Parietal Epithelial Cells Research Articles

Diabetic condition induces hypertrophy and vacuolization in glomerular parietal epithelial cells

Diabetic nephropathy (DN) is accompanied by characteristic changes in the glomerulus, but little is known about the effect of diabetes on parietal epithelial cells (PECs). In this study, a descriptive analysis of PECs was undertaken in diabetic db/db mice and in diabetic patients. PEC hypertrophy was significantly more prominent in diabetic mice than in nondiabetic mice, and this was evident even at the early stage. Additionally, the number of vacuoles in PECs was markedly increased in diabetic mice, suggesting the presence of cellular injury in PECs in DN. Although rare, binuclear cells were observed in mice with early diabetes. In cultured PECs, a high glucose condition, compared with normal glucose condition, induced cellular hypertrophy and apoptosis. Flow cytometry showed that some PECs in the G0 phase reentered the cell cycle but got arrested in the S phase. Finally, in human diabetic subjects, hypertrophy and vacuolization were observed in the PECs. Our data showed that PECs undergo substantial changes in DN and may participate in rearrangement for differentiation into podocytes.

Single cell RNA sequencing uncovers cellular developmental sequences and novel potential intercellular communications in embryonic kidney

Kidney development requires the coordinated growth and differentiation of multiple cells. Despite recent single cell profiles in nephrogenesis research, tools for data analysis are rapidly developing, and offer an opportunity to gain additional insight into kidney development. In this study, single-cell RNA sequencing data obtained from embryonic mouse kidney were re-analyzed. Manifold learning based on partition-based graph-abstraction coordinated cells, reflecting their expected lineage relationships. Consequently, the coordination in combination with ForceAtlas2 enabled the inference of parietal epithelial cells of Bowman’s capsule and the inference of cells involved in the developmental process from the S-shaped body to each nephron segment. RNA velocity suggested developmental sequences of proximal tubules and podocytes. In combination with a Markov chain algorithm, RNA velocity suggested the self-renewal processes of nephron progenitors. NicheNet analyses suggested that not only cells belonging to ureteric bud and stroma, but also endothelial cells, macrophages, and pericytes may contribute to the differentiation of cells from nephron progenitors. Organ culture of embryonic mouse kidney demonstrated that nerve growth factor, one of the nephrogenesis-related factors inferred by NicheNet, contributed to mitochondrial biogenesis in developing distal tubules. These approaches suggested previously unrecognized aspects of the underlying mechanisms for kidney development.

Expression profiles revealed potential kidney injury caused by SARS-CoV-2: a systematic analysis of ACE2 and clinical lessons learned from this discovery.

Background: Novel Coronavirus disease 2019 (COVID-19) was first detected in pneumonia patients in Wuhan, China in December 2019. Based on the current understanding, COVID-19 has become a global issue. Presumably, numerous studies have found that SARS-CoV-2 also transpires in kidney tissue with permanent viral loads. However, it is elusive as to whether SARS-CoV-2 can directly damage the kidney or induce acute renal failure. Hence, to comprehensively understand the impact of COVID-19 on kidney damage, we conducted a retrospective series of case studies to assess kidney functions. Additionally, ACE2 distribution in kidney tissue was analyzed through RNAseq data in open-access databases.Results: According to the findings from transcriptome analysis, we revealed higher ACE2 expression levels in females than males. Similar results were more noticeable in the elderly than in young adults. Furthermore, single-cell RNA sequencing data analysis showed high ACE2 expression in kidney tubule and collecting duct principal cells as well as glomerular parietal epithelial cells. On their admission, the patient's serum creatinine and blood urea nitrogen (BUN) were elevated to between 36.13% and 16.80%, respectively. The estimated glomerular filtration rate (EGFR) of < 60 ml/min per 1.73 m2 was reported in 10.92 % of the patients. Notably, at admission, increased BUN time varied linearly following the generalized additive mixed model. Thus, the hourly-increase of BUN in patients was 0.495 (95%CI: 0.263, 0.726).Conclusion: Based on clinical findings, it was ascertained that COVID-19 can damage renal function, but it seldom causes acute renal failure. Coronavirus may directly bind to ACE2-positive cells and damage kidney tissue in the analysis of scRNA-seq data in kidney tissue. Therefore, this evidence suggests that kidney tissue act as the SARS-CoV-2 infection site and the findings could provide insight into the pathophysiology of kidney damage.Methods: We systematically analyzed ACE2 expression profiles in organs based on open-access datasets for healthy individuals. Meanwhile, single-cell sequencing data for kidney samples were collected and analyzed. Assessments on kidney functions were conducted on 119 selected COVID-19 positive patients admitted from 10th February – 18th March 2020, in hospital in Wuhan City, Hubei Province. Consequently, their clinical records and laboratory findings, such as the estimated glomerular filtration rate (eGFR), Blood Urea Nitrogen (BUN), Creatinine, and Comorbidities, were collected.

