"Idiopathic" minimal change nephrotic syndrome: a podocyte mystery nears the end.

The discovery of zinc fingers and homeoboxes (ZHX) transcriptional factors and the upregulation of hyposialylated angiopoietin-like 4 (ANGPTL4) in podocytes have been crucial in explaining the cardinal manifestations of human minimal change nephrotic syndrome (MCNS). Recently, uncovered genomic defects upstream of ZHX2 induce a ZHX2 hypomorph state that makes podocytes inherently susceptible to mild cytokine storms resulting from a common cold. In ZHX2 hypomorph podocytes, ZHX proteins are redistributed away from normal transmembrane partners like aminopeptidase A (APA) toward alternative binding partners like IL-4Rα. During disease relapse, high plasma soluble IL-4Rα (sIL-4Rα) associated with chronic atopy complements the cytokine milieu of a common cold to displace ZHX1 from podocyte transmembrane IL-4Rα toward the podocyte nucleus. Nuclear ZHX1 induces severe upregulation of ANGPTL4, resulting in incomplete sialylation of part of the ANGPTL4 protein, secretion of hyposialylated ANGPTL4, and hyposialylation-related injury in the glomerulus. This pattern of injury induces many of the classic manifestations of human minimal change disease (MCD), including massive and selective proteinuria, podocyte foot process effacement, and loss of glomerular basement membrane charge. Administration of glucocorticoids reduces ANGPTL4 upregulation, which reduces hyposialylation injury to improve the clinical phenotype. Improving sialylation of podocyte-secreted ANGPTL4 also reduces proteinuria and improves experimental MCD. Neutralizing circulating TNF-α, IL-6, or sIL-4Rα after the induction of the cytokine storm in Zhx2 hypomorph mice reduces albuminuria, suggesting potential new therapeutic targets for clinical trials to prevent MCD relapse. These studies collectively lay to rest prior suggestions of a role of single cytokines or soluble proteins in triggering MCD relapse.

Cytokine storm-based mechanisms for extrapulmonary manifestations of SARS-CoV-2 infection.

Viral illnesses like SARS-CoV-2 have pathologic effects on nonrespiratory organs in the absence of direct viral infection. We injected mice with cocktails of rodent equivalents of human cytokine storms resulting from SARS-CoV-2/COVID-19 or rhinovirus common cold infection. At low doses, COVID-19 cocktails induced glomerular injury and albuminuria in zinc fingers and homeoboxes 2 (Zhx2) hypomorph and Zhx2+/+ mice to mimic COVID-19-related proteinuria. Common Cold cocktail induced albuminuria selectively in Zhx2 hypomorph mice to model relapse of minimal change disease, which improved after depletion of TNF-α, soluble IL-4Rα, or IL-6. The Zhx2 hypomorph state increased cell membrane to nuclear migration of podocyte ZHX proteins in vivo (both cocktails) and lowered phosphorylated STAT6 activation (COVID-19 cocktail) in vitro. At higher doses, COVID-19 cocktails induced acute heart injury, myocarditis, pericarditis, acute liver injury, acute kidney injury, and high mortality in Zhx2+/+ mice, whereas Zhx2 hypomorph mice were relatively protected, due in part to early, asynchronous activation of STAT5 and STAT6 pathways in these organs. Dual depletion of cytokine combinations of TNF-α with IL-2, IL-13, or IL-4 in Zhx2+/+ mice reduced multiorgan injury and eliminated mortality. Using genome sequencing and CRISPR/Cas9, an insertion upstream of ZHX2 was identified as a cause of the human ZHX2 hypomorph state.

The zinc fingers and homeoboxes 2 protein ZHX2 and its interacting proteins regulate upstream pathways in podocyte diseases.

