Non-Invasive Biomarkers for Diagnosis, Risk Prediction, and Therapy Guidance of Glomerular Kidney Diseases: A Comprehensive Review.

Effective management of glomerular kidney disease, one of the main categories of chronic kidney disease (CKD), requires accurate diagnosis, prognosis of progression, assessment of therapeutic efficacy, and, ideally, prediction of drug response. Multiple biomarkers and algorithms for the assessment of specific aspects of glomerular diseases have been reported in the literature. Though, the vast majority of these have not been implemented in clinical practice or are not available on a global scale due to limited access, missing medical infrastructure, or economical as well as political reasons. The aim of this review is to compile all currently available information on the diagnostic, prognostic, and predictive biomarkers currently available for the management of glomerular diseases, and provide guidance on the application of these biomarkers. As a result of the compiled evidence for the different biomarkers available, we present a decision tree for a non-invasive, biomarker-guided diagnostic path. The data currently available demonstrate that for the large majority of patients with glomerular diseases, valid biomarkers are available. However, despite the obvious disadvantages of kidney biopsy, being invasive and not applicable for monitoring, especially in the context of rare CKD etiologies, kidney biopsy still cannot be replaced by non-invasive strategies.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

CD4+ T cells produce GM-CSF and drive immune-mediated glomerular disease by licensing monocyte-derived cells to produce MMP12.

GM-CSF in glomerulonephritisDespite glomerulonephritis being an immune-mediated disease, the contributions of individual immune cell types are not clear. To address this gap in knowledge, Paust et al. characterized pathological immune cells in samples from patients with glomerulonephritis and in samples from mice with the disease. The authors found that CD4+ T cells producing granulocyte-macrophage colony-stimulating factor (GM-CSF) licensed monocytes to promote disease by producing matrix metalloproteinase 12 and disrupting the glomerular basement membrane. Targeting GM-CSF to inhibit this axis reduced disease severity in mice, implicating this cytokine as a potential therapeutic target for patients with glomerulonephritis. -CM.

Studies on the Role of the Transcription Factor Tcf21 in the Transdifferentiation of Parietal Epithelial Cells into Podocyte-Like Cells.

BACKGROUND/AIMS: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. METHODS: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. RESULTS: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin­1, ß-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. CONCLUSION: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Effects of Environmental Conditions on Nephron Number: Modeling Maternal Disease and Epigenetic Regulation in Renal Development.

A growing body of evidence suggests that low nephron numbers at birth can increase the risk of chronic kidney disease or hypertension later in life. Environmental stressors, such as maternal malnutrition, medication and smoking, can influence renal size at birth. Using metanephric organ cultures to model single-variable environmental conditions, models of maternal disease were evaluated for patterns of developmental impairment. While hyperthermia had limited effects on renal development, fetal iron deficiency was associated with severe impairment of renal growth and nephrogenesis with an all-proximal phenotype. Culturing kidney explants under high glucose conditions led to cellular and transcriptomic changes resembling human diabetic nephropathy. Short-term high glucose culture conditions were sufficient for long-term alterations in DNA methylation-associated epigenetic memory. Finally, the role of epigenetic modifiers in renal development was tested using a small compound library. Among the selected epigenetic inhibitors, various compounds elicited an effect on renal growth, such as HDAC (entinostat, TH39), histone demethylase (deferasirox, deferoxamine) and histone methyltransferase (cyproheptadine) inhibitors. Thus, metanephric organ cultures provide a valuable system for studying metabolic conditions and a tool for screening for epigenetic modifiers in renal development.

Endothelial-Specific Deletion of CD146 Protects Against Experimental Glomerulonephritis in Mice.

[Figure: see text].

C-C chemokine receptor type 2 mediates glomerular injury and interstitial fibrosis in focal segmental glomerulosclerosis.

BACKGROUND: Glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of chronic kidney injury leading to end-stage renal disease. Inflammatory mechanisms contribute to glomerular and interstitial scarring, including chemokine-mediated recruitment of leucocytes. In particular, accumulation of C-C chemokine receptor type 2 (CCR2)-expressing macrophages promotes renal injury and fibrotic remodelling in diseases like glomerulonephritis and diabetic nephropathy. The functional role of CCR2 in the initiation and progression of primary glomerulosclerosis induced by podocyte injury remains to be characterized. METHODS: We analysed glomerular expression of CCR2 and its chemokine ligand C-C motif chemokine ligand 2 (CCL2) in human focal segmental glomerulosclerosis (FSGS). Additionally, CCL2 expression was determined in stimulated murine glomeruli and glomerular cells in vitro. To explore pro-inflammatory and profibrotic functions of CCR2 we induced adriamycin nephropathy, a murine model of FSGS, in BALB/c wild-type and Ccr2-deficient mice. RESULTS: Glomerular expression of CCR2 and CCL2 significantly increased in human FSGS. In adriamycin-induced FSGS, progressive glomerular scarring and reduced glomerular nephrin expression was paralleled by induced glomerular expression of CCL2. Adriamycin exposure stimulated secretion of CCL2 and tumour necrosis factor-α (TNF) in isolated glomeruli and mesangial cells and CCL2 in parietal epithelial cells. In addition, TNF induced CCL2 expression in all glomerular cell populations, most prominently in podocytes. In vivo, Ccr2-deficient mice with adriamycin nephropathy showed reduced injury, macrophage and fibrocyte infiltration and inflammation in glomeruli and the tubulointerstitium. Importantly, glomerulosclerosis and tubulointerstitial fibrosis were significantly ameliorated. CONCLUSIONS: Our data indicate that CCR2 is an important mediator of glomerular injury and progression of FSGS. CCR2- targeting therapies may represent a novel approach for its treatment.

mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

A Novel Role for GATA3 in Mesangial Cells in Glomerular Development and Injury.

BACKGROUND: GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury. METHODS: To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/-) knockout mice, as well as injured human and rodent kidneys. RESULTS: We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis. CONCLUSIONS: These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.

Interleukin-1ß Inhibition for Chronic Kidney Disease in Obese Mice With Type 2 Diabetes.

Inflammasome-driven release of interleukin(IL)-1ß is a central element of many forms of sterile inflammation and has been evident to promote the onset and progression of diabetic kidney disease. We microdissected glomerular and tubulointerstitial samples from kidney biopsies of patients with diabetic kidney disease and found expression of IL-1ß mRNA. Immunostaining of such kidney biopsies across a broad spectrum of diabetic kidney disease stages revealed IL-1ß positivity in a small subset of infiltrating immune cell. Thus, we speculated on a potential of IL-1ß as a therapeutic target and neutralizing the biological effects of murine IL-1ß with a novel monoclonal antibody in uninephrectomized diabetic db/db mice with progressive type 2 diabetes- and obesity-related single nephron hyperfiltration, podocyte loss, proteinuria, and progressive decline of total glomerular filtration rate (GFR). At 18 weeks albuminuric mice were randomized to intraperitoneal injections with either anti-IL-1ß or control IgG once weekly for 8 weeks. During this period, anti-IL-1ß IgG had no effect on food or fluid intake, body weight, and fasting glucose levels. At week 26, anti-IL-1ß IgG had reduced renal mRNA expression of kidney injury markers (Ngal) and fibrosis (Col1, a-Sma), significantly attenuated the progressive decline of GFR in hyperfiltrating diabetic mice, and preserved podocyte number without affecting albuminuria or indicators of single nephron hyperfiltration. No adverse effect were observed. Thus, IL-1ß contributes to the progression of chronic kidney disease in type 2 diabetes and might therefore be a valuable therapeutic target, potentially in combination with drugs with different mechanisms-of-action such as RAS and SGLT2 inhibitors.

Exclusive expression of transmembrane TNF aggravates acute glomerulonephritis despite reduced leukocyte infiltration and inflammation.

Tumor necrosis factor-α (TNF) is a cytokine mediating inflammatory kidney diseases such as immune complex glomerulonephritis. Its two receptors, TNFR1 and TNFR2, play distinct roles in this process, with TNFR2 strongly required for induction of disease. In contrast to soluble TNF (sTNF), transmembrane TNF robustly activates TNFR2. Thus, we examined the functional role of transmembrane TNF by inducing heterologous nephrotoxic serum nephritis in wild-type and transgenic TNFΔ1-9,K11E knock-in mice expressing transmembrane TNF but no sTNF (memTNF mice). Compared to wild-type, nephritis was exacerbated in memTNF mice on day 5, indicated by increased albuminuria, higher serum urea levels, and more pronounced glomerular deposits, together with higher numbers of dying and proliferating glomerular cells. This was associated with greater loss of glomerular endothelial cells, increased podocyte stress, and signs of augmented necroptosis in memTNF kidneys. Aggravation of nephritis was dependent on transmembrane TNF expression in parenchymal cells, but not leukocytes. Surprisingly, increased kidney injury was associated with reduced renal leukocyte infiltration in memTNF mice, which correlated with decreased renal mRNA expression of pro-inflammatory mediators. This effect was also present in isolated memTNF glomeruli stimulated with interleukin-1ß in vitro. Thus, uncleaved transmembrane TNF is an important mediator of renal tissue damage characterized by increased renal cell death and loss of glomerular endothelial cells in murine glomerulonephritis. In contrast, sTNF predominantly mediates renal leukocyte recruitment and inflammation. These findings highlight the importance of transmembrane TNF in inflammatory kidney disease as a possible therapeutic target.

Metabolic pathways and immunometabolism in rare kidney diseases.

