Canagliflozin Improves Hypertensive Renal Damage Via SIRT 3

Hypertensive renal damage is the second leading cause of end-stage renal disease（ESRD）after diabetes nephropathy,and there is a lack of effective therapeutic agents to delay or prevent the deterioration of kidney function.Hypertensive renal damage is a long-term hypertension leading to renal function damage and renal fibrosis,histologically manifested as tubular atrophy,tubulointerstitial fibrosis and glomerulosclerosis.Several factors have been linked to the progression of hypertensive renal damage,including activation of the renin-angiotensin-aldosterone system（RAAS）;oxidative stress;immune system activation;endothelial dysfunction.The main drugs currently available for the treatment of hypertensive nephropathy include,angiotensin converting enzyme inhibitors（ACEI）and angiotensin receptor blockers（ARB）,direct renin inhibitors（aliskiren）,aldosterone receptor antagonists（MRA）,diuretics,calcium channel blockers（CCB）,beta-blockers,and alpha-blockers.There are also antihypertensive agents with renal protective effects,including sodium-glucose co-transporter 2（SGLT2）inhibitors and angiotensin receptor neprilysin inhibitor（ARNI）.Studies have shown both renal and cardiovascular benefits of long-term intervention with SGLT2 inhibitors,even though the initial treatment phase may briefly reduce the estimated glomerular filtration rate（e GFR）.Other therapeutic interventions include,blood pressure control,and lifestyle management such as body mass control and proper diet and scientific exercise.However,current therapies for hypertensive nephropathy can only delay,not reverse or stop,the progression of the disease.To further explore the possible mechanisms of renal damage in hypertension and the mechanism of action of canagliflozin（Cana）.High-salt diet was used to induce hypertensive renal damage to detect the antihypertensive and renoprotective effect of SGLT2i（canagliflozin,Cana）in Dahl salt-sensitive rats.To explore molecular mechanisms,AngⅡwas used to induce renal proximal tubular epithelial cell line（HK-2）injury to explore the effect of Cana.The effects of Cana were also compared to the ARB（Irbesartan）,a traditional treatment for hypertensive renal damage.In addition,abnormal activation of complement system is also considered to be a risk factor for the development of hypertension to hypertensive renal damage.We collected clinical case data of primary hypertension with renal damage to further explore the role of complement in the development of hypertensive renal damage and to provide new targets for the clinical management of hypertensive nephropathy.Part one Establishment and evaluation of hypertensive renal damage modelObjectives:This study aims to compare the antihypertensive and renoprotective properties of canagliflozin when combined with irbesartan,a renin-angiotensin system（RAS）blocker in Dahl salt-sensitive rats model of hypertensive renal damage induced by a high-salt diet.Methods:1.Male Dahl SS rats with free access to water and a custom AIN-76A purified rodent chow containing 0.3%sodium chloride（Na Cl）for 10 days of adaptive feeding,and then were randomly divided into 5 groups,each of 6animals:Group 1（Con）continued to served as 0.3%low-salt diet and received only the vehicles,Group 2（HSD）served as 8%high-salt diet,but not given any of the medications.Groups 3,4,and 5 served as 8%high-salt diet and were treated orally for 12 weeks with either canagliflozin 30 mg/kg/day（HSD+Cana group）,irbesartan 30 mg/kg/day（HSD+Irb group）,or both drugs（HSD+Cana+Irb group）;respectively.2.The rats were fed for 12 weeks and their blood pressure,weight,food intake,mental state,appearance,and signs were monitored.3.The Dahl SS rats were placed in the metabolic cage system to monitor24h water consumption,food intake,and 24h urine volume parallel oral glucose tolerance experiment.4.The serum of Dahl SS rats was collected after the experiment.An automatic biochemical analyzer was used to detect creatinine,urea nitrogen,uric acid,serum glucose,serum albumin,triglyceride,total cholesterol,LDL-C,HDL-C,ALT,AST,serum sodium,and random urine was collected and urine glucose and urine sodium were measured.5.The urine of Cyc-C,KIM-1,NGAL were detected by commercial kits.6.H&E,PAS and Masson staining were performed to analyze the damage of glomeruli and tubules and the area of renal fibrosis in five groups of rats,respectively.Results:1.Systolic blood pressure（SBP）was markedly increased in the HSD group as compared with that of the control