The glomerular podocyte as a target of growth hormone action: implications for the pathogenesis of diabetic nephropathy

Involvement of the growth hormone (GH) / insulin-like growth factor 1 (IGF-I) axis in the pathogenesis of diabetic nephropathy (DN) is strongly suggested by studies investigating the impact of GH excess and deficiency on renal structure and function. GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney. In the human a direct relationship has been noted between the activity of the GH/IGF-1 axis and renal hypertrophy, microalbuminuria, and glomerulosclerosis. Conversely, states of GH deficiency or deficiency or inhibition of GH receptor (GHR) activity confer a protective effect against DN. The glomerular podocyte plays a central and critical role in the structural and functional integrity of the glomerular filtration barrier and maintenance of normal renal function. Recent studies have revealed that the glomerular podocyte is a target of GH action and that GH's actions on the podocyte could be detrimental to the structure and function of the podocyte. These results provide a novel mechanism for GH's role in the pathogenesis of DN and offer the possibility of targeting the GH/IGF-1 axis for the prevention and treatment of DN.     

足细胞与糖尿病肾病关系的若干研究进展

足细胞在维持肾小球滤过屏障完整性及限制血浆蛋白的滤出方面发挥重要作用,足细胞的损伤可部分解释肾小球滤过屏障结构及功能的改变的原因。在糖尿病肾病早期,足细胞损伤已出现,并可进一步导致。肾脏损伤。本文就足细胞与糖尿病肾病中若干研究进展作简要综述。     

糖尿病肾病肾小球滤过膜超微结构改变及其与蛋白尿的关系

目的:观察2型糖尿病肾病(2TDN)患者肾小球滤过膜超微结构变化特点及与蛋白尿的关系。方法:将明确诊断的2TDN患者分为微量白蛋白尿组(尿白蛋白/24h30～300mg);蛋白尿组(尿蛋白0.5～2.0g/24h)和大量蛋白尿组(尿蛋白>3.5g/24h)。收集三组患者临床指标,并分别观察各组患者肾活检组织学及肾小球滤过膜超微结构变化,采用形态学计量分析方法分别测算肾小球体积、肾小球足细胞和内皮细胞的相对密度、绝对数目和足细胞足突宽度及肾小球基膜(GBM)厚度。结果:三组患者的年龄、性别、BMI、血压和糖尿病及肾脏病病程均无统计学差异。2TDN患者肾组织超微结构定量分析结果证实:(1)2TDN患者在微量白蛋白尿期肾小球毛细血管袢内皮细胞数目就已增加(实验组vs对照组P<0.01),内皮细胞相对密度则维持在一定水平(各组间P>0.05);(2)GBM改变包括三层结构消失、GBM增厚等,这些改变在微量白蛋白尿期就已发生,且GBM与病程密切相关(蛋白尿组,r=0.538,P<0.05)。(3)微量白蛋白尿组患者肾小球足细胞相对密度下降(微量白蛋白尿组vs对照组P<0.01)、足细胞足突宽度增加(微量白蛋白尿组vs对照组P<0.05)。随病程进展,足细胞相对密度和绝对数目呈进行性减少(微量白蛋白尿组vs大量蛋白尿组P<0.05)。(4)2TDN患者蛋白尿水平与肾小球足细胞数目(大量蛋白尿组,r=0.648,P<0.01)及GBM厚度(蛋白尿组,r=0.538,P<0.05)相关。结论:DN患者肾小球滤过膜超微结构发生了系列变化,这些变化与蛋白尿的发生、发展有着内在联系。     

Ultrastructural changes in the glomerular filtration barrier and occurrence of proteinuria in Chinese patients with type 2 diabetic nephropathy

