Podocytes Research Articles

Role of fibroblast specific protein 1 expression in the progression of adriamycin-induced glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a commonly occurring cause of steroid-resistant nephrotic syndrome and frequently progresses to renal failure. Podocyte epithelial-mesenchymal transition (EMT) is thought to induce podocyte detachment in glomerular diseases, and severe degeneration and shedding of glomerular podocytes plays a major role in the progression of FSGS. We showed that fibroblast specific protein 1 (FSP1), an EMT marker, is strongly expressed in podocytes of FSGS patients, but the significance of podocyte expression of FSP1 to the pathophysiology of FSGS remained unclear. Here, we investigated FSP1 expression in podocytes from mice with adriamycin (ADR)-induced nephropathy, a murine model of FSGS. The number of FSP1-positive (FSP1+) podocytes was increased in ADR-treated mice and positively correlated with the degree of proteinuria and glomerulosclerosis in ADR-treated mice. ADR-induced FSGS and the attendant proteinuria were significantly ameliorated in FSP1 knockout mice as compared to wild type mice. These findings indicate that podocyte expression of FSP1 plays a crucial role in the pathogenesis of FSGS, which makes FSP1 a potential target for treatment of FSGS.

Glomerular Endothelial Cells Are the Coordinator in the Development of Diabetic Nephropathy.

The prevalence of diabetes is consistently rising worldwide. Diabetic nephropathy is a leading cause of chronic renal failure. The present study aimed to explore the crosstalk among the different cell types inside diabetic glomeruli, including glomerular endothelial cells, mesangial cells, podocytes, and immune cells, by analyzing an online single-cell RNA profile (GSE131882) of patients with diabetic nephropathy. Differentially expressed genes in the glomeruli were processed by gene enrichment and protein-protein interactions analysis. Glomerular endothelial cells, as well as podocytes, play a critical role in diabetic nephropathy. A subgroup of glomerular endothelial cells possesses characteristic angiogenesis genes, indicating that angiogenesis takes place in the progress of diabetic nephropathy. Immune cells such as macrophages, T lymphocytes, B lymphocytes, and plasma cells also contribute to the disease progression. By using iTALK, the present study reports complicated cellular crosstalk inside glomeruli. Dysfunction of glomerular endothelial cells and immature angiogenesis result from the activation of both paracrine and autocrine signals. The present study reinforces the importance of glomerular endothelial cells in the development of diabetic nephropathy. The exploration of the signaling pathways involved in aberrant angiogenesis reported in the present study shed light on potential therapeutic target(s) for diabetic nephropathy.

Degree of foot process effacement in patients with genetic focal segmental glomerulosclerosis: a single-center analysis and review of the literature

Determining the cause of focal segmental glomerulosclerosis (FSGS) has crucial implications for evaluating the risk of posttransplant recurrence. The degree of foot process effacement (FPE) on electron micrographs (EM) of native kidney biopsies can reportedly differentiate primary FSGS from secondary FSGS. However, no systematic evaluation of FPE in genetic FSGS has been performed. In this study, percentage of FPE and foot process width (FPW) in native kidney biopsies were analyzed in eight genetic FSGS patients and nine primary FSGS patients. All genetic FSGS patients showed segmental FPE up to 38% and FPW below 2000 nm, while all primary FSGS patients showed diffuse FPE above 88% and FPW above 3000 nm. We reviewed the literature which described the degree of FPE in genetic FSGS patients and identified 38 patients with a description of the degree of FPE. The degree of FPE in patients with mutations in the genes encoding proteins associated with slit diaphragm and cytoskeletal proteins was varied, while almost all patients with mutations in other FSGS genes showed segmental FPE. In conclusion, the present study suggests that the degree of FPE in native kidney biopsies may be useful for differentiating some genetic FSGS patients from primary FSGS patients.

DACH1 protects podocytes and tubules from injury.

DACH1 protects podocytes and tubules from injury.

Modeling the Glomerular Filtration Barrier and Intercellular Crosstalk.