Transplantation of mesenchymal stem cells preserves podocyte homeostasis through modulation of parietal epithelial cell activation in adriamycin-induced mouse kidney injury model.

To determine the role of the transplantation of bone marrow-derived mesenchymal stem cells (MSCs) in podocyte renewal, we studied BALB/C mice with or without adriamycin-induced acute kidney injury. MSCs were transplanted ectopically under the capsule of the left kidney or into the peritoneal cavity after the onset of kidney injury to test testing their local or systemic paracrine effects, respectively. Adriamycin produced increases in urine protein: creatinine ratios, blood urea nitrogen, and blood pressure, which improved after both renal subcapsular and intraperitoneal MSCs transplants. The histological changes of adriamycin kidney changes regressed in both kidneys and in only the ipsilateral kidney after intraperitoneal or renal subcapsular transplants indicating that the benefits of transplanted MSCs were related to the extent of paracrine factor distribution. Analysis of kidney tissues for p57-positive parietal epithelial cells (PECs) showed that MSC transplants restored adriamycin-induced decreases in the abundance of these cells to normal levels, although after renal subcapsular transplants these changes did not extend to contralateral kidneys. Moreover, adriamycin caused inflammatory activation of PECs with coexpression of CD44 and phospho-ERK, which was normalized in both or only ipsilateral kidneys depending on whether MSCs were transplanted in the peritoneal cavity or subcapsular space, respectively.

Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern frequently found in patients with nephrotic syndrome that often progress to end-stage kidney disease. The initial step in development of this histologically defined entity is injury and ultimately depletion of podocytes, highly arborized interdigitating cells on the glomerular capillaries with important function for the glomerular filtration barrier. Since there are still no causal therapeutic options, animal models are needed to develop new treatment strategies. Here, we present an FSGS-like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission electron microscopy. By intravenous injection of fluorescent high-molecular weight dextran, we confirmed leakage of the size selective filtration barrier. Additionally, we observed severe podocyte foot process effacement of remaining podocytes, activation of proximal tubule-like parietal epithelial cells identified by ultrastructural cytomorphology, and expression of proximal tubule markers. These activated cells deposited extracellular matrix on the glomerular tuft which are all hallmarks of FSGS. Our findings indicate that glomerular response to podocyte depletion in larval zebrafish resembles human FSGS in several important characteristics. Therefore, this model will help to investigate the disease development and the effects of potential drugs in a living organism.

Parietal epithelial cell differentiation to a podocyte fate in the aged mouse kidney.

