Sumant S. Chugh

Diabetic Nephropathy : Mechanisms of Renal Disease Progression

Diabetic nephropathy is characterized by excessive amassing of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progress to glomerulosclerosis and tubulo-interstitial fibrosis. In view of this outcome, it would mean that all the kidney cellular elements, i.e., glomerular endothelia, mesangial cells, podocytes, and tubular epithelia, are targets of hyperglycemic injury. Conceivably, high glucose activates various pathways via similar mechanisms in different cell types of the kidney except for minor exceptions that are related to the selective expression of a given molecule in a particular renal compartment. To begin with, there is an obligatory excessive channeling of glucose intermediaries into various metabolic pathways with generation of advanced glycation products (AGEs), activation of protein kinase C (PKC), increased expression of transforming growth factor-β (TGF-β), GTP-binding proteins, and generation of reactive oxygen species (ROS). The ROS seem to be the common denominator in various pathways and are central to the pathogenesis of hyperglycemic injury. In addition, there are marked alterations in intraglomerular hemodynamics, i.e., hyperfiltration, and this along with metabolic derangements adversely compounds the hyperglycemia-induced injury. Here, the information compiled under various subtitles of this article is derived from an enormous amount of data summarized in several excellent literature reviews, and thus their further reading is suggested to gain in-depth knowledge of each of the subject matter.

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4−/− mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.

Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability

Neph1-deficient mice develop nephrotic syndrome at birth, indicating the importance of this protein in the development of a normal glomerular filtration barrier. While the precise subcellular localization of Neph1 remains unknown, its relationship with other components of the glomerular filtration barrier is of great interest in this field. In this paper, we localize the expression of Neph1 to the glomerular slit diaphragm by immunogold electron microscopy in rodents and describe its direct interaction with two other components of the slit diaphragm, nephrin and ZO-1. Both native and recombinant Neph1 associate with each other as dimers and multimers and interact with nephrin via their extracellular segments. Disruption of the Neph1-nephrin interaction in vivo by injecting combinations of individual subnephritogenic doses of anti-Neph1 and anti-nephrin results in complement- and leukocyte-independent proteinuria with preserved foot processes. This disruption modestly reduces Neph1 and nephrin protein expression in podocytes and dramatically reduces ZO-1 protein expression via the interaction of ZO-1 PDZ domains with the cytoplasmic tail of Neph1, independent of changes in mRNA expression of all three genes. The interaction between nephrin and Neph1 is specific and not shared by either protein with P-cadherin, another integral slit diaphragm protein. The interaction between nephrin and Neph1 therefore appears to be an important determinant of glomerular permeability.

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin.

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Circulating angiopoietin-like 4 links proteinuria with hypertriglyceridemia in nephrotic syndrome

The molecular link between proteinuria and hyperlipidemia in nephrotic syndrome is not known. We show in the present study that plasma angiopoietin-like 4 (Angptl4) links proteinuria with hypertriglyceridemia through two negative feedback loops. In previous studies in a rat model that mimics human minimal change disease, we observed localized secretion by podocytes of hyposialylated Angptl4, a pro-proteinuric form of the protein. But in this study we noted high serum levels of Angptl4 (presumably normosialylated based on a neutral isoelectric point) in other glomerular diseases as well. Circulating Angptl4 was secreted by extrarenal organs in response to an elevated plasma ratio of free fatty acids (FFAs) to albumin when proteinuria reached nephrotic range. In a systemic feedback loop, these circulating pools of Angptl4 reduced proteinuria by interacting with glomerular endothelial αvβ5 integrin. Blocking the Angptl4–β5 integrin interaction or global knockout of Angptl4 or β5 integrin delayed recovery from peak proteinuria in animal models. But at the same time, in a local feedback loop, the elevated extrarenal pools of Angptl4 reduced tissue FFA uptake in skeletal muscle, heart and adipose tissue, subsequently resulting in hypertriglyceridemia, by inhibiting lipoprotein lipase (LPL)-mediated hydrolysis of plasma triglycerides to FFAs. Injecting recombinant human ANGPTL4 modified at a key LPL interacting site into nephrotic Buffalo Mna and Zucker Diabetic Fatty rats reduced proteinuria through the systemic loop but, by bypassing the local loop, without increasing plasma triglyceride levels. These data show that increases in circulating Angptl4 in response to nephrotic-range proteinuria reduces the degree of this pathology, but at the cost of inducing hypertriglyceridemia, while also suggesting a possible therapy to treat these linked pathologies.

New Insights Into Human Minimal Change Disease: Lessons From Animal Models

The pathogenesis of minimal change disease (MCD), considered to be the simplest form of nephrotic syndrome, has been one of the major unsolved mysteries in kidney disease. In this review, recent landmark studies that have led to the unraveling of MCD are discussed. A recent study now explains the molecular basis of major clinical and morphologic changes in MCD. Overproduction of angiopoietin-like 4 (ANGPTL4) in podocytes in MCD causes binding of ANGPTL4 to the glomerular basement membrane, development of nephrotic-range selective proteinuria, diffuse effacement of foot processes, and loss of glomerular basement membrane charge, but is not associated with changes shown by light microscopy in the glomerular and tubulointerstitial compartments. At least some of this ability of ANGPTL4 to induce proteinuria is linked to a deficiency of sialic acid residues because oral supplementation with sialic acid precursor N-acetyl-d-mannosamine improves sialylation of podocyte-secreted ANGPTL4 and significantly decreases proteinuria. Animal models of MCD, recent advances in potential biomarkers, and studies of upstream factors that may initiate glomerular changes also are discussed. In summary, recent progress in understanding MCD is likely to influence the diagnosis and treatment of MCD in the near future.

