Influence of transient receptor potential cation channel 6 on renal tubulointerstitial inflammation in mice with diabetic kidney disease and its mechanism
Objective To investigate the influence of transient receptor potential cation channel 6(TRPC6)on renal tu-bulointerstitial inflammation in mice with diabetic kidney disease(DKD)and its mechanism.Methods A total of 36 male C57/BL6J mice,aged 6 weeks,were randomly divided into control group(group A),DKD model group(group B),DKD+normal sa-line intervention group(group C),DKD+mitophagy activator intervention group(group D),DKD+negative control lentivirus transfection group(group E),and DKD+TRPC6 knockdown lentivirus transfection group(group F),with 6 mice in each group.The mice in groups A and B were respectively given intraperitoneal injection of 0.1 mmol/L citrate buffer and 10 g/L streptozotocin(hereinafter referred to as administration);the mice in groups C and D were respectively given normal saline and 10 mmol/L urolithin A by gavage in addition to the treatment in group B;the mice in groups E and F were respectively injected with negative control lentivirus and TRPC6 knockdown lentivirus via the tail vein in addition to the treatment in group B.The levels of fasting blood glucose(FBG),urinary albumin-to-creatinine ratio(ACR),and blood urea nitrogen(BUN)were measured at week 12 after administration;PAS staining was used for the observation and scoring of renal tu-bular injury;RT-qPCR was used to measure the mRNA expression levels of inflammatory factors[interleukin-1β(IL-1β),mono-cyte chemoattractant protein-1(MCP-1),and tumor necrosis factor-α(TNF-α)]in renal tissue;immunohistochemical staining was used to measure the protein expression level of TRPC6 in renal tissue,and Western blotting was used to measure the relative expression levels of TRPC6,LC3B,P62,PINK1,and Parkin in renal tissue;transmission electron microscopy was used to observe the change in the number of mitophagosomes in renal tubular cells of mice.HK-2 cells were divided into high glucose+TRPC6 siR-NA transfection+DMSO intervention group(group G,treated with TRPC6 siRNA+35.0 mmol/L glucose+0.06％DMSO)and high glucose+TRPC6 siRNA transfection+mitophagy inhibitor intervention group(group H,treated with TRPC6 siRNA+35.0 mmol/L glucose+12 μmol/L oroxylin A),and then RT-qPCR was used to measure the mRNA expression levels of inflamma-tory factors(IL-1β,MCP-1,and TNF-α)in cells.Results At week 12 after administration,compared with group A,group B had significantly higher whole blood FBG,ACR,BUN,renal tubular injury score,mRNA expression levels of inflammatory fac-tors,and protein expression levels of TRPC6 and P62 in renal tissue(t=2.77-13.61,P＜0.05)and significantly lower protein ex-pression levels of LC3B-Ⅱ/LC3B-Ⅰ,PINK1,and Parkin in renal tissue(t=3.33-14.63,P＜0.05),withareduction in the num-ber of mitophagosomes in renal tubular cells.Compared with group C,group D had significantly lower levels of ACR and BUN,re-nal tubular injury score,mRNA expression levels of inflammatory factors,and protein expression level of P62 in renal tissue(t=2.40-23.50,P＜0.05)and significantly higher protein expression levels of LC3B-Ⅱ/LC3B-Ⅰ,PINK1,and Parkin in renal tissue(t=5.74-12.50,P＜0.05),with an increase in the number of mitophagosomes in renal tubular cells.Compared with group E,group F had significantly lower levels of ACR and BUN,renal tubular injury score,mRNA expression levels of inflammatory fac-tors,and protein expression levels of TRPC6 and P62 in renal tissue(t=2.45-7.09,P＜0.05)and significantly higher protein ex-pression levels of LC3B-Ⅱ/LC3B-Ⅰ,PINK1,and Parkin in renal tissue(t=7.91-13.18,P＜0.05),with an increase in the num-ber of mitophagosomes in renal tubular cells.Group H had significantly higher mRNA expression levels of inflammatory factors than group G(t=5.40-7.27,P＜0.05).Conclusion TRPC6 can aggravate renal tubulointerstitial inflammation by upregula-ting its expression and inhibiting mitophagy in renal tubular cells.Therefore TRPC6 inhibitors are promising for the treatment of DKD disease.
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