The glomerulus is a compact cluster of capillaries responsible for blood filtration and initiating urine production in the renal nephrons. A trilaminar structure in the capillary wall forms the glomerular filtration barrier (GFB), composed of glycocalyx-enriched and fenestrated endothelial cells adhering to the glomerular basement membrane and specialized visceral epithelial cells, podocytes, forming the outermost layer with a molecular slit diaphragm between their interdigitating foot processes. The unique dynamic and selective nature of blood filtration to produce urine requires the functionality of each of the GFB components, and hence, mimicking the glomerular filter in vitro has been challenging, though critical for various research applications and drug screening. Research efforts in the past few years have transformed our understanding of the structure and multifaceted roles of the cells and their intricate crosstalk in development and disease pathogenesis. In this review, we present a new wave of technologies that include glomerulus-on-a-chip, three-dimensional microfluidic models, and organoids all promising to improve our understanding of glomerular biology and to enable the development of GFB-targeted therapies. Here, we also outline the challenges and the opportunities of these emerging biomimetic systems that aim to recapitulate the complex glomerular filter, and the evolving perspectives on the sophisticated repertoire of cellular signaling that comprise the glomerular milieu.

The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes.

Podocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear. Genetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins. The study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins. Given the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance our understanding of SD regulation.

Activation of Rac1-Mineralocorticoid Receptor Pathway Contributes to Renal Injury in Salt-Loaded db/db Mice.

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Determination of the dynamic cellular transcriptional profiles during kidney development from birth to maturity in rats by single-cell RNA sequencing

Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into the gene expression profiles in early-stage kidney development. However, comprehensive gene expression profiles from mid- and late-stage kidney development are lacking. In the present study, by using the scRNA-seq technique, we analyzed 54,704 rat kidney cells from just after birth to adulthood (six time points: postnatal days 0, 2, 5, 10, 20, and 56) including the mid and late stages of kidney development. Twenty-five original clusters and 13 different cell types were identified during these stages. Gene expression in these 13 cell types was mapped, and single cell atlas of the rat kidney from birth to maturity (http://youngbearlab.com) was built to enable users to search for a gene of interest and to evaluate its expression in different cells. The variation trend of six major types of kidney cells—intercalated cells of the collecting duct (CD-ICs), principal cells of the collecting duct (CD-PCs), cells of the distal convoluted tubules (DCTs), cells of the loop of Henle (LOH), podocytes (PDs), and cells of the proximal tubules (PTs)—during six postnatal time points was demonstrated. The trajectory of rat kidney development and the order of induction of the six major types of kidney cells from just after birth to maturity were determined. In addition, features of the dynamically changing genes as well as transcription factors during postnatal rat kidney development were identified. The present study provides a resource for achieving a deep understanding of the molecular basis of and regulatory events in the mid and late stages of kidney development.

Role of autophagy and evaluation the effects of microRNAs 214, 132, 34c and prorenin receptor in a rat model of focal segmental glomerulosclerosis

AimsFocal segmental glomerulosclerosis (FSGS) is the common cause of chronic renal disease worldwide. Although there are many etiologic factors which have common theme of podocyte injury conclusive etiology is not clearly understood. In this study, we aimed to explore the role of autophagy in the pathogenesis of podocyte injury, which is the key point in disease progression, and the roles of intrarenal microRNAs and the prorenin receptor (PRR) in the 5/6 nephrectomy and adriamycin nephropathy models of FSGS. Main methodsFor experimental FSGS model, 5/6 nephrectomy and adriamycin nephropathy models were created and characterized in adult Sprague Dawley rats. Microarray analysis was performed on FSGS and control groups that was confirmed by q-RT-PCR. Beclin1, LC3B, PRR, ATG7 and ATG5 expression were evaluated by western blotting and immunohistochemistry. Also, Beclin1 and PRR expression were measured by ELISA. Glomerular podocyte isolation was performed and autophagic activity was evaluated in podocytes before and after transfection with miRNA mimic and antagonists. Key findingsGlomerular expression of Beclin1, LC3B, PRR, ATG7 and ATG5 were significantly lower in the 5/6 nephrectomy than adriamycin nephropathy group and in both groups lower when compared to control groups. Western blot results were consistent with immunohistochemical data. Electron microscopy revealed signs of impaired autophagy in FSGS. Autophagic activity decreased significantly after miR-214, miR-132 and miR-34c mimics and increased after transfection with antagonists. SignificanceThese results showed that the role of autophagic activity and decreased expression of PRR in FSGS pathogenesis and miR-34c, miR-132 and miR-214 could be a potential treatment strategy by regulating autophagy.

