| Diabetic kidney disease(DKD)continues to be a leading cause of end stage renal failure in the US and other developed countries.However,not every patient with diabetes mellitus develops diabetic nephropathy(DN),as evidenced by a number of long-term epidemiological follow-up studies.The reason for this heterogeneous outcome is not fully understood,but seemingly multifactorial and may involve a variety of causative factors,including genetics,environments,pre-existing kidney disease,severity of diabetes and others.Poor glycemic control has been a well-known risk factor for albuminuria and DN.Nevertheless,a significant portion of patients still progress to develop DN despite tight glycemic control.In clinical practice,it is necessary to adopt a sensitive and precise biomarker for stratifying diabetic patients at risk of progressing to renal impairment For years,albuminuria has been considered as a surrogate marker of diabetic kidney complications.Nevertheless,a growing body of evidence suggests that albuminuria may not be accurate.Significant discordance between albuminuria and renal impairment in DN has been noted.According to the United Kingdom Prospective Diabetes Study(UKPDS)data,of those patients who developed renal impairment,61%did not have albuminuria beforehand and 39%never developed albuminuria during the study.Likewise,of the patients that developed albuminuria,only 24%subsequently developed renal impairment during the study.In another word,for patients with microalbuminuria or macroalbuminuria,the prevailing majority may not develop renal impairment.These data thus challenge the classical paradigm of albuminuria always preceding renal impairment during the progression of diabetic kidney disease.As such,it is imperative to identify novel biomarkers for predicting the risk of DN or for early diagnosis of DN.In the past decade,a number of novel candidate molecules have been tested.Among these,glycogen synthase kinase(GSK)3 has emerged as an attractive molecule to serve in this role.GSK3 is a highly conserved,ubiquitously expressed serine/threonine protein kinase that was originally characterized to be a key transducer of insulin signaling cascade and govern glycogenesis.Interest in GSK3 expanded greatly with the realization that it also acts as a convergence point for multiple cell signaling pathways involved in inflammation,immunomodulation,embryogenesis,tissue injury,repair and regeneration.GSK3 exists as two isoforms,GSK3? and GSK3?.In different organ tissues,the two isoforms are differentially expressed.In kidney cortex,? rather than a isoform is predominantly expressed and located to glomeruli and proximal tubular cells.Renal GSK3? overactivity has been associated with diverse kidney disease,including proteinuric glomerulopathies and progressive chronic kidney disease.Nevertheless,as a key transducer of insulin signaling pathway,the role of GSK3? in pathogenesis of DN has been unknown.It remains to be delineated whether GSK3? dysregulation in the kidney is involved in DN.To address this issue,the present study examined the expression profiles of total and activated GSK3? in kidney specimens procured from db/db murine models of non-insulin dependent DN and in kidney biopsy tissues from patients with varying stages of type 2 DN.In addition,in a retrospective cohort of patients with type 2 diabetes,the expression levels of total and activated GSK3? were measured in urinary exfoliated cells serving as urine-based liquid kidney biopsy.We examined the relationship between progression of renal impairment in diabetic patients and expression levels of GSK3?or its activated form in baseline urinary exfoliated cells.PART ONE:The relationship between GSK3? and kidney injury in diabetic miceOBJECTIVETo observe the expression profiles of total and activated GSK3? in kidney specimens procured from db/db murine models of non-insulin dependent DN,and to examined the relationship between progression of renal impairment and expression levels of GSK3? or its activated form.METHODSThe db/db mice of leptin receptor deficiency are well-established models for non-insulin-dependent diabetes mellitus and DN.All mice were fed ad libitum and were sacrificed at indicated ages.The general conditions,body weight,fasten glucose,urine albumin/creatinine(ACR)were detected at regular intervals.Urine samples were collected and kidneys harvested for further examination.The age-matched db/m mice were used as control.1.Expression and location of GSK3? and its activated form(phosphorylated at Y216,p-GSK3?-Y216)in kidneys were examined by immunohistochemical staining,the protein expression of GSK3? and p-GSK3?-Y216 were analyzed by immunoblotting analysis at indicated ages.2.Pathological change comparison between db/db mice and db/m were observed by Periodic acid-Schiff staining.3.Immunofluorescence staining were adopted to detect the location of Fibronectin(FN),the main component of extracellular matrix.FN and Collagen ?(Col ?)expression in kidney specimens were analyzed by immunoblotting analysis.4.To determine whether GSK3? overexpression or hyperactivity is associated with DN progression featured by elevated urine albumin excretion rate and accumulation of Fibronectin,correlational analyses were conducted.RESULTS1.db/db mice were significantly obese,weight gain with decreased kidney weight/weight ratio and higher plasma glucose levels.Starting as early as 10 weeks old,db/db mice spontaneously develop overt diabetes as well as early signs of kidney injuries,characterized by progressive albuminuria and matrix accumulation in glomerulus.In contrast,age-matched control mice(db/m)have normal glycaemia and no noticeable evidence of kidney disease.2.Starting 10 weeks old,renal expression of totaland the activated form of GSK3?