Healthy aging is typified by a progressive and absolute loss of podocytes over the lifespan of animals and humans. To test the hypothesis that a subset of glomerular parietal epithelial cell (PEC) progenitors transition to a podocyte fate with aging, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice were generated. PECs were inducibly labeled with a tdTomato reporter, and podocytes were constitutively labeled with an EGFP reporter. With advancing age (14 and 24 months) glomeruli in the juxta-medullary cortex (JMC) were more severely injured than those in the outer cortex (OC). In aged mice (24m), injured glomeruli with lower podocyte number (41% decrease), showed more PEC migration and differentiation to a podocyte fate than mildly injured or healthy glomeruli. PECs differentiated to a podocyte fate had ultrastructural features of podocytes and co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of mesangial (Perlecan) or endothelial (ERG) cells. PECs differentiated to a podocyte fate did not express CD44, a marker of PEC activation. Taken together, we demonstrate that a subpopulation of PECs differentiate to a podocyte fate predominantly in injured glomeruli in mice of advanced age.

MAD2B contributes to parietal epithelial cell activation and crescentic glomerulonephritis via Skp2.

Mitotic spindle assembly checkpoint protein 2 (MAD2B), a well-known anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase-ζ, is critical for mitotic control and DNA repair. Previously, we detected a strong increase of MAD2B in the glomeruli from patients with crescentic glomerulonephritis and anti-glomerular basement membrane (anti-GBM) rats, which predominantly originated from activated parietal epithelial cells (PECs). Consistently, in vitro MAD2B was increased in TNF-α-treated PECs, along with cell activation and proliferation, as well as extracellular matrix accumulation, which could be reversed by MAD2B genetic depletion. Furthermore, we found that expression of S phase kinase-associated protein 2 (Skp2), an APC/CCDH1 substrate, was increased in the glomeruli of anti-GBM rats, and TNF-α-stimulated PECs and could be suppressed by MAD2B depletion. Additionally, genetic deletion of Skp2 inhibited TNF-α-induced PEC activation and dysfunction. Finally, TNF-α blockade or glucocorticoid therapy administered to anti-GBM rats could ameliorate MAD2B and Skp2 accumulation as well as weaken PEC activation. Collectively, our data suggest that MAD2B has a pivotal role in the pathogenesis of glomerular PEC activation and crescent formation through induction of Skp2 expression.

(Pro)renin receptor promotes crescent formation via the ERK1/2 and Wnt/β-catenin pathways in glomerulonephritis.

(Pro)renin receptor [(P)RR] has multiple functions, but its regulation and role in the pathogenesis in glomerulonephritis (GN) are poorly defined. The aims of the present study were to determine the effects of direct renin inhibition (DRI) and demonstrate the role of (P)RR on the progression of crescentic GN. The anti-glomerular basement membrane nephritis rat model developed progressive proteinuria (83.64 ± 10.49 mg/day) and glomerular crescent formation (percent glomerular crescent: 62.1 ± 2.3%) accompanied by increased macrophage infiltration and glomerular expression of monocyte chemoattractant protein (MCP)-1, (P)RR, phospho-extracellular signal-regulated kinase (ERK)1/2, Wnt4, and active β-catenin. Treatment with DRI ameliorated proteinuria (20.33 ± 5.88 mg/day) and markedly reduced glomerular crescent formation (20.9 ± 2.6%), induction of macrophage infiltration, (P)RR, phospho-ERK1/2, Wnt4, and active β-catenin. Furthermore, primary cultured parietal epithelial cells stimulated by recombinant prorenin showed significant increases in cell proliferation. Notably, while the ERK1/2 inhibitor PD98059 or (P)RR-specific siRNA treatment abolished the elevation in cell proliferation, DRI treatment did not abrogate this elevation. Moreover, cultured mesangial cells showed an increase in prorenin-induced MCP-1 expression. Interestingly, (P)RR or Wnt4-specific siRNA treatment or the β-catenin antagonist XAV939 inhibited the elevation of MCP-1 expression, whereas DRI did not. These results suggest that (P)RR regulates glomerular crescent formation via the ERK1/2 signaling and Wnt/β-catenin pathways during the course of anti-glomerular basement membrane nephritis and that DRI mitigates the progression of crescentic GN through the reduction of (P)RR expression but not inhibition of prorenin binding to (P)RR.