392-P: ROCK2-Mediated Podocyte Dysfunction in Diabetic Nephropathy

The small GTPase RhoA and its downstream effector, Rho-kinase (ROCK), are implicated in a variety of cellular functions (e.g., cell adhesion, migration) via effect on cytoskeletal reorganization. We and others have illuminated ROCK as an important pathogenic regulator of diabetic kidney disease. Renal ROCK activity is elevated in both type 1 and type 2 diabetic murine models, as well as in diabetic patients. ROCK has two isoforms, ROCK1 and ROCK2. ROCK1 has been shown to regulate mitochondrial fission and epithelial-to-mesenchymal transition in the setting of diabetic kidney disease: however, the role of renal ROCK2 remain unclear. In the present study, we have demonstrated the upregulation of ROCK2 in glomerular podocytes obtained from 3 established models of diabetic kidney disease, (i.e., streptozotocin injection, db/db, and high-fat diet treatment), and diabetic patients. In an attempt to elucidate the roles of podocyte ROCK2, we generated podocyte-specific ROCK2 knockout mice (PR2KO) by breeding ROCK2 flox mice with Podocin-Cre mice. We found that podocyte-restricted ROCK2 deletion did not lead to any defects in the kidney development and functions. Of note, PR2KO were protected against albuminuria and glomerular sclerosis in streptozotocin-induced diabetic mice. In addition, renoprotective actions of podocyte ROCK2 deletion were also observed in type 2 diabetic db/db mice and high-fat diet-fed animals. Mechanistically, RNA-Seq analysis in ROCK2-null podocytes revealed the recovery of fatty acid utilization. When considered alongside our previous observations, the current work highlights the importance of ROCK2 in diabetic podocytopathy. Disclosure K. Matoba: Research Support; Self; Eli Lilly Japan K. K. Y. Takeda: None. T. Akamine: None. Y. Nagai: None. R. Ukichi: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker’s Bureau; Self; Abbott Japan Co., Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K. K., Kissei Pharmaceutical Co., Ltd., Medtronic, MSD Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science (20K08645, 18K15985); Suzuken Memorial Foundation; Takeda Science Foundation; Yokoyama Foundation for Clinical Pharmacology; MSD Life Science Foundation; Ichiro Kanehara Foundation; Japan Diabetes Foundation; Ueh

MO030IDENTIFICATION OF REGULATED MRNAS AND MIRNAS IN GLOMERULI ISOLATED FROM AN FSGS-LIKE ZEBRAFISH MODEL

Abstract Background and Aims The zebrafish (Danio rerio) is a powerful animal model to study glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to become transparent, in vivo observation of these animals is possible. At 48 hours post fertilization, zebrafish larvae develop a single glomerulus which is attached to a pair of tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane and interdigitating podocyte foot processes bridged by a slit diaphragm. By using genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease like focal segmental glomerulosclerosis (FSGS) directly in vivo. FSGS is characterized by podocyte loss, the effacement of their foot processes as well as scarring of the glomerulus. To study FSGS in zebrafish larvae, we induced podocyte detachment by the use of a zebrafish strain expressing the enzyme nitroreductase converting metronidazole into a toxic substance specifically in podocytes. The aim of our study was to collect glomeruli for the identification of mRNAs as well as miRNAs by RNA_Seq that are up- and down-regulated in the glomeruli of this FSGS-like disease model. Method The transgenic zebrafish strain Cherry (Tg(nphs2:GAL4); Tg(UAS:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was utilized. After addition of metronidazole (MTZ) into the tank water, MTZ is converted into a cytotoxin by NTR leading to dose-dependent apoptosis exclusively in podocytes. Cherry larvae were treated at 4 days post fertilization (dpf) for 48 h with 80 µM MTZ. MTZ-treated and control larvae were homogenized at 6 dpf. The cell suspension was diluted, and red-fluorescent glomeruli were collected using a micropipette and a microscope. Total RNA was isolated, and integrity was checked by a Bioanalyzer. Libraries were generated with a MACE kit and True Quant small RNA seq kit by GenXPro. Constructs were amplified by PCR and sequenced on an Illumina Hiseq 2000. Normalization and statistical analysis for differential gene expression were done using DESeq2. Results Zebrafish larvae showed severe whole-body edema, proteinuria, loss of podocytes and an increased mortality rate after MTZ-treatment. The glomerular histology resembled mammalian FSGS. We found that only the RNA of manually collected glomeruli had an excellent quality. Using RNA_Seq, we identified a total of 16941 genes. DESeq2 analysis showed 494 up-regulated and 473 down-regulated genes. Gene ontology (GO) enrichment analysis of up-regulated genes revealed a total of 167 that are significantly enriched in GO terms (e.g. metabolic processes, immune response and ion transport). Down-regulated genes were enriched in 14 GO terms and most of them are linked to normal glomerular function and the slit diaphragm. DESeq2 analysis identified 200 miRNAs of 777 small RNAs. Some of these miRNA are already described to be regulated in different glomerular diseases like FSGS, lupus nephritis, IgA nephropathy and diabetic nephropathy. Conclusion We analyzed isolated glomeruli from transgenic zebrafish larvae that developed a FSGS-like disease. By sequencing, we have found mRNAs and miRNAs that were significantly regulated after the onset of disease. Detailed knowledge of these mRNAs and miRNA-based gene regulation will help to uncover the pathomechanism as well as to develop therapeutics for the treatment of FSGS.