OBJECTIVES: To characterise renal tissue metabolic pathway gene expression in different forms of glomerulonephritis. METHODS: Patients with nephrotic syndrome (NS), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), systemic lupus erythematosus (SLE) and healthy living donors (LD) were studied. Clinically indicated renal biopsies were obtained at time of diagnosis and microdissected into glomerular and tubulointerstitial compartments. Microarray-derived differential gene expression of 88 genes representing critical enzymes of metabolic pathways and 25 genes related to immune cell markers was compared between disease groups. Correlation analyses measured relationships between metabolic pathways, kidney function and cytokine production. RESULTS: Reduced steady state levels of mRNA species were enriched in pathways of oxidative phosphorylation and increased in the pentose phosphate pathway (PPP) with maximal perturbation in AAV and SLE followed by NS, and least in LD. Transcript regulation was isozymes specific with robust regulation in hexokinases, enolases and glucose transporters. Intercorrelation networks were observed between enzymes of the PPP (eg, transketolase) and macrophage markers (eg, CD68) (r=0.49, p<0.01). Increased PPP transcript levels were associated with reduced glomerular filtration rate in the glomerular (r=-0.49, p<0.01) and tubulointerstitial (r=-0.41, p<0.01) compartments. PPP expression and tumour necrosis factor activation were tightly co-expressed (r=0.70, p<0.01). CONCLUSION: This study demonstrated concordant alterations of the renal transcriptome consistent with metabolic reprogramming across different forms of glomerulonephritis. Activation of the PPP was tightly linked with intrarenal macrophage marker expression, reduced kidney function and increased production of cytokines. Modulation of glucose metabolism may offer novel immune-modulatory therapeutic approaches in rare kidney diseases.

Lymphotoxin expression in human and murine renal allografts.

The kidney is the most frequently transplanted solid organ. Recruitment of inflammatory cells, ranging from diffuse to nodular accumulations with defined microarchitecture, is a hallmark of acute and chronic renal allograft injury. Lymphotoxins (LTs) mediate the communication of lymphocytes and stromal cells and play a pivotal role in chronic inflammation and formation of lymphoid tissue. The aim of this study was to assess the expression of members of the LT system in acute rejection (AR) and chronic renal allograft injury such as transplant glomerulopathy (TG) and interstitial fibrosis/tubular atrophy (IFTA). We investigated differentially regulated components in transcriptomes of human renal allograft biopsies. By microarray analysis, we found the upregulation of LTß, LIGHT, HVEM and TNF receptors 1 and 2 in AR and IFTA in human renal allograft biopsies. In addition, there was clear evidence for the activation of the NFκB pathway, most likely a consequence of LTß receptor stimulation. In human renal allograft biopsies with transplant glomerulopathy (TG) two distinct transcriptional patterns of LT activation were revealed. By quantitative RT-PCR robust upregulation of LTα, LTß and LIGHT was shown in biopsies with borderline lesions and AR. Immunohistochemistry revealed expression of LTß in tubular epithelial cells and inflammatory infiltrates in transplant biopsies with AR and IFTA. Finally, activation of LT signaling was reproduced in a murine model of renal transplantation with AR. In summary, our results indicate a potential role of the LT system in acute renal allograft rejection and chronic transplant injury. Activation of the LT system in allograft rejection in rodents indicates a species independent mechanism. The functional role of the LT system in acute renal allograft rejection and chronic injury remains to be determined.

Renal biopsy-driven molecular target identification in glomerular disease.

Chronic kidney disease has severe impacts on the patient and represents a major burden to the health care systems worldwide. Despite an increased knowledge of pathophysiological processes involved in kidney diseases, the progress in defining novel treatment strategies has been limited. One reason is the descriptive disease categorization used in nephrology based on clinical findings or histopathological categories irrespective of potential different molecular disease mechanisms. To accelerate progress toward a targeted treatment, a definition of human disease extending from phenotypic disease classification to mechanism-based disease definitions is needed. In recent years, we have witnessed a major transition in biomedical research from a single gene research to an information rich and collaborative science. Tissue-based analysis in renal disease allows to link structure to molecular function. In our review, we introduce the concept of precision medicine in nephrology, describe several large cohort studies established for molecular analysis of kidney diseases, and highlight examples of renal biopsy-driven target identification by integrative systems biology approaches. Furthermore, we give an outlook on how the new disease definitions can be used for patient stratification in clinical trial design. Finally, we introduce the concept of an informational commons of renal precision medicine for joint analyses of large-scale data sets in renal failure.

Loss of endogenous thymosin ß4 accelerates glomerular disease.

Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin ß4 regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin ß4 improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin ß4 in the kidney is unknown. We demonstrate that thymosin ß4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin ß4 did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin ß4 in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin ß4 in the migration of these cells. Thymosin ß4 knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin ß4 is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression.

Keratins are novel markers of renal epithelial cell injury.

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros.

UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation.

Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN.