GPR43 Activation Mediates Podocyte Insulin Resistance In Diabetic Nephropathy

Objective:Diabetic nephropathy(DN)is a serious microvascular complication of diabetes mellitus.Glomerulosclerosis is the main pathological feature of DN.Glomerular podocyte injury is one of the early events of DN.Various causes lead to podocyte fusion,apoptosis and podocyte detachment,and eventually lead to impaired glomerular filtration barrier integrity and proteinuria.Podocyte insulin resistance is one of the important initiating factors of renal pathological damage in DN.In recent years,the role of GPR43 in regulating energy metabolism and insulin homeostasis has attracted extensive attention.However,the role of GPR43 activation and its potential mechanism in podocyte insulin resistance of DN are not clear.Therefore,using kidney tissues of DN patients,DN animal model,GPR43 gene knockout mouse model and in vitro podocyte culture model,this study aimed to explore the expression change of GPR43 in kidneys of DN,the role of GPR43 in podocyte injury,the effects of gut microbiota dysbiosis on GPR43 activation,and to further explore the potential mechanism of GPR43 activation in mediating podocyte injury and insulin resistance in DN.MethodsPart 1: The expression of GPR43 in DNWestern blotting and immunofluorescence staining were used to detect the expression of GPR43 in renal tissues of DN patients and diabetic mice models.The expression and co localization of podocyte specific proteins WT-1 and p Akt were detected by immunofluorescent staining.Part 2: The role of GPR43 in podocyte injuryIn vitro cultured podocytes,the effects of different concentrations of acetate and GPR43 si RNA on podocyte damage were analyzed.Real-time PCR and Western blotting were used to detect the expression of SCFAs receptors(GPR43,GPR41 and Oflr78),podocyte specific proteins and extracellular matrix proteins(nephrin,collagen-1 and α-SMA).Part 3: GPR43 activation mediates podocyte insulin resistanceReal-time PCR and Western blotting were used to detect the expression of insulin signaling pathway molecules(IR-β,IRS1 and p-Akt)after acetate stimulation with or without GPR43 specific inhibitor or GPR43 si RNA.The changes of 2-deoxyglucose uptake were detected by fluorescence enzyme labeled method.The translocation of GLUT4 was detected by immunofluorescent staining.Part 4: The effects of GPR43 gene knockout on kidney injury and podocyte insulin resistance in diabetic mice1.Construction of GPR43 gene knockout miceCRISPR/Cas9 system was used to construct GPR43 gene knockout mice.The tail DNA was extracted for genotyping.Immunofluorescent staining,real-time PCR and Western blotting were used to verify the efficiency of GPR43 gene knockout.2.Establishment of diabetic mouse modelSTZ(50 mg/kg body weight)was injected intraperitoneally for 5 consecutive days to establish type 1 diabetic mice model.After successful induction,the mice were divided into four groups:control group(Ctrl),GPR43 knockout group(GPR43 KO),diabetes mellitus(DM)and GPR43 gene knockout + DM group(GPR43 KO+DM).Urine samples were collected at Week 4,8 and12 to determine the ratio of microalbumin to creatinine.After 12 weeks,the mice were sacrified.The feces,blood,urine and kidney tissues of mice were collected for subsequent gut microbiota analysis,the change of serum biochemical indexes,urinary ACR,renal histopathological analysis and target protein expression detection.Part 5: The effects of depletion of gut microbiota treated by antibiotics and fecal microbiota transplantation on renal injury and podocyte insulin resistance in diabetic rats1.Construction of diabetic rat modelsA single intraperitoneal injection of STZ(100 mg/kg body weight)was used to establish the model of type 1 diabetic rats.2.The effects of depletion of intestinal microbiota on renal injury in diabetic rats After the successful establishment of diabetic rat model,the rats were divided into three groups:control group,DM group,and diabetes + antibiotics group(DM+AB group)and treated for 8weeks.The rats of DM+AB group were fed with drinking water containing with ampicillin(1g/L),neomycin(1 g/L)and vancomycin(0.5 g/L),metronidazole(1 g/L)and amphotericin B(1 g/L).The antibiotic solution was changed daily.3.The effects of fecal microbiota transplantation on renal injury in diabetic rats After the successful establishment of diabetic rat model,the rats were divided into three groups:control group,DM group and DM + FMT group and treated for 8 weeks.The rats of DM +FMT group were given fecal bacteria from wild type