FC 005DEPOSITION OF PLATELET-DERIVED MICRO-PARTICLES IN PODOCYTES CONTRIBUTES TO DIABETIC NEPHROPATHY

Abstract Background and Aims Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the developed world. Podocyte injury has been shown to be a critical cellular event in the progression of DN. Our previous studies demonstrated that platelet-derived microparticles (PMPs) mediated endothelial injury in diabetic rats. Therefore, this study aimed to investigate whether the PMPs deposit in podocytes and its potential effects on podocyte injury in DN. Method Deposition of PMPs in podocytes was checked by immunofluorescent staining and electron microscopy. Renal pathological change and ultra-microstructure were respectively checked by periodic acid-schiff staining and electron microscopy. The protein expression of inflammatory cytokines and extracellular matrix was measured by immunohistochemical staining and Western blot. Results The PMPs were found to be widely deposited in podocytes of glomeruli in diabetic patients and animal models and closely associated with progression of DN. Interestingly, aspirin treatment significantly inhibited accumulation of PMPs in glomeruli of diabetic rats, accompanied with alleviated mesangial matrix expansion, fusion of foot processes, decreased protein expression of inflammatory cytokines and extracellular matrix secretion. The in vitro study further confirmed the deposition of PMPs in podocytes. Moreover, PMPs stimulation induced phenotype transition of podocytes through decreased protein expression of podocin and increased protein expression of α-SMA and fibronectin, which was correlated with increased production of inflammatory cytokines. Conclusion Our findings for the first time demonstrated that deposition of PMPs in podocytes contributed to the development of DN.

