Inhibitory Effect Of SGLT2 On Diabetic Nephropathy And Its Mechanism

Diabetes mellitus (DM) is a common endocrine metabolic disease. With the changes of lifestyle, the number of diabetes mellitus patients, especially type2diabetes mellitus (T2DM) is growing rapidly all over the world. Diabetes mellitus is becoming a serious public health problem in modern society. Diabetes mellitus may cause a variety of complications, and one of the most severe complications is diabetic nephropathy (DN). It has been proven that more than40%of end-stage renal disease (EDRD) is related to diabetes. Therefore, revealing the mechanism of renal injury induced by diabetes mellitus has become an important topic in basic medical research.Although many medications for the treatment of diabetes mellitus have been developed, long-term clinical observation found that these drugs had no definite protective role on the occurrence of complications and the damage of target organ. even some of them had side effects, for example biguanide may cause digestive tract symptoms and lactic acidosis in senile patients; sulfonylureas and insulin may result in hypoglycemia and weight gain. Following the development of diabetes, the compensatory ability of pancreas exhaust and the production of insulin declines rapidly which may lead to the loss of glycemic control. Dose escalation or combination of different agents increases the complexity and risk of treatment. Therefore, there is urgent requirement to develop new agents or treatment strategies for the patient with diabetes.The kidney plays an important role in glucose homeostasis. Approximately180g of glucose is filtered daily in a healthy adult, and most of them is reabsorbed by the kidney, only less than0.5g being excreted in the urine. Studies on familial renal glycosuria(FRG) patients revealed that those patients exhibited a clinical manifestation of increasing urinary glucose excretion. However, most patients do not show obvious increase in blood glucose concentration or the occurrence probability of renal complication and urinary tract infections. The formation of urinary glucose excretion in FRG patients is caused by SLCA2gene mutation and the loss of SGLT2function.SGLT2is a high-capacity, low-affinity transmembrane protein, which is located on the luminal surface of the proximal tubule. SGLT2actively transport glucose into the cell against the concentration gradient by co-transporting with Na+. Then, on the basolateral surface, intracellular glucose is passively transported into the blood by facilitative glucose transporters (GLUTs). SGLT2is predominantly expressed in the proximal segments of the proximal tubule. It is responsible for reabsorbing over90%of the filtered glucose, while SGLT1, which is located in the distal segments, absorbs the remained10%.SGLT2is specifically expressed in kidney. So the inhibition of SGLT2has no effect on the glucose utilizations in other tissues or organs. Phlorizin, the natural SGLT2inhibitor, was first discovered in the extract of apple tree bark. However, it can not be used as anti-diabetic drug since its poor oral absorption and specificity. Currently, several new compounds of SGLT2inhibitors have been derived from phlorizin and some of them have been authorized to apply to pre-clinical and clinical investigations.SGLT2inhibitor is a new agent which reduces blood glucose levels by enhancing glucose excretion. Since the mechanism is independent of insulin secretion or action, it can be used as an addition of other oral anti-diabetic drug or a supplementary approach for comprehensive treatment in any type or stage of diabetes.Cells suffer from hyperglycemic condition in diabetic patients due to high blood glucose. High glucose mediated oxidative stress, non-enzymatic glycosylation, changes of inflammatory molecules and growth factors damage the target organ. So enhancing urinary glucose excretion which improves glucose control could reduce damages on organ, including DN.The aim of this study was to identify the effects of SGLT inhibitor on diabetes-induced renal injury in STZ-induced DM rats and db/db mice, and to explore the mechanisms involved. Results:Part I. The effects of SGLT2inhibitor on renal injury in STZ-induced DM rats and its mechanisms1. Type I diabetes model induced by STZ injection. After that, the rats were fed for2weeks before chemical application. Chemicals were applied orally for6weeks. The changes in blood glucose, urinary glucose, urine volume, kidney weight were recorded. The blood glucose, urinary glucose, urine volume, kidney weight were significantly higher in the diabetic rats. The blood glucose was significantly reduced in the diabetic rats administrating SGLT2inhibitor dapagliflozin, while the urinary glucose, urine volume were even