AimDiabetic nephropathy (DN) is one of the most important causes of end stage renal disease in the world. Its hallmark is proteinuria. Therefore, we set out to clarify the structural changes that occur in the glomerular filtration barrier in Chinese patients with true type 2 diabetic nephropathy, and to examine the relationship between these structural changes and proteinuria.Methods42 Chinese patients with true T2DN were divided into three groups according to urinary protein excretion. Glomerular volume, endothelial cell density, endothelial cell number, glomerular basement membrane (GBM) width, podocyte density, podocyte number and foot process width were evaluated using light and electron microscopic morphometry.ResultGlomerular volume and endothelial cell number were increased in diabetic patients, but there was no difference between patients with respect to the degree of proteinuria. As proteinuria progressed, endothelial cell density remained unchanged, while the glomerular basement membrane (GBM) and podocyte foot process width increased, podocyte density and number decreased.ConclusionsPodocyte and GBM change more obviously during the development of proteinuria. Besides, proteinuria was inversely related to podocyte density, and directly related to GBM and glomerular volume.     

糖尿病肾病肾小球滤过膜结构与肾功能及代谢指标的关系

目的:观察2型糖尿病肾病(T2DN)患者肾小球滤过膜超微结构改变与肾功能及代谢指标的关系.方法:将明确诊断的T2DN 75例患者分为:微量白蛋白尿组(尿白蛋白/24h 30～300 mg);蛋白尿组(尿蛋白0.5～2.0g/24h)和大量蛋白尿组(尿蛋白＞3.5g/24h).收集三组患者临床指标,并分别使用Cockcroft-Gault公式、简化的肾脏疾病饮食控制(MDRD)公式等计算患者的肾小球滤过率(eGFR);采用形态学计量分析方法分别测算肾小球体积、肾小球足细胞和内皮细胞的相对密度、绝对数目和足细胞足突宽度及肾小球基膜(GBM)厚度.结果:(1)肾小球滤过膜结构与GFR的关系:微量白蛋白尿组Ccr与肾小球足细胞密度及数日均负相关(分别为r=-0.480,P＜=0.05;r=-0.478,P＜0.05);大量蛋白尿组以SCr估算的eGFR与肾小球足细胞密度正相关(r=0.462,P＜0.05);余均未见明显相关.(2)多元回归分析结果:微量白蛋白尿组中肾小球足细胞密度、糖化血红蛋白、三酰甘油、尿酸与Ccr相关(R~2=0.616,P＜0.01);大量蛋白尿组肾小球足细胞密度、尿酸与以SCr估算的eGFR相关(R~2=0.613,P＜0.01).(3)肾小球滤过膜结构及肾小球体积与糖、脂质代谢指标的关系:①肾小球体积与血糖的关系:微量白蛋白尿组中肾小球体积和糖化血红蛋白正相关(r=0.425,P＜0.05);而在蛋白尿组则为负相关(r=-0.427,P＜0.05).②GBM厚度与血糖、血脂代谢指标的关系:微量白蛋白尿组中,以GBM厚度为因变量,糖及脂质代谢水平为自变量,可见空腹血糖水平和总胆固醇与基膜厚度相关(R~2=0.247,P＜0.05).结论:肾小球滤过膜结构与GFR及糖、脂质代谢水平间存在着密切联系,且与DN发展的不同阶段相关.     