MO005GENERATION OF NOVEL 3D CO-CULTURE SYSTEMS TO STUDY PODOCYTOPATHIES EX VIVO IN A PERSONALIZED MANNER*

Abstract Background and Aims The smallest filtration unit of the kidney, the glomerulus, is composed of capillaries formed by glomerular endothelial cells (GEC), glomerular basement membrane and podocytes. Furthermore, glomerular mesangial cells (GMC) between the capillary loops give structural support. It is known that loss of podocyte foot processes is leading to a dysfunctional glomerular filtration barrier and is seen in glomerular diseases like focal segmental glomerulosclerosis and other podocytopathies. Indeed, it is hard to investigate podocytes in culture because podocytes are terminally end-differentiated cells that do not proliferate, lack foot processes and cell type-specific markers. Although conditionally immortalized human podocytes regained the capacity of proliferation, marker expression and behaviour differ between cell lines. The overarching aims of this study are to generate a 3D glomerular co-culture model that better reflects the in vivo phenotype of glomerular cell types. We want to investigate cell-cell contact, interaction and communication and extracellular matrix production in 3D glomerular co-cultures. Furthermore, patient-derived hiPSC-podocytes will be used in the glomerular co-cultures to investigate podocyte disease in a personalized manner and to identify potential therapeutic targets. Method The hanging droplet method was used to produce 3D glomerular spheroids. Therefore, human differentiated immortalized podocytes, human GECs and human GMCs were inserted in a medium-droplet hanging from the lid of a petri dish and harvested at different time points. Fluorescent cell lines of the different glomerular cell types were tracked in a time-lapse experiment to study if cell attachment and spheroid formation undergoes a specific order and structure. Glomerular spheroids were further characterized regarding the expression of podocyte-specific markers and extracellular matrix synthesis by immunohistochemistry, electron microscopy and qPCR and were compared to human cells isolated from glomeruli. Furthermore, scRNA-sequencing analysis was performed in 2D mono-cultures of human GECs, GMCs and immortalized podocytes and on 3D co-cultures to see if this change in culture conditions leads to transcriptomic alterations. For the generation of patient-derived podocytes, skin fibroblast of patients with podocyte mutations (INF2 mutation and WT1 mutation) and from healthy controls were reprogramed in iPSCs and differentiated into podocytes that keep the patient’s mutation. Results First time-lapse experiments of glomerular co-cultures showed that human podocytes and human glomerular endothelial cells attach to each other (Fig. 1a) and histological sections revealed that the glomerular spheroids are encapsulated by a monolayer of cells (Fig. 1b). SEM allowed ultrastructural characterization of the 3D spheroid-like structures (Fig. 1c). TEM revealed cell protrusions of podocytes that were not seen in monocultures (Fig. 1d, e). We could also demonstrate production of extracellular matrix by the cells (Fig. 1f). Immunohistochemistry and qPCR showed expression of collagen-IV and laminin. During the reprogramming of patient-derived fibroblasts, size of the generated hiPSC decreased and the nuclei to cell body ratio increased. HiPSCs formed colonies with distinct boarders and the proliferation rate increased. Furthermore, generated hiPSC showed similar gene expression of pluripotency markers compared to a commercial hiPSC control cell line and podocytes derived from these hiPSC expressed synaptopodin (Fig. 2). Conclusion We generated a 3D co-culture model that better represents the complexity of the glomerulus ex vivo. It is indicated that this model provides better physiological conditions. By an insertion of patient-specific hiPSC-derived podocytes in the 3D co-culture we will investigate glomerular diseases in a personalized manner in the future.

MO011PODOCYTE NEPHRONECTIN IS REGULATED BY TRANSFORMING GROWTH FACTOR BETA VIA THE CANONICAL AND NON-CANONICAL PATHWAYS

Abstract Background and Aims The glomerular basement membrane (GBM), podocytes and glomerular endothelial cells (GEC) are composing the glomerular filtration barrier (GFB) within the glomerulus. Both podocytes and GEC are essential components for the synthesis of the extracellular matrix (ECM) of the GBM. One ECM protein of the GBM, which is mainly produced by podocytes is nephronectin (NPNT). An altered expression pattern of NPNT has been observed in different kidney diseases. While NPNT was shown to be down regulated in membranous glomerulonephropathy, its expression was elevated in diabetic glomerulopathy, compared to healthy controls. Using a morpholino-induced knockdown of npnt in zebrafish larvae, proteinuria, podocyte foot process effacement and thickening of the lamia rara interna of the GBM were observed. Mice that were intra peritoneally injected with a microRNA 378a (miR-378a) mimic showed a decrease in NPNT expression in the kidneys on both mRNA and protein level, suggesting a regulatory effect of miR-378a on NPNT. In addition, in cultured human podocytes treatment with transforming growth factor beta (TGFβ), as well as transfection with miR-378a mimic down regulated NPNT mRNA and protein expression. By blocking different parts of the TGFβ pathway, we want to further investigate the mechanisms by which TGFβ mediates the regulation of NPNT in podocytes. Method Our main model for this study are immortalized human podocytes, which are proliferating at 33°C and differentiating at 37°C due to a temperature sensitive SV40 large T antigen. Ten to 12 days differentiated podocytes were used for experiments. Differentiated cells were either transfected with a miR-192 or control miR mimic or pre-incubated with different inhibitors for components of both the canonical and the non-canonical TGFβ signaling pathways, followed by culture with or without additional TGFβ. Cell lysates were prepared to be used for Western Blot or qPCR analyses. Results Treatment of immortalized human podocytes with TGFβ decreased NPNT expression on mRNA and protein level. After transfecting immortalized human podocytes with a miR-192 mimic, a GEC-derived miR up regulated by TGFβ stimulation, we observed reduced NPNT expression, compared to control miR transfection. Blocking TGFβ receptor I signaling with the specific inhibitor SD208, caused higher NPNT protein abundance, while NPNT mRNA expression remained unchanged. Targeting downstream parts of both the canonical and non-canonical TGFβ pathways by using inhibitors for single molecules of the respective arms of the intricate TGFβ pathway showed ambiguous results. Suppression of either Smad2 and/or Smad3 tended to enhance NPNT protein levels, accompanied by mostly unaltered mRNA expression. Blockade of different parts of the non-canonical TGFβ pathway also up regulated protein expression of NPNT. However, NPNT mRNA expression was more variable. Therefore, regulation of podocyte NPNT might not be due to changes in mRNA transcription, but to modifications on posttranscriptional level. Conclusion Treating immortalized human podocytes with TGFβ or TGFβ-induced miR-192 reduced NPNT expression on both the mRNA and protein level. More detailed analysis with inhibition of different parts of the canonical and non-canonical TGFβ pathways hint that both pathways are involved in NPNT expression. Therefore, we suggest that the regulation of podocyte NPNT by TGFβ is fine-tuned via both the canonical and non-canonical pathways with additional modulation through TGFβ dependent miRs.