High glucose stimulates synthesis of fibronectin via a novel protein kinase C, Rap1b, and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser → Asn) or T61R (Thr → Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.

Rap1b GTPase Ameliorates Glucose-Induced Mitochondrial Dysfunction

The role of tubular injury in diabetic nephropathy is relatively unknown, despite that apoptosis of tubular epithelial cells is commonly observed in human renal biopsies. The GTPase Ras-proximate-1 (Rap1b) is upregulated in the hyperglycemic state and is known to increase B-Raf, an antiapoptotic effector protein. In this study, the effects of high glucose on renal tubular apoptosis and the potential ability for Rap1b to ameliorate these effects were investigated. In the kidneys of diabetic mice, apoptotic tubular cells and dysmorphic mitochondria were observed, Bcl-2 expression was decreased, and Bax expression was increased. Total Rap1b expression was slightly increased, but its associated GTPase activity was significantly decreased. In vitro, high extracellular glucose led to decreased Bcl-2 expression, reduced Rap1b GTPase activity, and increased levels of both Bax and GTPase activating protein in a proximal tubular cell line (HK-2). These changes were accompanied by increased DNA fragmentation, decreased high molecular weight mitochondrial DNA, altered mitochondrial morphology and function, disrupted Bcl-2-Bax and Bcl-2-Rap1b interactions, and reduced cell survival. Overexpression of Rap1b partially prevents these abnormalities. Furthermore, the BH4 domain of Bcl-2 was found to be required for successful protein-protein interaction between Bcl-2 and Rap1b. In summary, these data suggest that Rap1b ameliorates glucose-induced mitochondrial dysfunction in renal tubular cells.

ZHX Proteins Regulate Podocyte Gene Expression during the Development of Nephrotic Syndrome

Transcriptional regulation of podocyte gene expression in primary glomerular disease is poorly understood. In this study, we demonstrate a prominent role of members of the ZHX (zinc fingers and homeoboxes) family of proteins in regulating podocyte gene expression during the development of nephrotic syndrome. While studying mechanisms of glomerular disease, rat ZHX3 was cloned from a down-regulated gene fragment; its cellular localization, DNA binding, and transcriptional repressor properties were characterized; and its ability to influence podocyte gene expression directly or via ZHX1 and ZHX2 was studied. In eukaryotic promoters, ZHX3 bound to the CdxA binding motif. ZHX proteins were mostly sequestered in the non-nuclear compartment in the normal in vivo podocyte by virtue of heterodimer formation, and loss of heterodimerization was associated with entry into the nucleus. In experimental minimal change disease, ZHX3 was transiently down-regulated prior to the onset of proteinuria, and recovery of expression was associated with migration of ZHX3 protein into the nucleus and the development of proteinuria. This expression pattern mirrored the increased nuclear ZHX3 expression noted in vivo in the podocytes in human minimal change disease biopsies. In vitro, migration of ZHX3 protein into the nucleus during recovery from transient ZHX3 knockdown reproduced the gene expression profile of in vivo minimal change disease. Severe sustained knockdown of ZHX3 caused down-regulation of genes involved in focal sclerosis, including WT1, mediated mostly by increased nuclear entry of ZHX2 and ZHX1. In summary, ZHX proteins are major transcriptional mediators of podocyte disease.

Hyperglycemia: its imminent effects on mammalian nephrogenesis

A sustained exposure of the mammalian embryo to very high glucose ambience is associated with a multitude of congenital birth defects, including those of the cardiovascular, CNS, skeletal and urogenital systems during the first 6–8 weeks of gestation in humans. These urogenital abnormalities may be associated with “caudal regression syndrome” or may occur alone in the form of partial or total renal agenesis. Similarly, an increase in the incidence of morphogenetic defects is observed in the offspring of streptozotocin-induced diabetic rats and mice, and also in non-obese diabetic mice. In certain cases, failure during the growth of the lower parts of embryos or newborn mice involving the genitourinary system has been observed in animals with severe diabetes. Investigators have utilized whole organ culture systems to delineate the mechanisms relevant to dysmorphogenesis of the embryonic metanephros. A marked dysmorphogenesis of the metanephros is observed upon treatment with a high concentration of d-glucose. Associated with it are changes that include branching dysmorphogenesis of the ureteric bud iterations, reduced population of nascent nephrons, decreased expression of basement membrane proteoglycans, depletion of ATP stores, and fulminant apoptosis of the cells at the interface of mesenchyme and ureteric bud epithelium. The latter findings suggest that disruption of epithelial:mesenchymal interactions may be the major event responsible for the metanephric dysmorphogenesis induced by high glucose ambience.