Podocyte as the target for aldosterone: roles of oxidative stress and Sgk1

Accumulating evidence suggests that mineralocorticoid receptor blockade effectively reduces proteinuria in hypertensive patients. However, the mechanism of the antiproteinuric effect remains elusive. In this study, we investigated the effects of aldosterone on podocyte, a key player of the glomerular filtration barrier. Uninephrectomized rats were continuously infused with aldosterone and fed a high-salt diet. Aldosterone induced proteinuria progressively, associated with blood pressure elevation. Notably, gene expressions of podocyte-associated molecules nephrin and podocin were markedly decreased in aldosterone-infused rats at 2 weeks, with a gradual decrease thereafter. Immunohistochemical studies and electron microscopy confirmed the podocyte damage. Podocyte injury was accompanied by renal reduced nicotinamide-adenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced expression of aldosterone effector kinase Sgk1. Treatment with eplerenone, a selective aldosterone receptor blocker, almost completely prevented podocyte damage and proteinuria, with normalization of elevated reduced nicotinamide-adenine dinucleotide phosphate oxidase activity. In addition, proteinuria, podocyte damage, and Sgk1 upregulation were significantly alleviated by tempol, a membrane-permeable superoxide dismutase, suggesting the pathogenic role of oxidative stress. Although hydralazine treatment almost normalized blood pressure, it failed to improve proteinuria and podocyte damage. In cultured podocytes with consistent expression of mineralocorticoid receptor, aldosterone stimulated membrane translocation of reduced nicotinamide-adenine dinucleotide phosphate oxidase cytosolic components and oxidative stress generation in podocytes. Furthermore, aldosterone enhanced the expression of Sgk1, which was inhibited by mineralocorticoid receptor antagonist and tempol. In conclusion, podocytes are injured at the early stage in aldosterone-infused rats, resulting in the occurrence of proteinuria. Aldosterone can directly modulate podocyte function, possibly through the induction of oxidative stress and Sgk1.     

Cell biology of diabetic nephropathy: Roles of endothelial cells,tubulointerstitial cells and podocytes

Diabetic nephropathy is the major cause of end‐stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease.     

Mineralocorticoid receptors in the pathophysiology of chronic kidney diseases and the metabolic syndrome

Recent studies indicate that aldosterone/mineralocorticoid receptor (MR) is a major contributor of chronic kidney disease (CKD) progression. Aldosterone/MR induces glomerular podocyte injury, causing the disruption of the glomerular filtration barrier and proteinuria. Conversely, MR antagonists substantially reduce proteinuria, which can be partly attributable to the protective effects on podocytes. Aldosterone excess, caused by adipocyte-derived aldosterone-releasing factors and other mechanisms, can be pathologically important in the renal complication of metabolic syndrome. A rat model of metabolic syndrome exhibits podocyte injury and proteinuria with serum aldosterone elevation, and the renal damage is prevented by MR blockade. Accumulating data also indicate that MR inhibition can confer renoprotection in a subgroup with low or normal aldosterone levels. We have recently identified the cross-talk between MR and small GTPase Rac1, providing one theoretical basis for the renoprotective effects of MR antagonists in non-high-aldosterone subjects. MR blockade can be a promising strategy for preventing CKD progression, and future clinical trials will conclusively determine the efficacy and tolerability of selective MR inhibition in CKD and metabolic syndrome.     

糖尿病糖脂代谢紊乱与足细胞损害的实验研究

目的　观察糖脂代谢紊乱对糖尿病大鼠肾小球滤过屏障外层足细胞的影响 ,以探讨糖尿病肾病的发病机制。方法　采用链脲佐菌素诱导糖尿病大鼠模型 ,喂养 5周后 ,测定血糖、糖化血红蛋白、甘油三酯、总胆固醇、血肌酐、尿素氮和尿白蛋白排泄率 ,应用免疫组化检测肾小球足细胞损伤标志蛋白 -desmin的表达 ,同时利用透射电子显微镜观察肾小球足细胞超微结构。结果　糖尿病大鼠血糖、糖化血红蛋白、总胆固醇、血肌酐、尿素氮、尿白蛋白排泄率水平明显升高 (P <0 .0 5 ) ,肾小球内desmin蛋白表达上调 ,足细胞部分足突融合 ;同时 ,两组间血甘油三酯水平无显著性差异 (P >0 .0 5 )。结论　糖脂代谢紊乱可导致糖尿病大鼠肾小球滤过屏障外层足细胞明显损害 ,尿白蛋白排泄率增加 ,这可能是糖尿病肾脏损害、蛋白尿出现的机制之一     

Causes and consequences of proteinuria: the kidney filtration barrier and progressive renal failure

The past few years have witnessed a major breakthrough in the understanding of the molecular mechanisms and ultrastructural changes behind the development of proteinuria. The discovery of several proteins in the glomerular podocyte and slit diaphragm, where mutations lead to disease, has revealed the importance of this cell with its diaphragm as the major filtration barrier as opposed to the glomerular basement membrane (GBM) previously ascribed this function. Furthermore, accumulating clinical as well as experimental evidence points to the harmful effects of proteinuria, irrespective of the original damage. The purpose of this review is to shed light on what we know today about the two sides of this 'coin', the causes and the consequences of proteinuria.     

艾塞那肽对糖尿病肾病小鼠足细胞的保护作用

目的:探讨艾塞那肽对糖尿病肾病小鼠足细胞的作用。方法:通过给予C57BL/6J小鼠高脂饮食并注射链脲佐菌素建立糖尿病肾病模型,按随机数字表法将其分为糖尿病肾病对照组(DN组, n＝8)、艾塞那肽干预组(DN＋Ex组, n＝8)。同时将普通饲料喂养的C57BL/6J小鼠作为正常对照组(NC组, ...     

Endocrinological aspects of proteinuria and podocytopathy in diabetes: role of the aldosterone/mineralocorticoid receptor system

Aldosterone has emerged as a deleterious hormone in the kidney, for example as a potent inducer of proteinuria. We identified the podocyte, the final filtration barrier in the glomerulus, as a novel target of aldosterone. Activation of the mineralocorticoid receptor (MR) in the podocyte disrupts the filtration barrier and induces proteinuria. Recent clinical and experimental studies have shown the efficacy of MR antagonism in reducing albuminuria in patients or rodent models of type 1 and type 2 diabetes. We assessed the pathogenic role of aldosterone in SHR/NDmcr-cp, a rat model of type 2 diabetes/metabolic syndrome. Podocyte injury and proteinuria were early manifestations of nephropathy in this model, and were exacerbated by high-salt feeding. Inappropriate activation of the aldosterone/MR system, possibly via adipocyte-derived aldosterone releasing factors, underlay the renal damage. Furthermore, we identified Rac1, a Rho family small GTPase, as a novel ligand-independent activator of MR. This alternative pathway of MR activation, indeed, contributed to podocyte injury in proteinuric kidney disease. In conclusion, MR can be activated by several different pathways, both aldosterone-dependently and -independently, leading to podocyte impairment and progression of proteinuric kidney disease. MR antagonists are promising anti-proteinuric drugs in diabetes, although hyperkalemia is a concern.     

What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium?

Microalbuminuria is an important risk factor for cardiovascular disease and progressive renal impairment. This holds true in the general population and particularly in those with diabetes, in whom it is common and marks out those likely to develop macrovascular disease and progressive renal impairment. Understanding the pathophysiological mechanisms through which microalbuminuria occurs holds the key to designing therapies to arrest its development and prevent these later manifestations. Microalbuminuria arises from the increased passage of albumin through the glomerular filtration barrier. This requires ultrastructural changes rather than alterations in glomerular pressure or filtration rate alone. Compromise of selective glomerular permeability can be confirmed in early diabetic nephropathy but does not correlate well with reported glomerular structural changes. The loss of systemic endothelial glycocalyx—a protein-rich surface layer on the endothelium—in diabetes suggests that damage to this layer represents this missing link. The epidemiology of microalbuminuria reveals a close association with systemic endothelial dysfunction and with vascular disease, also implicating glomerular endothelial dysfunction in microalbuminuria. Our understanding of the metabolic and hormonal sequelae of hyperglycaemia is increasing, and we consider these in the context of damage to the glomerular filtration barrier. Reactive oxygen species, inflammatory cytokines and growth factors are key players in this respect. Taken together with the above observations and the presence of generalised endothelial dysfunction, these considerations lead to the conclusion that glomerular endothelial dysfunction, and in particular damage to its glycocalyx, represents the most likely initiating step in diabetic microalbuminuria.     

Nephrin is specifically located at the slit diaphragm of glomerular podocytes

We describe here the size and location of nephrin, the first protein to be identified at the glomerular podocyte slit diaphragm. In Western blots, nephrin antibodies generated against the two terminal extracellular Ig domains of recombinant human nephrin recognized a 180-kDa protein in lysates of human glomeruli and a 150-kDa protein in transfected COS-7 cell lysates. In immunofluorescence, antibodies to this transmembrane protein revealed reactivity in the glomerular basement membrane region, whereas the podocyte cell bodies remained negative. In immunogold-stained thin sections, nephrin label was found at the slit between podocyte foot processes. The congenital nephrotic syndrome of the Finnish type (NPHS1), a disease in which the nephrin gene is mutated, is characterized by massive proteinuria already in utero and lack of slit diaphragm and foot processes. These features, together with the now demonstrated localization of nephrin to the slit diaphragm area, suggests an essential role for this protein in the normal glomerular filtration barrier. A zipper-like model for nephrin assembly in the slit diaphragm is discussed, based on the present and previous data.     

Podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and is reversed by aldosterone blocker

Recent clinical studies implicate proteinuria as a key prognostic factor for renal and cardiovascular complications in hypertensives. The pathogenesis of proteinuria in hypertension is, however, poorly elucidated. Podocytes constitute the final filtration barrier in the glomerulus, and their dysfunction may play a pivotal role in proteinuria. In the present study, we examined the involvement of podocyte injury in Dahl salt-hypertensive rats, an animal model prone to hypertensive glomerulosclerosis, and explored the effects of inhibition of aldosterone. Four-week-old Dahl salt-resistant and salt-sensitive rats were fed a 0.3% or 8.0% NaCl diet. Some salt-loaded Dahl salt-sensitive rats were treated with a selective aldosterone blocker eplerenone (1.25 mg/g diet) or hydralazine (0.5 mmol/L). After 6 weeks, salt-loaded Dahl salt-sensitive rats developed severe hypertension, proteinuria, and glomerulosclerosis. Immunostaining for nephrin, a constituent of slit diaphragm, was attenuated, whereas expressions of damaged podocyte markers desmin and B7-1 were upregulated in the glomeruli of salt-loaded Dahl salt-sensitive rats. Electron microscopic analysis revealed podocyte foot process effacement. Podocytes were already impaired at as early as 2 weeks of salt loading in Dahl salt-sensitive rats, when proteinuria was modestly increased. Both eplerenone and hydralazine partially reduced systemic blood pressure as measured by indirect and direct methods in salt-loaded Dahl salt-sensitive rats, but only eplerenone dramatically improved podocyte damage and retarded the progression of proteinuria and glomerulosclerosis. Our findings suggest that podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and that inhibition of aldosterone by eplerenone is protective against podocyte damage, proteinuria, and glomerulosclerosis in this hypertensive model.     

Podocytes as target of vitamin D

Vitamin D deficiency is a prominent feature of chronic kidney disease (CKD) even in its early stages. While vitamin D deficiency leads to mineral imbalance and bone problems in CDK patients, it also accelerates the progression of kidney disease. Ever since the observation that vitamin D analogs reduce proteinuria in CKD patients, it has been postulated that podocytes are major target of the reno-protective action of vitamin D. Recent large randomized clinical trials have confirmed the potent anti-proteinuric activity of vitamin D therapy. Studies from various animal models of kidney disease have demonstrated that vitamin D prevents podocyte injury and cell loss, promotes the expression of slit diaphragm proteins and maintains the integrity of the glomerular filtration barrier. Emerging experimental data suggest that vitamin D may protect podocytes by targeting multiple pathways, including the renin-angiotensin system, Wnt/β-catenin pathway and pro-apoptotic pathway.     