FC 037NEUTROPHILS PLAY A KEY ROLE IN THE INITIATION OF GLOMERULAR HEMATURIA IN A POSTINFECTIOUS IGAN EXPERIMENTAL MODEL

Abstract Background and Aims Hematuria is a common finding in patients with IgA nephropathy (IgAN), occurring mainly after upper respiratory tract infections. Hematuria can lead to acute kidney injury and chronic loss of renal function in IgAN. However, the mechanisms involved in egression of erythrocytes from the glomerular capillaries into the urinary space are unknown. To answer this question, we developed an infection with Streptococcus pneumoniae (SP) in a humanized experimental IgAN model (α1KICD89tg mice) that resembles the pathological and clinical findings of disease (IgA1 and soluble CD89 mesangial deposits, complement activation, proteinuria and hematuria). Method α1KICD89tg mice (12 weeks old) received an intranasal instillation of SP (107 bacteria). Blood, urine and renal samples were obtained during 1 month after induction of respiratory infection. The presence of SP in lungs from these mice was confirmed by microbiological analysis. Hematuria was quantified in the urinary sediment and renal function was determined by biochemical analysis. Renal histological characteristics were evaluated by hematoxylin/eosin, masson's trichrome and PAS staining. IgA glomerular deposits, activation of complement system and infiltration of proinflammatory cells was examined by immunohistochemistry or immunofluorescence. Circulating leukocyte populations were studied on a hemocytometer. Renal inflammatory cytokines, metalloproteases, as well as markers of tubular and glomerular damage were determined in kidneys by RT-PCR and western-blot. To further validate the role of neutrophils in this pathological setting, we selective depleted these cells through a single injection of anti-Ly6G mAb (200 µg/kg i.p). Results SP-intranasal instillation in α1KICD89tg mice increased hematuria, microalbuminuria and proteinuria, peaking at 48h after induction of the respiratory infection. SP instillation caused disruption of the glomerular basement membrane, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin, as well as higher glomerular accumulation of IgA and proteins of complement system (C3, MBL). Hematuria intensity was positively correlated with the presence of interstitial F4/80+ macrophages, matrix metalloproteinase 9 (MMP-9), inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, CCL-2, CCL5 and CX3CL1/CX3CR1) as well as p65 NF-κB activation. Hematuria was negatively correlated with anti-inflammatory IL-10 mRNA expression, Factor H levels and collagen IV content. Notably, SP infection induced expression of the tubular injury markers N-GAL and KIM-1. Increased peripheral neutrophils levels were observed in the SP-infected α1KICD89tg mice. Mechanistically, anti-Ly6G-mediated neutrophil depletion reduced SP-mediated hematuria, proteinuria and albuminuria, prevented loss of synaptopodin and nephrin, decreased renal inflammation and MMP-9 expression in α1KICD89tg mice Conclusion In a humanized mouse model of IgAN, hematuria bouts following respiratory tract infections are caused by a neutrophil-mediated alteration of the glomerular filtration barrier (podocyte damage, complement deposits and loss of Collagen IV). These findings may help to unveil novel potential therapeutic approaches to combat one of the key elements in the progression of IgAN and related conditions.