higher compared with that of diabetic rats. The kidney weight decreased. No differences were observed in the blood glucose, urinary glucose and urine volume in the diabetic group after administrating AT1R inhibitor candesartan, but the kidney weight decreased. There was no significant difference in the body weight between diabetic rats and candesartan-treated rats. There was no significant change in body weight in the normal group after dapa administration. Our results suggested that the inhibition of SGLT2increases urine glucose excretion in turn reduces the blood glucose.2. Western blot results showed that the expression of SGLT2protein was increased in diabetic group compared with the normal group. Significant elevation of the SGLT2expression was observed in non-diabetic and diabetic rats after the administration of SGLT2inhibitor dapagliflozin. The expression of SGLT2protein after the administration of AT1R inhibitor candesartan did not change significantly in diabetic rats.3. The24h urinary protein, plasma BUN and plasma creatinine were significantly higher in the diabetic group. Significant reduction of24h urinary protein, plasma BUN, plasma creatinine was observed in diabetic rats after the administration of SGLT2inhibitor dapagliflozin and AT1R inhibitor candesartan. No significant changes in Periodic Acid-Schiff (PAS) stain, which showed basement membrane thickening, glomerular volume increasing and mesangial cell proliferation, was observed under microscope. Hematoxylin and eosin (H&E) staining showed that there were no obvious structural differences and inflammatory cell infiltration between groups. Masson staining showed that there was no difference in collagen deposition in each group. Electron microscopy results showed that mild thickness of the basement membrane, and foot process fusion occured in the diabetic rats. Western blot results showed that the expression of Collagen I protein was increased in the diabetic rats compared with normal group. Significant reduction of the expression of Collagen I protein was observed diabetic rats after the administration of SGLT2inhibitor dapagliflozin or AT1R inhibitor candesartan. These results suggested that the inhibition of SGLT2delayed diabetic renal injury.4. AGEs, MDA and TGF-β protein level were significantly increased in the diabetic rats. Treatment of SGLT2inhibitor dapagliflozin and AT1R inhibitor candesartan inhibited the up-regulation of AGEs、MDA and TGF-β protein level, indicating that AGEs、MDA、TGF-β are involved in renal protection by SGLT2inhibition.Part Ⅱ. The effects of SGLT2inhibitor on renal injury in db/db mice and its mechanisms1. The blood glucose and weight gain were significantly higher in db/db mice compared with C57BL/6mice. Significant reductions in blood glucose and body weight gain were observed in db/db mice after the administration of SGLT2inhibitor dapagliflozin. No changes were observed in the blood glucose, body weight gain in db/db mice after administrated of AT1R inhibitor candesartan. This result proved that the SGLT2inhibitor reduces blood glucose and weight gain in type II diabetes animals models.2. Western blot results showed that the expression of SGLT2protein was elevated in db/db mice compared with C57BL/6mice. Significant elevation in the expression of SGLT2was observed in both C57BL/6and db/db mice after the administration of SGLT2inhibitor dapagliflozin. The administration of AT1R inhibitor candesartandid did not change the expression of SGLT2in db/db mice significantly.3. There were no obvious differences in plasma BUN and creatinine between groups. Periodic Acid-Schiff (PAS) stain showed that there was mild basement membrane thickening in db/db mice. Western blot results showed that the expression of Collagen I protein was increased in db/db mice compared with C57BL/6mice. Significant reduction in the expression of Collagen I protein was observed in diabetic rats after the administration of SGLT2inhibitor dapagliflozin or AT1R inhibitor candesartan. These results suggested that inhibition of SGLT2delays diabetes-induced renal injury in db/db mice.4. AGEs, MDA and TGF-β protein level were significantly increased in db/db mice. SGLT2inhibitor dapagliflozin or ATIR inhibitor candesartan treatment inhibited the up-regulation of AGEs、MDA and TGF-β production, indicating that the protective effect of dapagliflozin or candesartan are probably mediated by attenuating the increase in AGEs、MDA、TGF-p.In summary, our results demonstrated that SGLT2inhibitor decreases blood glucose by enhancing the urine glucose excretion. SGLT2inhibitor attenuates diabetic renal damage in both type1diabetes and type2diabetes. The possible mechanism may be related to the reduction of generation of AGEs, MDA, TGF-β induced by hyperglycemia.