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Single-cell RNA sequencing analysis of human kidney reveals the presence of ACE2 receptor: A potential pathway of COVID-19 infection.

BackgroundA novel coronavirus called SARS‐Cov‐2, which shared 82% similarity of genome sequence with SARS‐CoV, was found in Wuhan in late December of 2019, causing an epidemic outbreak of novel coronavirus‐induced pneumonia with dramatically increasing number of cases. Several organs are vulnerable to COVID‐19 infection. Acute kidney injury (AKI) was reported in parts of case‐studies reporting characteristics of COVID‐19 patients. This study aimed at analyzing the potential route of SARS‐Cov‐2 entry and mechanism at cellular level.MethodSingle‐cell RNA sequencing (scRNA‐seq) technology was used to obtain evidence of potential route and ACE2 expressing cell in renal system for underlying pathogenesis of kidney injury caused by COVID‐19. The whole process was performed under R with Seurat packages. Canonical marker genes were used to annotate different types of cells.ResultsTen different clusters were identified and ACE2 was mainly expressed in proximal tubule and glomerular parietal epithelial cells. From Gene Ontology (GO) & KEGG enrichment analysis, imbalance of ACE2 expression, renin‐angiotensin system (RAS) activation, and neutrophil‐related processes were the main issue of COVID‐19 leading kidney injury.ConclusionOur study provided the cellular evidence that SARS‐Cov‐2 invaded human kidney tissue via proximal convoluted tubule, proximal tubule, proximal straight tubule cells, and glomerular parietal cells by means of ACE2‐related pathway and used their cellular protease TMPRSS2 for priming.

Pathological characteristics of BK polyomavirus-associated nephropathy with glomerular involvement.

BackgroundThis study aimed to investigate the pathological characteristics of BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) with glomerular involvement in kidney transplant recipients.MethodsForty-four patients with glomerular BKPyV infection were retrospectively included for analysis. Immunohistochemical (IHC) staining was performed on paraffin sections using monoclonal mouse anti-SV40 large T antigen antibody.ResultsIn BKPyV-infected glomeruli, the glomerular parietal epithelial cells (GPECs) were swollen, hyperchromatic, and enlarged, with an increased nuclear to cytoplasm (N/C) ratio and smudgy basophilic intra-nuclear viral inclusions. IHC staining revealed the distribution of BKPyV involvement in GPECs, podocytes, and shedding cells within Bowman’s space. Notably, BKPyV affected GPEC proliferation and caused crescent formation (7 biopsies, 15.9%). Three biopsies exhibited fibrous crescents and the absence of viral inclusions. The other 4 biopsies exhibited cellular and fibro-cellular crescents, with viral cytopathic changes and positive IHC staining in the proliferative GPECs. Electron microscopy showed viral particles in both GPECs and podocytes. BKPyV-infected GPECs were degenerative, with mitochondrial swelling, endoplasmic reticulum expansion, and multi-layered membranous structure formation. Twelve (27.3%) patients received repeat biopsies within 1.6 to 39.5 months (median: 13.5 months), but none revealed persistent glomerular BKPyV infection.ConclusionsDistinct glomerular changes in BKPyVAN biopsies should raise the possibility of glomerular involvement.

Single-Cell Transcriptome Profiling of the Kidney Glomerulus Identifies Key Cell Types and Reactions to Injury.

The glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability. Single-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency). All cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis. Generation of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.

Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

CD44 impacts glomerular parietal epithelial cell changes in the aged mouse kidney.