MO625INHIBITION OF SGLT-2 CANNOT RESCUE NEPHRIN EXPRESSION IN DIABETIC NEPHROPATHY

Abstract Background and Aims Early glomerular damage in diabetes is induced by high blood glucose level (hyperglycemia) which affects the glomerular filtration barrier and leads to proteinuria. Podocyte-specific proteins like the transmembrane protein nephrin form the slit diaphragm that is important for proper function of the glomerular filtration barrier. Sodium-glucose co-transporter 2 (SGLT2) specific proteins are involved in glucose reabsorption in the kidney and maintain the normal glucose level in the blood. Recent studies showed remarkable success of SGLT2-inhibition in patients with diabetic nephropathy. Therefore we wanted to study if hyperglycemia induced reduction of nephrin expression is affected bySGLT-2 inhibition. Therefore we induced hyperglycemia in zebrafish larvae by knockdown of Pancreatic duodenal homeobox 1 (Pdx1) transcription factor and treated zebrafish larvae with Empagliflozin. In parallel we treated Bl/6 mice with streptozotozin and treated them with Empagliflozin. We then analyzed nephrin expression in both model systems. Method Zebrafish is an ideal model to study glomerular diseases, because the zebrafish larvae develops a pronephros with high homology to the human glomerulus. In order to inhibit SLGT-2 after Pdx1-knockdown, we treated both control and diabetic zebrafish larvae with 10µM Empagliflozin from 1dpf to 5dpf. We used a zebrafish line that expresses a fluorescent Vitamin D binding plasma protein Tg(l-fabp:DBP:eGFP) to measure proteinuria by measuring the GFP signal in both retinal and glomerular vessels of 120 hpf larvae. Immunohistochemistry against nephrin was performed using a specific zebrafish antibody. Results Conclusion Despite the promising effects of SLGT-2 inhibitor treatment in patients with diabetic nephropathy the early effects on nephrin expression are not addressed and remain an unchanged problem by this novel treatment option.

FC 029PODOCYTE AND GLOMERULAR ENDOTHELIAL CELL DERIVED MICRORNAS REGULATE PODOCYTE NEPHRONECTIN IN MEMBRANOUS GLOMERULONEPHRITIS

Abstract Background and Aims Autoantibodies binding to podocyte antigens cause idiopathic membranous glomerulonephritis (iMGN). However, it remains elusive how autoantibodies reach the subepithelial space because the glomerular filtration barrier is normally size selective and impermeable for antibodies. Method Kidney biopsies from patients with MGN, cell culture, zebrafish and mice models were used to investigate the role of nephronectin (NPNT) regulating microRNAs (miRs) for the glomerular filtration barrier. Results We found that endothelial cell-derived miR-192-5p and podocyte-derived miR-378a-3p are upregulated in patients with anti-phospholipase A2 receptor antibody positive (PLA2R-ab+) iMGN and regulate glomerular NPNT expression (Fig. 1). Overexpression of miR-378a-3p and miR-192-5p as well as morpholino mediated npnt knockdown in zebrafish induced edema, proteinuria, loss of podocyte markers and podocyte effacement. The most prominent phenotype however were structural changes of the glomerular basement membrane (GBM) with increased lucidity, slicing and lamellation especially of the lamina rara interna (Fig. 2, Fig. 3). The phenotype was comparable to ultrastructural findings seen in iMGN. IgG sized nanoparticles accumulated in lucidity areas of the lamina rara interna and lamina densa of the GBM in npnt knockdown zebrafish models. Loss of slit diaphragm proteins and severe structural impairment of the GBM were further confirmed in podocyte specific Npnt knockout mice (Fig. 4). Conclusion Podocyte NPNT is important for proper glomerular filter function and GBM structure and is regulated by podocyte and glomerular endothelial cell derived miRs. We hypothesize that loss of NPNT in the GBM is part of the pathophysiology of iMGN and enables subepithelial immune complex deposition in iMGN.