Zinc fingers and homeoboxes (ZHX) proteins are heterodimeric transcriptional factors largely expressed at the cell membrane in podocytes in vivo. We found ZHX2-based heterodimers in podocytes, with ZHX2-ZHX1 predominantly at the cell membrane of the podocyte cell body, and ZHX2-ZHX3 at the slit diaphragm. In addition to changes in overall ZHX2 expression, there was increased podocyte nuclear ZHX3 and ZHX2 in patients with focal segmental glomerulosclerosis, and increased podocyte nuclear ZHX1 in patients with minimal change disease. Zhx2 deficient mice had increased podocyte ZHX1 and ZHX3 expression. Zhx2 deficient mice and podocyte specific Zhx2 overexpressing transgenic rats develop worse experimental focal segmental glomerulosclerosis than controls, with increased nuclear ZHX3 and ZHX2, respectively. By contrast, podocyte specific Zhx2 overexpressing transgenic rats develop lesser proteinuria during experimental minimal change disease due to peripheral sequestration of ZHX1 by ZHX2. Using co-immunoprecipitation, the interaction of ZHX2 with aminopeptidase A in the podocyte body cell membrane, and EPHRIN B1 in the slit diaphragm were noted to be central to upstream events in animal models of minimal change disease and focal segmental glomerulosclerosis, respectively. Mice deficient in Enpep, the gene for aminopeptidase A, and Efnb1, the gene for ephrin B1 developed worse albuminuria in glomerular disease models. Targeting aminopeptidase A in Zhx2 deficient mice with monoclonal antibodies induced albuminuria and upregulation of the minimal change disease mediator angiopoietin-like 4 through nuclear entry of ZHX1. Thus, podocyte ZHX2 imbalance is a critical factor in human glomerular disease, with minimal change disease disparities mediated mostly through ZHX1, and focal segmental glomerulosclerosis deviations through ZHX3 and ZHX2.

Novel therapeutic approaches for chronic kidney disease due to glomerular disorders.

Improved understanding of glomerular disease mechanisms over the past decade has led to the emergence of new and targeted therapeutic strategies for chronic kidney disease (CKD). Most promising among these are the administration of recombinant mutated human angiopoietin-like 4, sialic acid-related sugars that induce sialylation in vivo, compounds related to Bis-T-23, and immune depletion of the soluble urokinase receptor from the circulation. Taking these therapeutic strategies into clinical trials will be the first step away from repurposed and relatively toxic drugs currently used for treating kidney disease.

The proteinuria-hypertriglyceridemia connection as a basis for novel therapeutics for nephrotic syndrome.

The development of new and specific treatment options for kidney disease in general and glomerular diseases in specific has lagged behind other fields like heart disease and cancer. As a result, nephrologists have had to test and adapt therapeutics developed for other indications to treat glomerular diseases. One of the major factors contributing to this inertia has been the poor understanding of disease mechanisms. One way to elucidate these disease mechanisms is to study the association between the cardinal manifestations of glomerular diseases. Because many of these patients develop nephrotic syndrome, understanding the relationship of proteinuria, the primary driver in this syndrome, with hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, edema, and lipiduria could provide valuable insight. The recent unraveling of the relationship between proteinuria and hypertriglyceridemia mediated by free fatty acids, albumin, and the secreted glycoprotein angiopoietin-like 4 (Angptl4) offers a unique opportunity to develop novel therapeutics for glomerular diseases. In this review, the therapeutic potential of mutant forms of Angptl4 in reducing proteinuria and, as a consequence, alleviating the other manifestations of nephrotic syndrome is discussed.

Nephrotic syndrome: components, connections, and angiopoietin-like 4-related therapeutics.

Nephrotic syndrome is recognized by the presence of proteinuria in excess of 3.5 g/24 h along with hypoalbuminemia, edema, hyperlipidemia (hypertriglyceridemia and hypercholesterolemia), and lipiduria. Each component has been investigated individually over the past four decades with some success. Studies published recently have started unraveling the molecular basis of proteinuria and its relationship with other components. We now have improved understanding of the threshold for nephrotic-range proteinuria and the pathogenesis of hypertriglyceridemia. These studies reveal that modifying sialylation of the soluble glycoprotein angiopoietin-like 4 or changing key amino acids in its sequence can be used successfully to treat proteinuria. Treatment strategies on the basis of fundamental relationships among different components of nephrotic syndrome use naturally occurring pathways and have great potential for future development into clinically relevant therapeutic agents.

Glomerular disease: looking beyond pathology.