rats.The feces of the control group were placed in the pre-cooled sterile PBS solution containing 10% glycerol,and the suspension was slowly poured into the stomach of diabetic rats through the catheter during transplantation,and the other groups were given sterile PBS solution containing 10% glycerol by gavage.Three days after gavage,feces were collected and 16 S r DNA sequencing was performed to evaluate the composition and abundance of gut microbiota in diabetic rats after fecal transplantation.The feces,blood,urine and kidney tissues of mice were collected for subsequent gut microbiota analysis,the assay of serum biochemical indexes,urinary ACR,renal histopathological analysis and target protein expression detection.In the above-mentioned animal experiments,blood glucose,BUN and Scr were detected by clinical biochemical assay.Urinary microalbumin level was detected by a mouse ELISA kit.Plasma acetate level was detected by gas chromatography.PAS staining was used to observe the pathological damage of kidney.Ultrastructural changes of podocytes were observed by transmission electron microscopy.The expression of GPR43,nephrin,collagen I,fibronectin,and insulin pathway related molecules were repectively detected by immunofluorescent staining,real-time PCR and Western blotting.16 S r DNA sequencing was used to analyze the changes of gut microbiota diversity.Part 6: The mechanisms of GPR43 mediating podocyte insulin resistance in DN1.GPR43 activation induced insulin resistance in podocytes through the suppression of AMPKα activity in podocytes.Western blotting was used to detect the effects of GPR43 knockout in vivo and podocyte GPR43 knockdown on AMPK phosphorylation in podocytes.Western blotting was used to detect the inhibitory effect of AMPK agonist AICAR on the decrease of Akt phosphorylation mediated by acetate.2.PKC-PLC mediated inhibition of AMPKα activity by GPR43 in podocytes Western blotting was used to detect the effects of PKC inhibitor Go 6983 and PLC inhibitor U73122 on Akt phosphorylation mediated by GPR43 activation in podocytes.Results:1.Increased GPR43 expression in the kidneys of DN was involved in podocyte insulin resistanceImmunofluorescent staining and Western blotting demonstrated that the GPR43 protein expression was increased in podocytes of DN patients and in the kidneys of diabetic mice.Colocalization of WT-1 with phosphorylated Akt protein was gradually decreased in the kidneys at various stages of DN in patients.2.Acetate induced podocyte injuty via the activation of GPR43Acetate remarkably increased the m RNA and protein expression of GPR43 in podocytes in a dose-dependent manner.Acetate at the concentration of 40 mmol/L also activated Oflr78 in podocytes.However,the increase of Oflr78 m RNA expression was less pronounced compared with GPR43 m RNA expression.Acetate stimulation induced phenotypic transformation of podocytes,characterized by dramatically increased α-SMA and collagen I expression and decreased nephrin expression at the m RNA and protein levels in podocytes.These effects were dramatically attenuated after the treatment with GPR43 si RNA.3.The activation of GPR43 downregulated insulin signalling in podocytes Acetate treatment at the concentrations of 3,5,and 10 mmol/L downregulated the PI3K-Akt signaling in podocytes via a decrease in the Akt protein phosphorylation in the absence or in the presence of high concentrations of glucose.Acetate inhibited the Akt protein phosphorylation in podocytes induced by insulin stimulation.In addition,acetate inhibited the m RNA and protein expression of insulin receptor substrate 1(IRS1)and insulin receptor β(IRβ)with or without high concentrations of glucose.Furthermore,acetate inhibited glucose uptake of podocytes stimulated by insulin,which was correlated with the disruption of the insulininduced GLUT4 translocation to the cellular membrane mediated by acetate as shown by immunofluorescent staining.GPR43 si RNA or GLPG0974,an antagonist of GPR43,were able to override acetate-mediated decrease in Akt protein phosphorylation in podocytes.4.GPR43 deficiency alleviated insulin resistance-mediated podocyte injury in DN Real-time PCR confirmed successful construction of the GPR43 KO model.The protein expression of GPR43 were significantly decreased in the kidneys of GPR43 KO+DM mice compared with that in the DM group.There was no difference in the blood glucose level and kidney weight to body weight ratio between the controls and the GPR43 KO mice.There was no difference in the blood glucose levels and body weight in