but as a consequence of hyperfiltration and not of significant hyperglycemia [25].

**Figure 2.** Renal glucose handling. TmG, transport maximum for glucose. Adapted from [26]

In a given day, the kidneys can produce, via gluconeogenesis, 15–55g glucose and it can metabolize 25–35g glucose. Regarding the glucose metabolic pathways, it is obvious that renal reabsorption represents the main mechanism by which the kidney is involved in glucose homeostasis. Therefore, the change in tubular glucose reabsorption may have a considerable impact on glucose homeostasis [4].

#### *3.3.1. Renal glucose transporters*

Glucose is a polar compound with positive and negative charged areas; therefore it is soluble in water. Its transport into and across cells is dependent on two specialized carrier protein families: the GLUTs (facilitated glucose transporters) and the SGLTs (sodium-coupled glucose cotransporters). These transporters are responsible for glucose passage and reabsorption in several tissue types, including the proximal renal tubule, blood-brain barrier, small intestine [27]. GLUTs are responsible for the passive transport of glucose across cell membranes, in order to equilibrate its concentrations across a membrane. SGLTs, on the other hand, are involved in active transport of glucose against a concentration gradient by means of sodium-glucose cotransport [27].

There are six members of the SGLT family indicated in Table 1.


**Table 1.** The sodium glucose co-transporter family (adapted from [27])

occurs (Figure 2). Nevertheless, there can be slight differences between the nephrons and the inaccurate nature of biological systems may potentially lead to the development of glucosuria when blood glucose is below TmG. Glucosuria may occur at lower plasma glucose levels in certain conditions of hyperfiltration (eg. pregnancy), but as a consequence of hyperfiltration

**Figure 2.** Renal glucose handling. TmG, transport maximum for glucose. Adapted from [26]

There are six members of the SGLT family indicated in Table 1.

In a given day, the kidneys can produce, via gluconeogenesis, 15–55g glucose and it can metabolize 25–35g glucose. Regarding the glucose metabolic pathways, it is obvious that renal reabsorption represents the main mechanism by which the kidney is involved in glucose homeostasis. Therefore, the change in tubular glucose reabsorption may have a considerable

Glucose is a polar compound with positive and negative charged areas; therefore it is soluble in water. Its transport into and across cells is dependent on two specialized carrier protein families: the GLUTs (facilitated glucose transporters) and the SGLTs (sodium-coupled glucose cotransporters). These transporters are responsible for glucose passage and reabsorption in several tissue types, including the proximal renal tubule, blood-brain barrier, small intestine [27]. GLUTs are responsible for the passive transport of glucose across cell membranes, in order to equilibrate its concentrations across a membrane. SGLTs, on the other hand, are involved in active transport of glucose against a concentration gradient by means of sodium-glucose

and not of significant hyperglycemia [25].

8 Treatment of Type 2 Diabetes

impact on glucose homeostasis [4].

*3.3.1. Renal glucose transporters*

cotransport [27].

SGLT2 is considered the most important because, based on animal studies, it is responsible for the reabsorption of 90% of the glucose filtered at the glomerulus [24]. The other 10% of glucose reabsorbed in the proximal tubule is ensured by SGLT1. Of the family of GLUT proteins expressed in the kidneys, GLUT2 is the major transporter and it releases into circulation the glucose reabsorbed by SGLTs in the proximal tubular cells [28].

The renal glucose transport was investigated by analyzing the gene mutations within SGLT family. These can lead to several inherited diseases presenting renal glucosuria that include familial renal glucosuria (FRG) and glucose-galactose malabsorption (GGM). FRG represents an autosomal recessive or autosomal dominant disorder caused by several SGLT2 mutations. Its main characteristic is persistent glucosuria without hyperglycemia or renal tubular dysfunction. Most of the patients with FRG do not have any clinical manifestations; this is why FRG is not commonly described as a "disease" but as a condition known as benign glucosuria. Nevertheless, there is a severe form of FRG, known as type O, where mutations of the SGLT2 gene lead to a complete lack of renal tubular glucose reabsorption. This condition is still associated with a good prognosis. Due to the fact that FRG is mainly asymptomatic, subjects with this condition are discovered through routine urinalysis [24].

GGM represents a more serious disease. It is inherited autosomal recessive and is caused by mutation of the SGLT1 transporter. Its main characteristics are represented by intestinal symptoms. They appear in the first few days of life and determine glucose and galactose malabsorption. The consequences are severe; diarrhea and subsequent dehydration may become fatal unless a special diet (glucose- and galactose-free) is initiated. Some patients with GGM may present glucosuria but it is typically mild, and some other subjects have no sign of urinary glucose excretion. This confirms that SGLT1 has a minor role in renal reabsorption of glucose [24]. The mutations involving the GLUT family are associated with more severe consequences, because these transporters are more widespread throughout the major organ systems. SGLT2 and SGLT1 are located mainly in the renal system, but GLUT2 is present almost everywhere in the organism, having an important role in glucose homeostasis through its involvement in intestinal glucose uptake, renal reabsorption of glucose, and hepatic uptake and release of glucose [24].

**Figure 3.** Glucose filtration and reabsorption in the proximal tubule of the kidney (adapted from [28])

Direct *in vivo* experiments of Vallon et al. on gene targeted mice lacking *Sglt2* gene, demon‐ strated that the SGLT2 protein is responsible for all glucose reabsorption in the proximal tubule and for the bulk of glucose reabsorption in the kidney overall [29]. According to this study, in wild-type mice, 99.7 ± 0.1% of fractional glucose is reabsorbed and in Sglt2−/− mice (not expressing SGLT2), only 36 ± 8% is reabsorbed. It was also found that in Sglt2−/− mice, even if SGLT1 glucose reabsorption is increased (SGLT1 transporters reach their transport maximum), up regulation of SGLT1 expression does not occur (both SGLT1 mRNA and protein expression are reduced by ~40%) when the amount of glucose in proximal tubule is increased. The results of the study of Gorboulev et al. [30] are in correspondence with those of Vallon et al., indicating that wild-type mice do not use the maximal transport capacity of SGLT1 at normoglycemic conditions but when glucose load to the SGLT1 is increased (for instance, diabetes and SGLT2 inhibition), SGLT1 may operate at full transport capacity [30].

Molecular structure of SGLTs has been studied thoroughly on SGLT1, which is the first described member of SGLTs family [31]. SGLT2 is 59% identical to SGLT1 and has almost the same architecture. Its secondary structure consists of 14-transmembrane helices (TM1–TM13) with both the NH2 and COOH termini facing the extracellular side of the plasma membrane [32]. The first kinetic model of Na+/glucose co-transporters was proposed by Parent et al. [33].