The National Institute of Diabetes and Digestive and Kidney Diseases-supported Kidney Research National Dialogue asked the scientific community to formulate and prioritize research objectives aimed at improved understanding of kidney function and disease progression. Over the past 2 years, 1600 participants posted almost 300 ideas covering all areas of kidney disease. An overriding theme that evolved through these discussions is the need to move beyond pathology to take advantage of basic science and clinical research opportunities to improve diagnostic classification and therapeutic options for people with primary glomerular disease. High-priority research areas included focus on therapeutic targets in glomerular endothelium and podocytes, regenerating podocytes through developmental pathways, use of longitudinal phenotypically defined disease cohorts to improve classification schemes, identifying biomarkers, disease-specific therapeutics, autoantibody triggers, and changing the clinical research culture to promote participation in clinical trials. Together, these objectives provide a path forward for improving clinical outcomes of glomerular disease.

Angiopoietin-like 4 based therapeutics for proteinuria and kidney disease.

Current drugs used to treat proteinuric disorders of the kidney have been borrowed from other branches of medicine, and are only partially effective. The discovery of a central, mechanistic role played by two different forms of the secreted glycoprotein angiopoietin-like 4 (Angptl4) in human and experimental glomerular disease has opened new treatment avenues. Localized upregulation of a hyposialylated form (lacks sialic acid residues) of Angptl4 secreted by podocytes induces the cardinal morphological and clinical manifestations of human minimal change disease, and is also being increasingly recognized as a significant contributor toward proteinuria in experimental diabetic nephropathy. Oral treatment with low doses of N-acetyl-D-mannosamine, a naturally occurring precursor of sialic acid, improves sialylation of Angptl4 in vivo, and reduces proteinuria by over 40%. By contrast, a sialylated circulating form of Angptl4, mostly secreted from skeletal muscle, heart and adipose tissue in all major primary glomerular diseases, reduces proteinuria while also causing hypertriglyceridemia. Intravenous administration of recombinant human Angptl4 mutated to avoid hypertriglyceridemia and cleavage has remarkable efficacy in reducing proteinuria by as much as 65% for 2 weeks after a single low dose. Both interventions are mechanistically relevant, utilize naturally occurring pathways, and represent new generation therapeutic agents for chronic kidney disease related to glomerular disorders.

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 αvß5 integrin. Blocking the Angptl4-ß5 integrin interaction or global knockout of Angptl4 or ß5 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.

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury.

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.

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.

HIV-associated nephropathy: experimental models.