group.Following the administration of Cana,Irb,or Cana combined with Irb,the SBP of rats in HSD+Cana,HSD+Irb,and HSD+Cana+Irb groups were found to be significantly reduced when compared with that of rats in the HSD group.The reduction in SBP in the HSD+Cana+Irb group was significantly greater than reductions observed for HSD+Cana and HSD+Irb groups.No statistical difference was observed between HSD+Cana and HSD+Irb groups with regards to reductions in SBP.2.Diastolic blood pressure（DBP）was markedly increased in the HSD group as compared to the control group.Following administration of Cana,Irb,or Cana combined with Irb,the DBP of rats in HSD+Cana,HSD+Irb,and HSD+Cana+Irb groups were found to be significantly reduced when compared with that of rats in the HSD group.The reduction in DBP in the HSD+Cana+Irb group was significantly greater than reductions observed for HSD+Cana and HSD+Irb groups in the third week of receiving the corresponding drug.Interestingly,when DBP was measured at the sixth week,no statistical differences were observed between the HSD+Cana,HSD+Irb and HSD+Cana+Irb groups with regards to reductions in DBP.3.It was observed that rats in the HSD group and drug administration groups（HSD+Cana,HSD+Irb,and HSD+Cana+Irb）consumed more water than those in the control group,and consequently had increased urine outputs.This was particularly marked in HSD+Cana and HSD+Cana+Irb groups.There was a marked increase in the consumption of food in HSD+Cana and HSD+Cana+Irb groups when compared to the HSD group.However,there were decreases in body weight in HSD+Cana and HSD+Cana+Irb groups as compared to the HSD group.4.Additionally,the urine protein-creatinine ratio was found to be significantly higher in the HSD group when compared with that in the control group,but after administration of Cana combined with Irb,the urine protein-creatinine ratio was significantly reduced when compared with that in the HSD group.Similarly,at 12 weeks,the rats in the HSD group exhibited a higher level of urine cystatin C,kidney injury molecule-1（KIM-1）,and neutrophil gelatinase-associated lipocalin（NGAL）as compared with those in the control group.Administration of Cana as both a monotherapy and in combination with Irb markedly reduced the level of urine cystatin C,KIM-1,and NGAL.Urinary sodium excretion was significantly higher in the HSD group compared with that observed in the control group.Administration of Cana as both a monotherapy and in combination with Irb significantly reduced urinary sodium excretion when compared to the HSD group with no drug treatment.Urinary glucose excretion was found to be significantly higher in HSD+Cana+Irb and HSD+Cana groups as compared with that in the control and HSD groups,and urinary glucose excretion in the HSD+Cana+Irb group was significantly greater than that in the HSD+Cana group.Kidney weight was significantly elevated in the HSD group compared with that in the control group.Administration of Cana+Irb resulted in a significant reduction in kidney weight in HSD rats,as compared to the HSD group which received no drug administration.In addition,no significant differences were observed in serum glucose,TG,TC,LDL-C,HDL-C,serum albumin,or serum sodium,between any group.5.Hematoxylin and eosin（H&E）staining revealed normal renal histology in the control group.However,in the HSD group,remarkable glomerular atrophy,tubular granular degeneration,were observed.PAS staining demonstrated cell proliferation and infiltration of both the mesangial matrix and basement membrane in the glomerulus;protein casts in renal tubules;hyaline degeneration;in the HSD group as compared with the control group.In addition,Masson’s trichrome staining identified significant increases of glomerular and tubule-interstitial fibrosis in the kidneys of the HSD group as compared with the control group.However,HSD+Cana,HSD+Irb and HSD+Cana+Irb groups demonstrated marked decreases of cell proliferation and infiltration of mesangial matrix and basement membrane in the glomerulus;glomerular atrophy;tubular swelling;and renal interstitial fibrosis,in the kidneys.No statistical differences were observed between HSD+Cana,HSD+Irb and HSD+Cana+Irb groups in renoprotective effects.Conclusions:1.The model of hypertensive renal damage was established after feeding Dahl SS rats with 8%high salt diet for 12 weeks,which was consistent with the model characteristics of hypertensive renal damage.2.Cana combined with Irb had a more pronounced effect on lowering SBP than Cana or Irb administration.At week 3 of receiving the corresponding pharmacological administration,Cana combined with Irb was significantly more effective in lowering DBP than Cana or Irb administration,but as the duration of drug administration increased,no statistical differences were observed between the Cana combined with Irb,Cana or Irb administration with regards to reductions in DBP.3.Cana can also reduce weight and diuresis while lowering blood pressure,but does not affect normal serum glucose and serum sodium.4.Dahl SS rats developed hypertensive renal damage and renal fibrosis after12 weeks of 8%high-salt diet induced,while Cana ameliorated these changes.Part two Effect of Cana on Epithelial Mesenchymal Transition（EMT）and Oxidative Stress in Dahl Salt-Sensitive RatsObjective:We observed renal EMT and oxidative stress after 12 weeks of8%high-salt diet induced,and further compared the effects of Cana,Irb,Cana combined with Irb on renal EMT and oxidative stress in Dahl SS rats.Methods:1.The protein expression levels of epithelial calmodulin（E-cadherin）,vimentin andα-SMA in kidney tissues by Western Blotting.2.The protein expression levels of E-cadherin,vimentin,α-SMA in kidney tissues were detected by immunohistochemical staining.3.The level of MDA,GSH in the kidney cortex was determined by a commercial kit using a protein removal method.Results:1.Western blot analysis identified that Cana administration restored SIRT3expression and blocked HSD-induced EMT in the kidney by concurrently decreasing vimentin andα-SMA protein levels whilst increasing levels of E-cadherin.However,decreased levels of vimentin protein levels were observed in the Cana+Irb group as compared to the Cana monotherapy group.These results were in accordance with results obtained by immunohistochemical staining.No statistically significant changes were observed between HSD+Cana,HSD+Irb and HSD+Cana+Irb groups with regards to the expression ofα-SMA,SIRT3 and E-cadherin.The reduction in vimentin in the HSD+Cana+Irb group was significantly greater than reductions observed for HSD+Cana and HSD+Irb groups.2.Cana administration ameliorated HSD-induced oxidative stress in the kidneys and significantly attenuated levels of MDA in renal tissues.In contrast,Cana increased expression of GST and catalase in the kidneys of HSD-induced rats.No statistically significant changes were observed between HSD+Cana,HSD+Irb and HSD+Cana+Irb groups with regards to the expression of MDA,GST and catalase.Conclusions:1.EMT is an important component of renal fibrosis,manifested by reduced expression of E-cadherin and increased expression of vimentin andα-SMA.EMT occurred in Dahl SS rats induced by 8%high salt diet for 12 weeks,and Cana,Irb and Cana combined with Irb administration all improved renal damage by increasing SIRT3 and decreasing EMT.2.The kidney oxidative stress indexes in Dahl SS rats induced by 8%high-salt diet for 12 weeks were significantly higher in MDA levels,while GSH level and catalase protein expression were significantly lower.Cana,Irb and Cana combined with Irb administration could improve renal damage by reducing oxidative stress,and there was no statistical difference in the effects of the three groups of drug administration.Part three SIRT3 mediates the effect of canagliflozin on Ang II-induced epithelial mesenchymal transition of HK-2Objective:The aim of this part was to investigate whether canagliflozin can improve EMT of Ang-II induced HK-2,and the effect of canagliflozin on SIRT3.Methods:1.HK-2 was inoculated on 96-well plates at a density of 6×10⁴ cells/well,and the concentration of Ang-II was screened by CCK-8.2.The concentration of Cana was screened by CCK-8.3.siRNA-SIRT3 was synthesized,and SIRT3 gene silencing was tested by q RT-PCR and Western Blotting.4.HK-2 cells were divided into 7 groups:Con,AngⅡgroup,AngⅡ+Cana25,AngⅡ+siRNA SIRT3,AngⅡ+si-NC,AngⅡ+Cana25+siRNA SIRT3 and AngⅡ+Cana25+si-NC groups.5.After 24 hours of cell culture for each group,cells were collected and Western Blotting was used to detect the protein expression level of SIRT3,FOXO3a,catalase,E-cadherin,vimentin,α-SMA.Results:1.HK-2 was inoculated on the 96-well plate with a density of 6×10⁴cells/well,and the Ang-Ⅱconcentrations were 10^-8M,10^-7M,10^-6M,and 10^-5M,respectively.The optimal Ang-Ⅱconcentration was screened by CCK-8,and finally 10^-6M was selected.2.HK-2 was inoculated on 96-well plates at a density of 6×10⁴ cells/well,Cana concentration was 5μM,10μM,15μM,20μM,25μM,30μM was added into cell medium with an Ang-Ⅱconcentration of 10^-6M,the optimal