Exploring The Role Of Kidney Tubule AMPK Pathway In Female Protection Against Diabetic Kidney Disease

Background:China has witnessed an unprecedented increase in diabetes incidence in the last few decades.It was estimated that there is over 11% of adult in China are affected by diabetes,which accounts for one-quarter of worldwide diabetic patients.Diabetes could cause damage to both small and large vessels,which gives rise to multi-organ complications.Diabetic complications include diabetic kidney disease(DKD),coronary atherosclerosis,and diabetes-induced liver injury,among which DKD is the most common complication that troubles more than 30% diabetic patients.Epidemiology studies indicate the sexual dimorphism of DKD,and some researchers speculated that was caused by sex hormones.Similarly,a similar pattern is found in liver study,and inhibition of kidney tubular sodium-glucose cotransporter 2(SGLT2)demonstrated hepatic protection against obesity-induced liver damage,which indicates the connection between kidney and liver.AMPK is the key energy sensor in cell and regulates the metabolism at cell and whole-body levels.Many studies have shown the protective effect of AMPK against kidney injury in diabetic patients.However,it remains to be known whether AMPK plays a role in the sexual dimorphism of DKD.To explore the role of renal AMPK in gender disparity of DKD,we used Cre/lox P technique to generate the kidney tubule specific AMPKγ2 knock out(KTAMPKγ2KO)mice,and verified AMPKγ2 expression in different tissues by PCR and Western Blot.These wild type phenotype and transgenic mice were treated with high fat diet and streptozotocin to induce diabetes.We measured the physiological parameters and collected the 24-hour urine monthly.Urine samples and tissue samples were analysis to determine the severity of organ damage.To further explore the connection of sex hormone and AMPK in high glucose induced renal cell injury,17 β-estradiol was applied to treat murine proximal tubule(MCT)cell.This study depicted the role of renal AMPK in diabetic complications would help us better understand mechanism of diabetes-related kidey and liver injury and develop new therapeutic or preventional strategies.Methods:1.Applying Cre/lox P technique to establish KTAMPKγ2KO mice.And q PCR and Western-Blot techniques were applied to detect the expression of AMPK subunits in different tissues.2.Diabetes was established by a combination of HFD and STZ injection.To evaluate the kidney injury,urinalysis was applied to measure the content of albumin,kidney injury molecular 1(KIM-1),and hydrogen peroxide in the urine,and Periodic Acid Schiff Staining(PAS)was also used to depict the histopathological change in the kidney.3.q PCR,western blot,and immunofluorescence were applied to analyze the content of inflammation molecules,matrix protein,and AMPK pathway in the kidney to evaluate the mechanism of diabetesinduced kidney injury.4.Western blot and immunofluorescence were applied to analyze the expression of matrix protein in the liver.5.MCT cells were incubated with high-glucose,17β-estradiol and AMPK inhibitor Compound C,and western blot was used to check the activity of AMPK pathway and the content of matrix proteins.Results:1.The renal expression of AMPKγ2 in KTAMPKγ2KO mice was dramatically decreased in both RNA and protein levels,while AMPKγ2expression were unchanged in liver and muscle.There was no change of expression of other subunits in kidney cortex.2.Elevated hydrogen peroxide,KIM-1 and albumin levels in urine,dilated renal tubules,and cell shedding were observed in male WT and KTAMPKγ2KO mice after HFD/STZ treatment,which was absent in female WT mice,but KTAMPKγ2KO caused increased urinary hydrogen peroxide,KIM-1,and albumin,and dilated renal tubules and cell shedding according to histopathological analysis in female diabetic mice3.Increased levels of p21cip1/cdkn1 a and mcp-1/ccl2,accumulation of matrix proteins and AMPK inhibition in the kidney were observed in male WT and KTAMPKγ2KO mice after HFD/STZ treatment,which were absent in female WT mice,but KTAMPKγ2KO gave rise to increased levels of p21cip1/cdkn1 a and mcp-1/ccl2,accumulation of matrix protein and AMPK inhibition in the liver.4.Accumulation of matrix protein in the liver was observed in male WT and KTAMPKγ2KO mice after HFD/STZ treatment,which was absent in female WT mice,but KTAMPKγ2KO caused an increased level of matrix protein in the liver.5.High glucose can inhibit the activity of AMPK and increase the expression of matrix protein in MCT cells,while 17β-estradiol can activate AMPK and inhibit the increase of matrix protein caused by high glucose,and the effect of 17β-estradiol can be inhibited by AMPK inhibitor Compound C.Conclusion:1.Female mice were resistant to diabetes-induced renal AMPK suppression,kidney injury and liver change.2.KTAMPKγ2KO could induce inhibition of renal AMPK activity,leading to increased levels of renal inflammation molecules and accumulation of hepatic and renal matrix proteins,which eventually contribute the diabetes-induced kidney injury and change in liver.3.17β-estradiol could alleviate matrix protein accumulation induced by high glucose in MCT cells.