Since 1984 reports of renal involvement in AIDS patients have been presented in the literature. Different forms of renal disease were noted in the AIDS population including those related to systemic and local renal infections, tubulointerstitial disease, renal involvement by neoplasm and glomerular disease including collapsing glomerulopathy (CG). HIV-associated nephropathy (HIVAN) has been demonstrated to be more severe in the black population. HIVAN is the most common cause of renal failure in HIV-1-seropositive patients. The term HIVAN is reserved for the typical histopathological form of focal and segmental glomerulosclerosis (FSGS) characterized by the findings of coexistent glomerular and severe tubulointerstitial disease. In both humans and the murine model, glomerular lesions include FSGS, glomerular collapse and podocyte hyperplasia. The tubulointerstitial damage as well as the glomerular collapse can also be seen in non-HIV primary collapsing GN, raising the question of common mechanisms to HIV and other non-identified viral agents related to the development of the disease. Although controversial, increasing evidence supports a direct effect of the virus on renal cells either as a result of exposure to viral proteins or direct renal parenchyma infection. The use of a HIV-1 transgenic mouse model has demonstrated a direct etiologic link between HIV-1 expression in kidney and the development of HIVAN with unique viral-host interactions, which depend at the same time on stimulating features of the virus and the individual nature of the host response. The infection of renal cells by HIV-1 could be detected by reverse transcription-polymerase chain reaction (RT-PCR) of gag RNA at a low level. Some studies using an HIV-1 transgenic mouse model have demonstrated that expression of HIV- 1 in the kidney is required for the development of HIVAN. The final common pathway in the development of HIV-associated nephropathy is likely to involve alterations in the patterns of gene expression of renal parenchyma cells by cytokines and growth factors, leading to interstitial fibrosis and enhanced glomerular matrix synthesis. The nature of the host response to viral infection is critical to the development of nephropathy.HLA-linked responses particular to a subset of blacks may explain some of the epidemiologic features of HIVAN. There may also be biological heterogeneity in the strains of HIV-1 that could account for a particular renotropic strain. HIV strains from different parts of the world may vary by as much as 15% at the level of nucleotide sequence. The infectivity of human immunodeficiency virus (HIV-1) in human glomerular cells has been evaluated by exposing homogeneous cultures of human glomerular capillary endothelial, mesangial and epithelial cells to HIV in vitro. The mechanism of access of HIV into glomerular endothelial and mesangial cells is unknown up to now; HIV is generally infectious for cells expressing the CD4 antigen in their cell membrane. Other modes of HIV entry into cells independent of the CD4 receptor are possible through mechanisms involving Fc-receptors or coinfection with other enveloped viruses such as HTLV-l. Our understanding of the pathogenesis of HIVAN has been aided by the development of a transgenic model. The curious fact that only 3 of 8 founded transgenic lines developed nephropathy emphasizes that the expression of viral gene products per se is not sufficient to produce nephropathy. Human renal epithelium does not express CD4 receptors and in vitro attempts to infect glomerular epithelial cells using laboratory strains of HIV-1 have proven fruitless. The striking morphologic and phenotypic similarities between HIVAN and collapsing idiopathic FSGS raise the question whether the altered podocyte gene expression in collapsing idiopathic FSGS may also be due to a viral infection. This hypothesis is further supported by de novo occurrence of collapsing idiopathic FSGS in immunosuppressed renal transplantation patients and by epidemiologic data. In conclusion, there are likely to be common mechanisms in the pathogenesis for collapsing idiopathic glomerulosclerosis and HIVAN. A primary injury of the podocyte leading to dysregulation of the cellular phenotype appears to mediate the glomerular tuft collapse in both conditions. Primary collapsing glomerulopathy recurs post-transplantation, raising the possibility of circulating factors implicated in the pathogenesis of visceral epithelial cell damage in steroid-resistant minimal change disease or recurrent FSGS. Recurrence of CG can occur hours after transplantation, suggesting that the plasma of CG patients contains one or more factors capable of inducing proteinuria due to the damage of the podocyte that results in the increase in glomerular permeability. In a rat model of CG developed by our group, the injection of serum from CG patients resulted in proteinuria, glomerular tuft retraction and podocyte damage at the ultrastructural level (visceral epithelial cell foot-process effacement). No ultrastructural or light microscopy abnormalities were seen in rats injected with serum from non-collapsing FSGS or healthy subjects. Based on the experience of our group, circulating factors play a dominant role in the pathogenesis of idiopathic CG.

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.

A case of infection-associated antiproteinase-3-negative cytoplasmic antineutrophil cytoplasmic antibody pauci-immune focal necrotizing glomerulonephritis.

We present the case of a man with Gram-negative sepsis and exposure to oral silica who developed pauci-immune focal necrotizing glomerulonephritis (PI-FNGN) in the setting of a subacute polymicrobial central venous line (CVL) infection. He developed a cytoplasmic antineutrophil cytoplasmic autoantibody (C-ANCA) that was antiproteinase-3 (PR-3) and antimyeloperoxidase (MPO) antibody negative. We believe this is the first reported case of Gram-negative sepsis-associated PI-FNGN. Chronic silica exposure is a leading environmental risk factor in the development of ANCA vasculitis. Oral silica is a common pharmaceutical additive and its bioavailability is being recognized. Oral silica, therefore, may also be a risk for development of autoreactivity. The PI-FNGN resolved with antibiotic therapy alone. The C-ANCA titer declined as the PI-FNGN resolved. The case supports experimental and observational research that environmental exposures act as adjuvants for an immune response and also provide epigenetic triggers for autoreactivity. The C-ANCA was negative for PR-3, its major antigen. C-ANCA antigen specificity may depend on the pathogenesis of the underlying disease, potentially elicited by a cross-reaction of an antibody to foreign and self target antigen sequence homology or alternatively elicited by antigenic epitope spread.

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.