(phosphorylated at Y216)in db/db mice versus the control db/m mice was progressively elevated,and mainly localized to podocytes and mesangial cells in glomeruli and to tubular epithelial cells in tubulointerstitium,as shown by immunohistochemistry staining.Even at the early.stage of diabetes in db/db mice,such as at 10 or 14 weeks old,when albuminuria was trivial,the activity of GSK3?has been prominently elevated as reflected by significant induction of phosphorylated GSK3? in the kidney.3.The morphologic findings were corroborated by immunoblot analysis of whole kidney homogenates.4.Regression analysis demonstrated that the staining intensity of total and activated GSK3? in glomeruli positively correlated with urinary excretion of albuminuria in db/db mice at different ages.5.As shown by fluorescent immunohistochemistry staining,db/db mouse kidneys demonstrated a progressively increased expression of FN in both glomeruli and interstitium.This was further corroborated by immunoblot analysis of kidney homogenates for FN and Col ?.6.Expression levels of total and activated GSK3? were positively correlated with that of fibronectin in kidney tissues.CONCLUSIONS1.GSK3? is overexpressed and hyperactive in renal glomerular and tubular parenchymal cells in db/db mice2.GSK3? hyperactivity in the kidneys of db/db mice was largely attributable to post-translational modifications,like phosphorylation at Y216 at the early stage,but mainly due to increased expression of total GSK3? at the late stage.3.GSK3? overexpression and hyperactivity is associated with albuminuria and diabetic kidney injury.PART TWO:The relationship between GSK3? and cell injury in renal intrinsic cells under high-glucose conditionOBJECTIVE1.To determine if GSK3? plays a role in diabetic kidney injury,in vitro cultures of diverse kidney cell lines,including murine podocytes,mesangial cells and tubular epithelial cells,were employed and exposed to a diabetic milieu that consists of high ambient glucose and TGF?1.Mannitol group was set up to exclude the effect of hypertonia on cells.2.To explore if a causal relationship exists between GSK3? hyperactivity and cytopathic changes of kidney cells,GSK3? activity in cultured kidney cells was artificially manipulated by RNA interference or gene overexpression.METHODS1.Conditionally immortalized mouse podocytes between passages 21 to 25,mouse glomerular mesangial cells(MMC)and Murine proximal tubular epithelial(TKPT)cells were cultured in vitro and divided into 3 groups,normal milieu/culture media group(NG),diabetic milieu consisting of higher glucose and TGF?1(HG)and high osmolality control media containing mannitol(MG).At indicated time points,cells were fixed or cell lysates collected for further investigation.2.The expression and intracellular localization of GSK3? and p-GSK3?-Y216 in podocytes,mesangial cells and renal tubular epithelial cells were observed by immunofluorescence co-staining.The expression levels of GSK3? and p-GSK3?-Y216 in these cells were determined by Western blotting.Quantitatively analysis of arbitrary values was followed.3.The expression and intracellular localization of F-actin and SYNPO in podocytes,FN in mesangial cells and ZO-1 in renal tubular epithelial cells were observed by immunofluorescence co-staining.The expression levels of SYNPO,FN and ZO-1 were determined by immunoblot analysis,the Image J software was used for intensity measurements.4.To determine whether GSK3? overexpression or hyperactivity is associated with cytopathic changes of kidney cells,correlational analyses were conducted.5.To discern if GSK3P overexpression and hyperactivity is sufficient for the cytopathic effects induced by the diabetic milieu,cells were transiently transfected with either an empty vector or a vector encoding the constitutively active mutant of GSK3?(S9A).Immunoblot analysis were used to detect GSK3?,p-GSK3?-Y216,SYNPO,FN and ZO-1 expression.6.GSK3?,p-GSK3?-Y216,SYNPO,FN and ZO-1 expression in kidney cells were also tested after transfection of scramble siRNA or GSK3? siRNA.RESULTS1.Exposure of podocytes to the diabetic milieu resulted in striking podocyte injury,marked by loss of podocyte marker protein synaptopodin.This was concurrent with prominent induction of total and activated GSK3?,as shown by fluorescent immunocytochemistry staining and confirmed by immunoblot analysis.2.In cultured mesangial cells,expression of total and activated GSK3? was likewise augmented following exposure to the diabetic milieu,in parallel with increased expression of FN.3.In cultured renal tubular epithelial cells also overexpressed total and activated GSK3? after diabetic milieu treatment and this was accompanied with tubular cell dysfunction and dedifferentiation,marked by diminished and disrupted expression of epithelial tight junction molecules like ZO-1.4.While empty vector transfection barely exerted any effects,ectopic overexpression of S9A resulted in loss of synaptopodin expression in podocytes,elevated fibronectin production in mesangial cells and reduced ZO-1 expression in renal tubular cells even in the normal milieu,reminiscent of the cytopathic