CD44 contributes to the activation of glomerular parietal epithelial cells (PECs). Although CD44 expression is higher in PECs of healthy aged mice, the biological role of CD44 in PECs in this context remains unclear. Accordingly, young (4 months) and aged (24 months) CD44−/− mice were compared to age‐matched CD44+/+ mice, both aged in a nonstressed environment. Parietal epithelial cell densities were similar in both young and aged CD44+/+ and CD44−/− mice. Phosphorylated ERK 1/2 (pERK) was higher in aged CD44+/+ mice. Vimentin and α‐SMA, markers of changes to the epithelial cell phenotype, were present in PECs in aged CD44+/+ mice, but absent in aged CD44−/− mice in both outer cortical (OC) and juxtamedullary (JM) glomeruli. Because age‐related glomerular hypertrophy was lower in CD44−/− mice, mTOR activation was assessed by phospho‐S6 ribosomal protein (pS6RP) staining. Parietal epithelial cells and glomerular tuft staining for pS6RP was lower in aged CD44−/− mice compared to aged CD44+/+ mice. Podocyte density was higher in aged CD44−/− mice in both OC and JM glomeruli. These changes were accompanied by segmental and global glomerulosclerosis in aged CD44+/+ mice, but absent in aged CD44−/− mice. These results show that the increase in CD44 in PECs in aged kidneys contributes to several changes to the glomerulus during healthy aging in mice, and may involve ERK and mTOR activation.

P0110DPP4- INHIBITOR LINAGLIPTIN ACCELERATES RESOLUTION OF CRESCENT FORMATION IN A RAT MODEL OF ANTI-GBM GLOMERULONEPHRITIS

Abstract Background and Aims Crescent formation is a severe glomerular damage in the pathogenesis of different kidney diseases. However the exact pathomechanism of crescent formation is unclear up to now. In human kidney biopsies we observed high dipeptidyl peptidase 4 (DPP4) expression in early crescent formation. Therefore, we investigated the potential involvement of DPP4 in the pathogenesis of crescentic anti-GBM nephritis in a rat model using a DPP4- inhibitor independent of his present use in the treatment of type 2 diabetes. Method Anti-GBM nephritis was induced in Wistar Kyoto rats for investigation of short and long term effects of the DPP4- inhibitor linagliptin treatment (3 mg/kg bw). In the short term experiment (endpoint 2 weeks) linagliptin treatment was started either with model induction or one week later. In the long term experiment (endpoint 8 weeks) linagliptin treatment was started either on day 0 or on week 4. In both experiments we included an untreated healthy and anti-GBM nephritic group. All groups consisted of 11 animals. Kidney function was monitored on weeks 1, 2, 3, 5 and 8. Kidney tissue was used to determine DPP4 expression and activity, morphologic changes and mRNA expression. Results Similarly to our observation in human kidney biopsies the in situ DPP4 activity and DPP4 expression were both increased in nephritic glomeruli especially within the crescents. Linagliptin could almost completely inhibit all renal DPP4 activity without any effect on DPP4 expression. Short-term treatment had no significant effect on crescent formation. However, long-term preventive treatment resulted in reduced number of crescents (51±3% vs 65±3%), glomerulosclerosis (score 1.2±0.07 vs 1.6±0.1), tubulointerstitial injury (score 1.2±0.1 vs 1.8±0.2), renal fibrosis (score 1.3±0.13 vs 1.9±0.14) and proteinuria (265±29 vs 363±22 mg/24h) compared to untreated nephritic rats. Therapeutic intervention with linagliptin resulted in weaker amelioration of renal disease at week 8, but also significantly reduced renal fibrosis (score 1.4±0.13 vs. 1.9±0.14) and crescent formation (52±4% vs. 65±3%) compared with vehicle. About 20% of all glomeruli showed Pax8+ parietal epithelial cells (PECs) on the tuft in short term treatment, being significantly reduced by therapeutic linagliptin treatment on day 14. In the long term experiment the number of Pax8+ PECs on glomerular tuft was reduced by more than 50% in both treatment groups and confirmed by quantitative mRNA analysis using isolated glomeruli. Despite the number of Pax8-positive cells on glomerular tuft was reduced by linagliptin therapy, a subpopulation of PECs positive for the transcription factor SOX9 was significantly increased on glomerular tuft compared to healthy and non-treated rats on day 14. Conclusion DPP4- inhibition with linagliptin reduced Pax8+ cells on glomerular tuft, indicating accelerated resolution of formed crescents and improvement in renal injury. This DDP4 effect might be mediated by differential regulation of a subpopulation of PECs expressing the transcription factor SOX9.

