The Role Of Autophagy Regulation In The Progression Of Acute Kidney Injury And Chronic Kidney Disease

Objective:Klotho gene was originally identified in 1997 as an anti-aging gene,and it showed anti-oxidative,anti-apoptotic and a lot more benefits in various organs.It is thought to be mainly expressed in renal distal tubules and it plays a renoprotective role in kidney injury.Thiazide is commonly used to treat hypertension and extracellular fluid volume expansion in clinical work.It is known to block thiazide-sensitive NaCl cotransporter(rTSCI/NCC)as to reduce NaCl reabsorption in the distal convoluted tubule(DCT).It has been reported that treatment of thiazide in rats provokes apoptosis in distal tubular cells.We studied the expression of aKlotho after thiazide treatment,so as to establish novel methods of investigating the predominant source of aKlotho.Methods:Metolazone,furosemide or vehicle were applied to SD rats(n=18,3 groups)for 3 days intraperitoneally via osmotic minipumps.Histology was studied by light microscope with HE staining.Expression of aKlotho and markers of tubules were examined by immunoblotting and immunohistochemistry.In addition,immunoprecipitation was used to study soluble aKlotho expression in the sera of rats.Results:Compared with vehicle and furosemide group,metolazone treated rats had significantly reduced NCC,aKlotho and calbindin D28k expression in the kidney.Immunoprecipitation results also showed remarkable reduced aKlotho levels in the sera of rats with matolazone treatment.HE staining showed marked renal tubular cell proliferation,inflammatory cell filtration and collapsed distal tubules in thiazide group.Conclusion:Distal renal tubule-derived aKlotho may mainly contribute to the circulating aKlotho levels.At the same time,thiazide treatment-related morphopathological changes in kidney could be potencially associated with decreased aKlotho expression in the distal renal tubules and circulation.Objective:AKI confers increased risk of progression to CKD.aKlotho is a cytoprotective protein,the expression of which is reduced in both AKI and CKD,but the relationship of aKlotho expression level to AKI progression to CKD has not been studied.We sought to explore the the role of aKlotho in the prognosis after AKI.We also want to investigate if aKlotho participates in renal recovery after ischemic/oxidative injury and extracellular matrix metabolism through regulating autophagic flux.Methods:We established two AKI mice models:bilateral ischemia-reperfusion injury(Bi-IRI)and unilateral nephrectomy plus contralateral ischemia-reperfusion injury(Npx-IRI).We altered systemic aKlotho levels by genetic manipulation,phosphate loading,or aging and examined the effect on long-term outcome after AKI,based on creatinine clearance(Clcr),plasma phosphate(Ppi),urine albumin creatinine ratio(ACR),pathology,fibrosis marker etc.In vitro,we transfected OKP cells with GFP-LC3 or GFP-Col I plasmid and treated with H2O2,aKlotho,autophagy inducer,or autophagy suppressor,to test the role of aKlotho in autophagy,oxidative stress and in collagen I metabolism..Results:Despite apparent initial complete recovery of renal function,both types of AKI eventually progressed to CKD,with decreased creatinine clearance,hyperphosphatemia,and renal fibrosis.Compared with wild-type mice,heterozygous aKlotho-hypomorphic mice(aKlotho haplo insufficiency)progressed to CKD much faster,whereas aKlotho-overexpressing mice had better preserved renal function after AKI.High phosphate diet exacerbated aKlotho deficiency after AKI,dramatically increased renal fibrosis,and accelerated CKD progression.Recombinant aKlotho administration after AKI accelerated renal recovery and reduced renal fibrosis.Compared with wild-type conditions,aKlotho deficiency and overexpression are associated with lower and higher autophagic flux in the kidney,respectively.Upregulation of autophagy protected kidney cells in culture from oxidative stress and reduced collagen I accumulation.Conclusion:We propose that aKlotho upregulates autophagy,attenuates ischemic injury,mitigates renal fibrosis,and retards AKI progression to CKD.Objective:C-reactive protein(CRP)was recently reported tobe closely associated with poor renal function in patients with acute kidney injury(AKI),but whether CRP is pathogenic or a mere biomarker in AKI remains unclear.Impaired autophagy is known toexacerbate renal ischemia-reperfusion injury(IRI).We examined whether CRP is pathogenic in AKI via reduction of autophagy.Methods:We mated transgenic rabbit CRP over-expressing mice(Tg-CRP)with Tg-GFP-LC3mice(LC3)and Tg-RFP-GFP-LC3 mice(RG-LC3)respectively to generate Tg-CRP-GFP-LC3 mice(PLC3)and Tg-CRP-RFP-GFP-LC3 mice(PRG-LC3).AKI was induced by IRI.Results:Compared with LC3mice,PLC3 mice developed more severe kidney damage after IRI.Renal tubules were isolated from LC3 mice at baseline for primary culture.OKP cells were transiently transfected with GFP-LC3 plasmid.CRP addition exacerbated lactate dehydrogenaserelease from both cell types.Immunoblots showed lower LC-3 Ⅱ/Ⅰ ratiosand higher levels of p62,markers of reduced autophagic flux,in the kidneys of PLC3 mice compared to LC3 mice after IRI,and in primary cultured renal tubules and OKP cells treated with CRP and H2O2 compared to H2O2 alone.Immunohistochemistry showed much fewerLC-3 puncta in kidneys ofPLC3 mice compared to LC3 mice after IRI.Similarly,CRP addition reduced GFP-LC3 puncta induced by H2O2 in primary cultured proximal tubules and in GFP-LC3 plasmid transfected OKP cells.Rapamycin,an autophagy inducer,rescued impaired autophagy and reduced renal injury in vivo.Conclusion:We provide further evidence that CRP is more than mere biomarker in AKI;it renders the kidney more susceptible to ischemic/oxidative injury by down-regulating autophagic flux.