effect of the diabetic milieu.5.RNA interference using scramble siRNA barely affect the cytopathic effect of the diabetic milieu in all kidney cells.In contrast,GSK3? silencing substantially abrogated the diabetic milieu-instigated SYNPO suppression in podocytes,induction of FN in mesangial cells and ZO-1 disruption in renal tubular cells.CONCLUSIONS1.GSK3? was likely essential for triggering diabetes-related cytopathic changes in kidney cells.2.GSK3? hyperactivity is sufficient and essential for kidney cell injury and dysfunction elicited by diabetic milieu.PART THREE:The relationship between GSK3? in urine exfoliated cells and kidney injury in diabetic patientsOBJECTIVE1.To further validate if the above findings in animal and cell culture models are also applicable to clinical DN,that GSK3? hyperactivity participates in progression of diabetic kidney injury.2.To verify if measuring GSK3? or activated GSK3? in urinary exfoliated cells could be harnessed for predicting progression of human diabetic kidney disease.METHODS1.The expression of GSK3? gene in glomeruli of DN patients and healthy controls was retrieved and compared by Nephroseq database.2.Archived kidney biopsy tissues from patients with varying stages of DN and normal human kidney tissues were processed for peroxidase immunohistochemistry staining for GSK3? and p-GSK3?.Pathological change were observed by Periodic acid-Schiff staining.3.The biobank of diabetes was utilized and a retrospective cohort study was conducted.Patients with type 2 diabetes mellitus,who had been followed up at our hospital were screened.The demographic and clinical characteristics of these patients at baseline and those after 5-year follow-up were collected for comparison.Multivariate Cox proportional hazards regression analysis was used to find out risk factors associated with diabetic kidney injury.4.To determine if GSK3? was overexpressed or over-activated in renal cells and if this preceded the progression of diabetic kidney injury,urinary exfoliated cells that had been banked at baseline were retrieved and examined by immunoblot analysis.5.To assess the ability of total or activated GSK3? in baseline urinary exfoliated cells to serve as biomarkers for predicting the progression of diabetic kidney disease,receiver operating characteristic(ROC)curve analysis was carried out.RESULTS1.A significantly increased GSK3? expression in kidney biopsy tissues from DN was showed from the Nephromin-based data.2.The increased expressions of total and activated GSK3? were predominantly located to podocytes and mesangial cells in glomerulus and to renal tubular epithelial cells in the tubulointerstitium.3.Renal expressions of total and activated GSK3? were increasingly augmented along with DN progression as graded by pathologic classification based on PAS staining.Morphologic results revealed a positive association between the relative expression levels of total or activated GSK3? and the severity of DN.4.Based on the magnitude of albuminuria,the patients were stratified into 2 subgroups:normoalbuminuria[16.00(10.00,25.00)]and microalbuminuria[102.90(53.45,184.92)]groups.Except for urine albumin excretion levels and systolic pressure,the 2 subgroups of patients were not statistically different at baseline in all other clinical,biochemical and physiological parameters,including duration of diabetes,glycated hemoglobin levels,kidney function,blood pressure and prescribed treatments(P>0.05).5.After 5 years' follow-up,patients with progression of renal impairment,as compared with those without progression,exhibited significant reduction in eGFR(P=0.010)and marked increment in urine albumin excretion(P<0.001).In addition,patients with progression of renal impairment showed higher systolic pressure(P=0.001),lower serum albumin(P=0.029),and higher proportion of ACEI and ARB(P=0.008).6.Both total and activated forms of GSK3? could be evidently detected in all urinary exfoliated cell samples.Patients with progression of renal impairment had higher levels of total GSK3? and relative activity of GSK3? in baseline.7.In multivariate Cox proportional hazards regression analysis that was adjusted for other covariables like age,sex,BMI,etc.,only total GSK3? levels as well as the relative activity of GSK3? in baseline urinary exfoliated cells were identified to be potential,risk factors independently affecting the progression of renal impairment in patients with type 2 diabetes mellitus.8.While total GSK3? levels alone in baseline urinary exfoliated cells failed to show a discriminating ability,the relative activity of GSK3? in urinary exfoliated cells,expressed as pGSK3?/GSK3p ratios,demonstrated an excellent accuracy and power in discriminating patients with progressive renal impairment from those with stable kidney function,with AUC being 0.844(95%CI 0.747-0.941,P=0.001),which was much better than the AUC for baseline albuminuria 0.750(95%CI 0.626-0.874).The cutoff that maximized Youden's index was 1.24 for relative GSK3?activity and 76 mg/24h for UAE.At these cutoffs,the sensitivity and specificity were 69%and 87.1%for pGSK3?/GSK3? ratios and 69%and 77.4%for UAE.CONCLUSIONS1.GSK3? hyperactivity plays a role in diabetic kidney injury.2.Measurements of relative GSK3? activity in urinary exfoliated cells may provide much better accuracy than UAE in predicting the progression of renal impairment in diabetic patients. |
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