P0420PARTIAL PHARMACOGENETIC PODOCYTE DEPLETION IN A HIGH-THROUGHPUT ZEBRAFISH MODEL RESEMBLES HUMAN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS

Abstract Background and Aims Although focal and segmental glomerulosclerosis (FSGS) has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, glomerular podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Therefore, novel drug-screening assays are needed. Unfortunately, simple cellular in vitro-based screening assays are not ideal as glomerular architecture and crosstalk between glomerular cells is insufficiently modelled. Therefore, reliable animal models are still required for drug development, which unfortunately are not ideal for high-throughput applications. To date, due to its size, easy maintenance and breeding, zebrafish larvae are the simplest vertebrate model that are used in high-content screenings. Until today, it was unclear whether zebrafish can be used as a model for human FSGS. We therefore aimed to investigate whether partial podocyte-depletion in larval zebrafish leads to formation of FSGS-like disease and if the model can be used for screening purposes. Method We used a transgenic zebrafish model of pharmacogenetic podocyte depletion: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express the bacterial nitroreductase under control of the podocin promotor and can be dose-dependently ablated upon administration of metronidazole. Proteinuria was quantified using in vivo confocal laser scanning microscopy of intravenously administered high-molecular-weight fluorescent dextran. Plastic-embedded larvae where histologically and morphometrically assessed using HE, PAS and Jone’s silver staining after metronidazole washout. Glomerular ultrastructure was assessed using transmission electron microscopy of ultrathin sections. Immunofluorescence staining was carried out on kryosections to investigate extracellular matrix deposition (collagen-1, laminin), cellular proliferation (pcna) as well as parietal cell origin and activation (pax2a). Results To partially deplete podocytes, larvae where treated with 80 µM metronidazole from 4-6 days post fertilization, so that a subset of podocytes was depleted. In contrast to controls, podocyte-depleted larvae developed severe whole-body edema (Fig. A). Dynamic in vivo imaging of intravascular 500 kDa fluorescent dextran revealed massive leakage of the glomerular filtration barrier. Ultrastructural and immunofluorescent evaluation showed broad foot process effacement of remaining podocytes (Fig. D) and massive decrease of the slit diaphragm component podocin. Moreover, we found numerous sub-podocyte space pseudocysts (asterisk in Fig. D), microvillous transformation and formation of podocytic tight junctions as well as parietovisceral adhesions of the two layers of Bowman’s capsule. Parietal epithelial cells where activated, changed their phenotype towards a cuboidal shape, began to proliferate as demonstrated by pcna immunofluorescence and where recruited to cellular lesions on the glomerular tuft as demonstrated by the presence of cuboidal pax2a+ cells on the glomerular tuft (arrowheads Fig. B). Moreover, we found significant extracellular matrix deposition by the pax2a+ cells as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C). Conclusion Herein we show that upon podocyte-depletion, zebrafish larvae develop important functional and morphological features of human FSGS such as severe proteinuria and edema, podocyte foot process effacement, activation of parietal epithelial cells which contribute to cellular lesions and deposit extracellular matrix on the glomerular tuft. We conclude that this model resembles the human disease in important features and therefore propose its applicability for a high-throughput drug screening assay for FSGS.

