Dapagliflozin Delays Renal Fibrosis In Diabetic Kidney Disease By Inhibiting YAP/TAZ Activation

Background: Diabetes kidney disease(DKD)is the most common complication of diabetes and has become the main cause of end-stage renal disease,which will cause great economic burden and seriously affect the quality of life of patients.Although great progress has been made in the treatment of DKD,renal failure and end-stage renal disease are still a major social problem.Studies have shown that sodium glucose cotransporter 2(SGLT2)inhibitors,such as dapagliflozin and empagliflozin,not only have good cardiorenal protective effects in diabetes patients,but also can improve the renal adverse events in non-diabetes patients,but the mechanism of action is still unclear.Long-term overactivation of YAP/TAZ has been found to lead to tubulointerstitial fibrosis in multiple animal models of fibrosis-related kidney injury.SGLT2 inhibition has recently been shown to downregulate YAP expression in pancreatic cancer,but the role of SGLT2 inhibition in regulating Hippo-YAP/TAZ in fibrotic kidney disease models including DKD has not been reported.Objective: This study aims to explore whether inhibition of SGLT2 can alleviate renal fibrosis in DKD rats by regulating Hippo-YAP/TAZ pathway and provide new evidence and ideas for SGLT2 inhibitor as a strategy to delay renal fibrosis.Method:1.Clinical study: We selected 58 patients with DKD confirmed by renal biopsy in the Second Affiliated Hospital of the Air Force Military Medical University from 2017 to2022 for study and selected 8 patients with renal tumor resection as control.The clinical and laboratory data of all patients receiving renal biopsy were recorded systematically,including age、gender、blood pressure、diabetes history、24-hour urinary protein(24-h UP)、blood urea nitrogen(BUN)、serum creatinine(Scr)、estimated glomerular filtration rate(e GFR)、cystatin C、fasting blood glucose(FBG)and haemoglobin A1c(Hb A1c).Immunohistochemical or immunofluorescent staining of YAP、TAZ、α-SMA and TGF β1 was quantified and analyzed.2.Cell experiment: human renal tubular epithelial cells(HK-2)were divided into five groups: normal glucose group(NG,5.56 m M),high glucose group(HG,30 m M),HG+dapagliflozin group(HG+DAPA,10 μM),HG+verteporfin group(HG+Vert,250 n M)and HG+DAPA(10 μM)+Vert(250 n M)group.The activity of each group was determined by Cell Counting Kit-8(CCK-8),and the expression of YAP/TAZ was detected by immunofluorescence staining.Extract cell protein and use Western Blotting to semi-quantitatively detect and analyze YAP/TAZ,p-YAP/p-TAZ,and downstream signal factors CTGF and AREG.Then SGLT2 was further verified by si RNA knockout.3.Animal experiment: Six-to eight-week-old male SD rats were fed adaptively for1 week,and then randomly divided into a control group(n=6)and DKD group(n=24).The control group was fed with a normal diet,and the DKD group was fed with a highfat diet.8 weeks later,diabetes was induced by intraperitoneal injection of 35mg/kg STZ in the DKD group.The animal model of DKD was established successfully when the 24-h UP > 30 mg/d.The DKD rats were randomly divided into 4 groups: DKD,DKD+DAPA group,DKD+Vert group and DKD+DAPA+Vert group,and were treated by Vert intraperitoneal injection or DAPA gavage for 8 weeks.The rats were weighed every day.Moreover,the blood glucose level was measured every week,and 24-h urine was collected using metabolic cages.At the end of the experiment,the rats were sacrificed,and their serum,urine and kidney tissue were collected.Scr,BUN,24-h UP,inflammatory factors,indicators of oxidative stress,renal pathological score(H&E staining,PAS staining,Masson staining),immunohistochemistry staining(CTGF,AREG,α-SMA,TGF-β1),immunofluorescence staining(YAP/TAZ)and extraction of tissue proteins were used for western blotting analysis