concentration of Cana was screened by CCK-8,and finally the 25μM concentration of Cana was selected,which showed no cytotoxicity and showed drug activity.3.After transfection of siRNA into HK-2 cells,the expression of SIRT3m RNA and SIRT3 protein in si-SIRT3-1 group and si-SIRT3-2 group were significantly decreased compared with si-NC control group,and there was no statistical significance between si-SIRT3-1 group and si-SIRT3-2 group.4.Western Blotting results showed that the protein expression levels of SIRT3,FOXO3a,catalase,E-cadherin in the AngⅡ+Cana25 group were significantly higher than those in the AngⅡgroup.The protein expression levels of vimentin andα-SMA were significantly decreased.5.After the SIRT3 gene silence,the protein expression of SIRT3,FOXO3a,catalase,E-cadherin in the AngⅡ+Cana 25+siRNA SIRT3 group was significantly reduced compared with the AngⅡ+Cana 25+si-NC group and the AngⅡ+Cana 25 group.The protein expression of vimentin andα-SMA in the AngⅡ+Cana 25+siRNA SIRT3 group was significantly increased compared with the AngⅡ+Cana 25+si-NC group and the AngⅡ+Cana 25 group.Conclusions:1.Western Blotting results showed that the protein expression levels of SIRT3,FOXO3a,catalase and E-cadherin were significantly increased and the protein expression levels of vimentin andα-SMA were significantly decreased after Cana administration compared with Ang II group.2.After SIRT3 gene silencing,the increase in E-cadherin protein after Cana administration was largely attenuated by SIRT3 silencing,whereas the decrease in protein of vimentin andα-SMA was significantly eliminated by SIRT3silencing.The results suggest that Cana may inhibit Ang II-induced EMT in HK-2 cells through a SIRT3-dependent pathway.Part four The Correlation study of Circulatory Complement Activation and Hypertensive Renal DamageObjective:The expression levels of complement C3,complement bypass pathway activity（AP50）and complement regulatory factor H（CFH）were analyzed in patients with essential hypertension and hypertension combined with renal damage to investigate their correlation with hypertensive renal damage.Methods:Serum samples from 66 participants with established essential hypertension with renal damage（RD）were assessed the functional capability of the complement components C3,CFH and the AP activation.Groups of age and gender matched participants included 59 essential hypertension without renal damage（NRD）and 58 health participants（normal）.Serum samples were collected from the participants,and the levels of complement C3 and CFH,and the AP50 activity were measured in the serum.Results:Our study found C3 and AP50 exhibit a continued increase from normal to NRD to RD.The RD group had a profound increase in circulating C3and AP50（（178.52±18.82）mg/d L,20.97（20.22,22.14）U/ml,respectively）compared to NRD（（166.71±21.31）mg/d L;P=0.004,19.20（18.59,20.50）U/ml;P<0.001,respectively）and healthy participants（（152.15±17.80）mg/d L;p<0.001,18.27（17.16,19.03）U/ml;P<0.001,respectively）,while CFH was significantly lower（239.78（212.93,255.50）μg/L）compared to NRD（269.23（239.43,292.21）μg/L;P<0.001）and healthy participants（319.36（292.69,353.07）μg/L;P<0.001）.Spearman correlation analysis:The serum level of C3,AP50,CFH was significantly correlated with serum creatinine（r_s=0.301,P<0.001;r_s=0.474,P<0.001;and r_s=-0.443,P<0.001,respectively）,e GFR（r_s=-0.322,P<0.001;r_s=-0.534,P<0.001;and r_s=0.513,P<0.001,respectively）.In normal and NRD groups,multivariate logistic regression analysis revealed that the serum of C3,CFH,SBP were independent risk factors for hypertension[odds ratio 1.100（95%confidence interval 1.047,1.155）,P<0.001 for SBP;1.052（1.018,1.087）,P=0.003 for C3;0.961（0.945,0.977）,P<0.001 for CFH)].In normal and RD groups,multivariate logistic regression analysis revealed that the serum of C3,AP50,CFH,SBP were independent risk factors for hypertensive renal damage[odds ratio 1.101（95%confidence interval 1.011,1.198）,P=0.026 for C3;4.251（1.086,16.649）,P=0.038 for AP50;0.924（0.869,0.983）,P=0.012 for CFH;1.170（1.010,1.356）,P=0.036for SBP].In RD and NRD groups,multivariate logistic regression analysis revealed that the serum of C3,AP50,CFH,BMI were independent risk factors from hypertension to hypertensive renal damage[odds ratio 1.465（95%confidence interval 1.175,1.827）,P=0.001 for BMI;2.219（1.514,3.253）,P<0.001 for AP50;0.974（0.960,0.988）,P<0.001 for CFH)].Conclusion:Abnormal activation of complement,especially of complement AP,may be a risk factor for the development and progression of hypertensive renal damage.