Study On The Mechanism Of Capsaicin And Metabolic Surgery In Improving Diabetic Nephropathy

Background and objective:Diabetic nephropathy is the most common,primary and serious manifestation of target organ damage of diabetes and has become the main cause of chronic kidney disease and end-stage kidney disease.The incidence rate of diabetes and diabetic nephropathy is increasing worldwide,and the treatment of diabetic nephropathy has been a huge social economy burden.At present,the main treatment principle for diabetic nephropathy is to intensively control blood glucose.Although intensively control blood glucose can partially delay the occurrence of albuminuria,it cannot prevent or improve the occurrence and development of diabetic nephropathy in long-term observation and follow-up.After many years,the glomerular filtration rate is still significantly reduced,or even increase the death rate of all-cause or cardiovascular causes.Using insulin to control blood glucose in type 1 or type 2 diabetic patients even increases the risk of hypoglycemia.Therefore,it is necessary to explore the treatment method of diabetic nephropathy beyond the glycemic control or insulin.It has been reported that spicy food consumers have different degrees of slowing down the occurrence and progress of kidney disease compared with non-spicy food consumer,but there is no relevant mechanism research.Capsaicin takes effects through activating TRPV1,a transient receptor potential?TRP?channel in vivo.TRP channels are non-selective for cation,which are involved in regulating many cell behaviors related to Ca²⁺.TRPs are involved in the regulation of renal function.TRPV1 can be activated by a variety of endogenous and exogenous factors including capsaicin.After the activation of TRPV1 by dietary capsaicin,the transient intracellular Ca²⁺influx is increased,which plays a role in regulating the physiological functions of various tissues.It has been reported that TRPV1 is involved in the regulation of acute renal injury,but its effect on diabetic nephropathy has not been studied.TRPV1 can activate AMPK in fat and other tissues.However,it has not been reported whether TRPV1 has this effect in kidney.In addition,in recent years,people also pay attention to another treatment,metabolic surgery,for treatment of diabetic nephropathy.Metabolic surgery was initially used in the treatment of obesity,now it has been widely used in the treatment of type 2 diabetes.In the world and in China,the main purpose of metabolic surgery in recent years has changed from obesity to metabolic diseases,including diabetes,and the most important operation has also changed to Roux-en-Y gastric bypass?RYGB?.The improvement effect of metabolic surgery on diabetic nephropathy has been confirmed in some retrospective studies.In some patients with diabetic nephropathy after surgery,diabetic nephropathy was significantly improved,urinary protein decreased,glomerular filtration rate recovered.And in the patients whose renal function has been seriously damaged before the operation,the operation also has significant effect and is very safe.However,the mechanism of surgical improvement of diabetic nephropathy has not been fully studied.In the occurrence and development of diabetic nephropathy,mitochondrial dysfunction of podocytes is a very important event.Under the condition of diabetes,the mitochondrial function of podocytes is seriously defective.The Ca²⁺homeostasis of mitochondria is the basis of mitochondrial function maintenance.Ca²⁺overload of mitochondria will lead to serious damage of mitochondrial function.Mitochondrial associated ER membrane?MAM?is a connecting channel between mitochondria and ER.It allows Ca²⁺to enter mitochondria from ER,which is very important to regulate Ca²⁺homeostasis of mitochondria.In some target organs of diabetes mellitus,the formation of MAM is involved in the regulation of mitochondrial Ca²⁺homeostasis.However,the role of MAM in diabetic nephropathy has not been studied.It has been reported that AMPK can regulate the formation of MAM in the heart,but in the kidney,the effect of AMPK on MAM has not been reported.AMPK has been widely studied in the treatment of diabetic nephropathy and the improvement of mitochondrial function.It is widely believed that the activity of AMPK is inhibited in diabetic state.However,the mechanism of the inhibition of AMPK activity under the condition of diabetes is rarely studied.This is of great significance for the treatment of diabetes and diabetic nephropathy.The purpose of this paper is:1.The mechanism of capsaicin improving diabetic nephropathy.2.The role of AMPK and MAM in the improvement of diabetic nephropathy of capsaicin.3.To study the mechanism of the inactivation of AMPK in diabetic state.4.The mechanism of metabolic surgery to improve diabetic nephropathy.Materials and