MO252PLA2R -VE MEMBRANOUS GLOMERULONEPHRITIS PATIENTS SHOWS MORE EFFECTIVE RESPONSE THAN PLA2R +VE ON TACROLIMUS PLUS LOW DOSE PREDNISOLONE THERAPY*

Abstract Background and Aims PLA2R is an autoantigen present in glomerular podocytes of Membranous Nephropathy (MN) patients. Several drugs have been tried which included nonspecific anti-proteinuric agents; corticosteroids, alone or with alkylating agents; cyclosporine; intravenous Ig; mycophenolate mofetil; and rituximab. There is no standard therapy for patients with frequent relapsing or steroid-dependent MN. We propose the efficacy of low dose Tacrolimus (TAC) plus prednisolone and associated changes in anti-PLA2R in adult IMN. Method Total 101 membranous nephropathy patients were treated with combination of prednisolone 1mg/kg alt-day) and Tac 0.1mg/kg/day (trough 6-10 ng/ml first 6M and 4-6 ng/ml for next 3M) then both taper by 1/3 every month up to 12M. Out of 101 patients; 15 diabetic; 7 lupus; 1HBV and 1 ankylosing spondylitis patients were excluded. Finally; 77 Patients were followed and evaluated for the anti-PLA2R level at baseline; 3M; 6M; 12M and end of follow-up (17-61 ; median 38 months). CR; PR; relapse; and side-effects were recorded.Of the 77 patients; at 3M 60(77.92%; CR-37; PR-23); at 6M 61(79.22%; CR-53; PR-8); at 12M 53(68.86%; CR-47; PR-6) achieved remission. Eight (10.38%) relapsed and 16(20.77%) showed no response at 12M. At end of follow-up; out of 54 responsive patients 37(68.51%; CR-36; PR-1) remained in remission and 17(31.48%) patients relapsed. Results Out of 77 patients; 51 (66.3%) were anti-PLA2R positive. Remission rate was significantly low in PLA2R+ve than PLA2R-ve (36/51 vs 24/26; p=0.03) at 3M; (36/51 vs 25/26; p=0.009) at 6M and (31/51 vs 22/26; p=0.03) at 12M. PLA2R level was decreased by 60.38% and 77.56% at 3M and 6M respectively (1A & 1B). There were significant correlations between PLA2R level and 24h proteinuria at baseline; 3M and at 6M (1C). During therapy 4 patients develop cutaneous tenia; 1 osteonecrosis of the femur head; 1 corpus tunnel syndrome; 4 onset diabetes; 3 tremor; and 14 patients experienced GI symptoms. The eGFR was decreased significantly (p=0.003) by 26.5% at the end of therapy and was normalized after stopping Tac; and 5 non-responsive patients had doubling of serum creatinine and progressively deteriorated eGFR. To note; 4 females had pregnancy and successful delivery in our cohort of patients. Conclusion PLA2R+ve patients showed poor response compare to PLA2R-ve patients. Remission with Tacrolimus and prednisolone therapy is comparable to historical Ponticelli (Pred plus CYP) regimen. Successful pregnency was ovserved on Tac based regimen.

Renal Handling of Albumin-From Early Findings to Current Concepts.

Albumin is the main protein of blood plasma, lymph, cerebrospinal and interstitial fluid. The protein participates in a variety of important biological functions, such as maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver, as well as in the kidney tubular epithelium. Long-lasting investigation in this area has delineated the principal route of its catabolism involving glomerular filtration, tubular endocytic uptake via the multiligand scavenger receptor tandem—megalin and cubilin-amnionless complex, as well as lysosomal degradation to amino acids. However, the research of the last few decades indicates that also additional mechanisms may operate in this process to some extent. Direct uptake of albumin in glomerular podocytes via receptor for crystallizable region of immunoglobulins (neonatal FC receptor) was demonstrated. Additionally, luminal recycling of short peptides into the bloodstream and/or back into tubular lumen or transcytosis of whole molecules was suggested. The article discusses the molecular aspects of these processes and presents the major findings and controversies arising in the light of the research concerning the last decade. Their better characterization is essential for further research into pathophysiology of proteinuric renal failure and development of effective therapeutic strategies.

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement

Homozygous ITGA3 Missense Mutation in Adults in a Family with Syndromic Epidermolysis Bullosa (ILNEB) without Pulmonary Involvement