the DM and GPR43 KO+DM mice groups.However,the ratio of kidney weight to body weight in the GPR43 KO+DM mice was decreased compared with that in the DM group.Urinary albumin excretion rates at Week 8 and Week 12 were decreased in the GPR43 KO+DM group compared with that in the DM group.However,there was no difference in the levels of BUN and Scr between the four groups.We subsequently evaluated the role of GPR43 KO in the pathological renal injury.The PAS staining showed that mesangial expansion was significantly decreased in the GPR43 KO+DM mice compared with that in the DM mice,which was in accordance with the decrease in the m RNA and protein expression of collagen I and fibronectin.Moreover,electron microscopy observation demonstrated that there was a significant improvement in podocyte foot process effacement accompanied with reduced thickness of the glomerular basement membrane(GBM)in the diabetic GPR43 KO mice compared with that in the DM mice.The density of podocytes was further assessed by immunofluorescent staining of Wilms’ tumor-1(WT-1),a specific biomarker of podocytes.The results showed that podocyte number was decreased in the kidneys of the DM group.However,podocyte density was increased in the kidneys of diabetic mice with GPR43 knockout.GPR43 KO increased Akt protein phosphorylation in podocytes in diabetic mice,suggesting that GPR43 depletion ameliorated the insulin signalling in podocytes with the hyperglycaemia background.Gut microbiota had significantly different composition and distribution in the DM group versus the controls.These characteristics were not substantially influenced by GPR43 KO as illustrated by the principal component analysis.5.Activation of GPR43 induced by acetate released from gut microbiota contributed to the progression of DNThe results showed that the serum levels of glucose in the antibiotics-treated diabetic rats were decreased compared to that in diabetic rats.However,there was no difference after the FMT treatment.Interestingly,the serum level of acetate was increased in diabetic rats compared with that in the controls,whereas antibiotics or FMT treatment decreased the serum acetate level.These results suggest that an increase in serum acetate could be produced from gut microbiota,which mediated the activation of GPR43 in the kidney.Moreover,antibiotic or FMT treatment decreased the GPR43 protein expression in the kidneys or podocytes of diabetic rats accompanied with a decrease in urinary albumin protein excretion,mesangial matrix expansion,podocyte foot process effacement,and increased podocyte number.Moreover,antibiotics or FMT treatment restored the podocyte insulin signalling in the kidneys of diabetic rats through an increase in Akt phosphorylation as demonstrated by the immunofluorescent co-staining and Western blotting.Thus,these findings suggest that dysbiosis of gut microbiota in DN could be involved in the impairment of insulin signalling in podocytes via microbiota metabolites.6.Activation of GPR43 mediated podocyte insulin resistance in DN through the inhibition of AMPKα activityAcetate inhibited AMPKα phosphorylation in podocytes in a dose-dependent manner.However,the acetate-mediated inhibitory effect on AMPKα phosphorylation was overridden by GPR43 si RNA and GLPG0974,an inhibitor of GPR43.The level of AMPKα phosphorylation in the kidney of GPR43 KO diabetic mice was increased compared with that in diabetic mice.These results suggest that GPR43 could be a key regulator for the inhibition of AMPKαphosphorylation.To clarify the role of AMPKα in activation of GPR43-mediated podocyte insulin resistance,we determined the effect of AMPKα activity restoration induced by an AMPK agonist(5-aminoimidazole-4-carboxamide ribonucleotide,AICAR)on podocyte insulin resistance mediated by GPR43 activation.The AICAR treatment reversed the acetatemediated inhibition of Akt phosphorylation in podocytes.The PKC inhibitor Go 6983 and the PLC inhibitor U73122 effectively blocked the GPR43-mediated inhibition of Akt phosphorylation in podocytes with or without insulin.These findings suggest that acetatemediated activation of GPR43 suppresses the bioactivity of insulin in podocytes via the suppression of Akt phosphorylation mediated by the PKC-PLC-AMPKα pathway.Conclusions:Our findings for the first time demonstrated that the GPR43 was a critical modulator of insulin signaling.The GPR43 activation induced by acetate produced from gut microbiota contributed to podocyte insulin resistance in diabetic nephropathy through the suppression of AMPK activity.