methods:This study consists of two parts.In the first part,in db/db mice and TRPV1 knockout mice,to explore the effect of TRPV1 on diabetic nephropathy,and the role of AMPK and MAM in it.The second part,RYGB operation was performed in diabetic rats and AMPK?2 knockout mice to explore the mechanism of inhibition of AMPK in diabetic state and RYGB operation to improve diabetic nephropathy.1.In db/db mice and TRPV1KO and control mice,the renal function indexes,urinary albumin,glomerular filtration rate,serum creatinine,blood urea nitrogen,and renal pathological structure were observed.2.To detect the changes of mitochondrial function and quantity in WT and TRPV1KO mice.The Ca²⁺homeostasis of mitochondria and endoplasmic reticulum,and the formation of MAM in podocytes were observed under electron microscope.3.The expression of AMPK,nephrin,podocin and CaMKK?protein was detected after TRPV1 was activated in the glomeruli and podocytes.The effect of capsaicin on AMPK phosphorylation was detected by transfection of CaMKK?siRNA and intervention of Ca MKK?inhibitor on podocytes.4.To detect the expression of fundc1 protein and MAM protein and the integrity of the complex in the glomeruli of mice with capsaicin intervention.After overexpression of Func1 in podocyte lines,the expression of fundc1 protein and MAM protein and the integrity of the complex were detected.5.To detect the effect of AMPK on the expression of Fundc1 and the effect of binding of fundc1 promoter to ZF5 protein on the transcription of Fundc1.6.Diabetes was induced by STZ in SD rats.RYGB operation was performed on diabetic rats 4 weeks later.Renal function indexes,including urinary albumin,glomerular filtration rate,serum creatinine,blood urea nitrogen,and renal pathological structure were observed.The changes of mitochondrial function and quantity were observed after operation.The expression and phosphorylation of AMPK,PGC1-?were detected.7.To verify the role of AMPK,in WT and AMPK?2 knockout mice,STZ was used to induce diabetes,and then RYGB was carried out and in the diabetic rats after operation,AMPK inhibitor CC was given by micro osmotic pump to observe the renal function.8.To detect the changes of AMP/ATP ratio in renal cortex of diabetic rats and rats after operation.9.Microarray m RNA chip was used to detect the renal cortex of diabetic rats and post-operative rats to explore the change pathway related to AMPK.10.To detect the changes of NADPH content,NADPH synthetase H6PD in renal cortex of diabetic rats and rats after operation,and to verify the effect of NADPH on AMPK activity in cells.The effect of NADPH content on AMPK activity and kidney function was verified by injecting r AAV of H6PD into rats.Results:1.The renal function of db/db mice and STZ induced diabetic WT mice declined.The renal function of db/db and WT mice treated with capsaicin improved,but that of TRPV1KO mice did not.2.In diabetic mice,mitochondrial calcium overload and MAM formation increased significantly,while after capsaicin intervention,mitochondrial calcium overload improved and MAM decreased.3.After activation of TRPV1,AMPK phosphorylation and the expression of nephrin and podocin increased in glomeruli and podocytes.Then the administration of si RNA and inhibitors of CaMKK?,AMPK phosphorylation and the expression of nephrin and podocin were down regulated.4.After activation of TRPV1,the expression of fundc1 in glomeruli and podocytes was down regulated.After overexpression of fundc1,MAM formation increased,and mitochondrial calcium overload showed.5.AMPK significantly inhibited the binding of fundc1 promoter to ZF5 protein.6.In diabetic rats,the renal function and mitochondrial function,morphology and quantity were significantly improved after RYGB.AMPK phosphorylation and PGC1-?expression were up-regulated by RYGB.7.The improvement effect of operation on diabetic nephropathy was eliminated after the treatment of AMPK inhibitor CC or the knockout of AMPK?2.8.The ratio of AMP/ATP increased both in diabetic rats and RYGB treated rats.9.The NADPH synthesis pathway was down regulated and the content of NADPH in renal cortex was decreased in RYGB treated rats.In cultured podocytes,NADPH significantly inhibited AMPK activity.10.In diabetic rats,AMPK phosphorylation was up-regulated and renal function was significantly restored after administration of H6PD sh RNA rAAV.Conclusion:1.Capsaicin can activate TRPV1,improve mitochondrial function of podocyte and diabetic nephropathy.2.Capsaicin can activate TRPV1,promote transient Ca²⁺influx,up-regulate CaMKK?expression,activate AMPK,inhibit Fundc1 expression and MAM formation,and improve mitochondrial Ca²⁺homeostasis.3.Metabolic surgery can improve glomerular mitochondrial function and diabetic nephropathy by downregulating H6PD expression leaded inhibition of production of NADPH and activation of AMPK.