Molecular Mechanisms Involved in Intrarenal Renin-Angiotensin and Alternative Pathways in Diabetic Nephropathy - A Review

Uncontrolled or chronic hyperglycemia causes kidney failure induced by the dysfunction of biomolecules and upregulation of inflammatory cytokines and growth factors. The reninangiotensin system (RAS) is incorporated in the regulation of renal hemodynamics. In a healthy state, local RAS is independent of systemic RAS. However, in pathological conditions such as chronic hyperglycemia, angiotensin II (Ang II) increases locally and causes tissue damage, mainly through the induction of oxidative stress, inflammation, and upregulation of some growth factors and their receptors. Such tissue events may cause disruption of the glomerular filtration barrier, thickening and hypertrophy of the glomerular basement membrane, microvascular hyperpermeability, proteinuria, and finally decrease in the glomerular filtration rate (GFR). Reduced GFR causes the kidney to sense falsely a low blood pressure condition and respond to it by stimulating systemic and local RAS. Therefore, patients with diabetic nephropathy (DN) suffer from chronic hypertension. In contrast to local RAS, there are alternative pathways in the kidney that act protectively by reducing tissue Ang II. Such autoregulatory and protective mechanisms are weakened in chronic kidney disease. Previously, it was presumed that systemic RAS inhibitors such as ACE inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) could prevent renal damage by controlling blood pressure and proteinuria. However, the progression of renal failure to end-stage renal disease (ESRD), despite such treatments, indicates the presence of factors other than Ang II. This review highlights the molecular mechanism in renal disease and discusses pharmaceutical and therapeutic approaches.     

Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy

Microalbuminuria is the earliest detectable clinical abnormality in diabetic glomerulopathy. On a molecular level, metabolic pathways activated by hyperglycemia, glycated proteins, hemodynamic factors, and oxidative stress are key players in the genesis of diabetic kidney disease. A variety of growth factors and cytokines are then induced through complex signal transduction pathways. Transforming growth factor-beta 1 (TGF-beta1) has emerged as an important downstream mediator for the development of renal hypertrophy and the accumulation of mesangial extracellular matrix components, but there is limited evidence to support its role in the development of albuminuria. The loss of proteoglycans in the glomerular basement membrane (GBM) has been recently questioned as causative of the albuminuria, and current research has focused on the podocyte as a central target for the effects of the metabolic milieu in the development and progression of diabetic albuminuria. Podocyte-derived vascular endothelial growth factor (VEGF), a permeability and angiogenic factor whose expression is increased in diabetic kidney disease, is perhaps a major mediator of the increased protein filtration. Decreased podocyte number and/or density as a result of apoptosis or detachment, GBM thickening with altered matrix composition, and a reduction in nephrin protein in the slit diaphragm with podocyte foot process effacement, all comprise the principal features of diabetic podocytopathy that clinically manifests as albuminuria and proteinuria. Many of these events are mediated by angiotensin II whose local concentration is stimulated by high glucose, mechanical stretch, and proteinuria itself. Angiotensin II in turn stimulates podocyte-derived VEGF, suppresses nephrin expression, and induces TGF-beta1 leading to podocyte apoptosis and fostering the development of glomerulosclerosis. Proteinuria can then induce in tubular cells a genetic program leading to tubulointerstitial inflammation, fibrosis and tubular atrophy. Besides direct effects of albuminuria on tubular cells, pathophysiological changes in the ultrafiltration barrier lead to an increased tubular filtration of various growth factors (TGF-beta1, insulin-like growth factor I) that may further alter the function of tubular cells. Moreover, angiotensin II also stimulates uptake of ultrafiltered proteins into tubular cells and enhances the production of proinflammatory and profibrotic cytokines within the cells. Migration of macrophages and other inflammatory cells into the tubulointerstitium occurs. Increased synthesis and decreased turnover of extracellular matrix proteins in tubular cells and interstitial fibroblasts contribute to interstitial fibrosis. In addition, under locally high concentrations of angiotensin II and TGF-beta1, tubular cells may change their phenotype and become fibroblasts by a process called epithelial to mesenchymal transition (EMT) which contributes to interstitial fibrosis and tubular atrophy because of vanishing epithelia cells. An alternative explanation for the development of albuminuria in diabetic nephropathy that involves primarily an abnormality in tubular handling of ultrafiltered proteins has also been suggested, but these changes are not necessarily exclusive of the altered properties of glomerular ultrafiltration barrier.