of the above indicators.Result:1.With the exacerbation of DKD stage,the expression of YAP/TAZ in kidneys increased.There was no significant difference in the expression of YAP and TAZ in renal tissue between control group and DKD I group.Compared with patients with DKD I,the expression levels of YAP and TAZ in renal tubular epithelial cells of patients with DKD II and DKD Ⅲ increased significantly(P<0.001).Patients with DKD Ⅲ were also accompanied by an increased in the involved area of tubular atrophy,but there was no significant difference in the levels of FBG,Hb A1 c,24h-UP or u ACR between the two groups.Furthermore,compared with that in patients with DKD I,the IOD/area of fibrosis factors TGFβ1 and α-SMA in the kidneys of patients with DKD II and DKD III were significantly increased,especially in patients with DKD III(P<0.001).2.Dapagliflozin inhibited high glucose-induced activation of YAP/TAZ in HK-2cells.The expression of YAP/TAZ in HK-2 cells treated with HG increased(P<0.001),and the ratio of p-YAP/YAP and p-TAZ/TAZ decreased significantly(P<0.001),indicating that HG significantly increased YAP/TAZ expression and nuclear translocation in HK-2 cells in vitro.The results of immunofluorescence and western blot showed that both DAPA and Vert could reverse the decrease in cell viability caused by HG(P<0.01,P<0.05)and inhibit increased HG-induced YAP/TAZ expression and nuclear translocation in HK-2 cells(P<0.01,P<0.001).3.Dapagliflozin reversed the high expression and nuclear translocation of YAP/TAZ in kidneys of DKD rats.The results of immunofluorescence and western blot showed that DAPA could reduce the expression of YAP(P<0.001,P<0.01),TAZ(P<0.001,P<0.01)and increase the proportion of p-YAP/YAP(P<0.001)and p-TAZ/TAZ(P<0.01).The results of the addition of the YAP/TAZ inhibitor Vert and DAPA were similar,but the difference between the combination and use alone was not statistically significant.4.Dapagliflozin reduced the expression of CTGF and AREG in vitro and in vivo.The western blot results showed that the expression of CTGF and AREG in HK-2cells was significantly increased after culture in 30 m M HG(P<0.001),while DAPA and Vert reversed this phenomenon in vitro(P<0.001).DAPA and Vert reversed the IOD/area of CTGF and AREG in the kidneys of DKD rats(P<0.01),either alone or in combination in vivo.5.SGLT2 interference blocked the activation of YAP/TAZ.The loss of SGLT2 significantly reduced the expression of YAP/TAZ(P<0.05)and downstream factors CTGF and AREG(P<0.01)in HK-2 cells under HG conditions,while increasing the ratios of p-YAP/YAP and p-TAZ/TAZ(P<0.001).6.Dapagliflozin has stronger anti-renal fibrosis ability than verteporfin.Dapagliflozin treatment reduced RBG,24-h UP,BUN,Scr,tubulointerstitial injury scores,and the IOD/area of fibrosis-related factors TGFβ1 and α-SMA in DKD rats with significant differences(P<0.01,P<0.001).Verteporfin treatment can reduce Scr,the degree of kidney damage and fibrosis in DKD rats(P<0.05)but has no effect on other indicators.In addition,the combination of dapagliflozin and verteporfin did not show a superior effect than dapagliflozin alone in improving all the above indexes.7.Dapagliflozin has stronger antioxidative stress and anti-inflammatory effect than verteporfin.Compared with the control group,the levels of TNF-α,IL-1β and IL-6 in the kidneys of DKD rats were significantly increased(P<0.001).DAPA reduced the levels of the above inflammatory factors in the kidneys of DKD rats more than Vert.Compared with the control rats,DKD rats had significantly increased MDA levels(P<0.001),and decreased SOD activity(P<0.001)and GSH levels(P<0.01),which were reversed by DAPA and/or Vert treatment,and DAPA had a stronger effect.Conclusion: SGLT2 inhibition could reverse the progression of renal fibrosis in DKD by inhibiting Hippo-YAP/TAZ pathway.