P0336INHIBITION C-C CHEMOKINE RECEPTOR TYPE 2 (CCR2) ON ACTIVATED KIDNEY PARIETAL EPITHELIAL CELLS REVEALS A NEW THERAPEUTIC APPROACH FOR CHRONIC KIDNEY DISEASES

Abstract Background and Aims We have recently demonstrated that small molecule inhibitors of CCR2 rapidly reduced proteinuria and preserved renal structure in animal models of diabetic nephropathy (DN) and focal segmental glomerular sclerosis (FSGS). Similar beneficial effects of CCR2 inhibition were also observed in DN patients in a large diabetic nephropathy Phase 2 clinical trial. Despite its rapid and robust effects in these kidney diseases, the mechanism by which CCR2 inhibition affords such renal protection remains unclear. CCR2 is well-known to be expressed on cells of the immune system, where it mediates inflammation and immunological functions by controlling the trafficking of monocytes and T cells. However, immune cell expression of CCR2 may not adequately explain the rapid pharmacologic benefits of CCR2 inhibitors in DN and FSGS because these diseases do not exhibit appreciable kidney inflammation. To better understand the protective mechanism(s) of CCR2 inhibition in renal diseases, we have used a variety of in vivo nephropathy models to determine if CCR2 is expressed on kidney parenchymal cells. Our results suggest that a subset of CD45 negative, renal parietal epithelial cells express CCR2. Method To characterize CCR2 expression in the kidney we used CCR2 RFP heterozygous mice (CCR2RFP/+) in which one copy of CCR2 was replaced by red fluorescent protein (RFP), whose expression is under the control of the endogenous CCR2 promoter. This approach provided a highly sensitive method for CCR2 detection by both flow cytometry and immunofluorescence, and compensated for the lack of validated, commercially available anti-CCR2 antibodies. Enriched glomerular cells were obtained by a non-enzymatic disruption of kidneys, followed by centrifugation at low speed (80gs), and sieving through 300µm and 150µm meshes. The Adriamycin (ADR)-nephropathy model with CCR2 RFP heterozygous mice was used to determine CCR2 expression on renal cells. 5/6 nephrectomy models of FSGS, as well as the db/db diabetic nephropathy model were used to access the change of activated parietal epithelial cells and proteinuria, both in the presence and absence of a CCR2 inhibitor in the setting of kidney injury. Results We observed dramatic increases in the number of activated parietal epithelial cells (PECs, identified as CD133+CD24+CD44hi cells by flow cytometry) in all three renal disease models. In the ADR nephropathy model, we found that in 30-40% of activated PEC cells within an enriched population of glomerular cells expressed CCR2/RFP. CD44hi RFP+ increased much more dramatically than CD44hi RFP- cells in response to ADR, and this was highly correlated with proteinuria, suggesting that CCR2 plays an important role in PEC cell activation and kidney pathology in ADR induced kidney injury. Furthermore, we found that blocking CCR2 with the CCR2-specific antagonist CCX872 significantly reduced both proteinuria and the number of activated PECs in the 5/6 nephropathy and db/db diabetic nephropathy models. Conclusion This is the first report that CCR2 is present on a subset of activated renal PECs. This population is markedly upregulated during kidney injury in a variety of murine models of nephropathy. Changes in the abundance CCR2+ activated PECs in the glomerular enriched preparations in response to CCR2 inhibition suggests that CCR2 antagonism may represent a novel approach for the treatment of chronic kidney disease.

Spatio-temporal patterning of different connexins in developing and postnatal human kidneys and in nephrotic syndrome of the Finnish type (CNF)

Connexins (Cxs) are membrane-spanning proteins which enable flow of information important for kidney homeostasis. Changes in their spatiotemporal patterning characterize blood vessel abnormalities and chronic kidney diseases (CKD). We analysed spatiotemporal expression of Cx37, Cx40, Cx43 and Cx45 in nephron and glomerular cells of developing, postnatal kidneys, and nephrotic syndrome of the Finnish type (CNF) by using immunohistochemistry, statistical methods and electron microscopy. During kidney development, strong Cx45 expression in proximal tubules and decreasing expression in glomeruli was observed. In developing distal nephron, Cx37 and Cx40 showed moderate-to-strong expression, while weak Cx43 expression gradually increased. Cx45/Cx40 co-localized in mesangial and granular cells. Cx43 /Cx45 co-localized in podocytes, mesangial and parietal epithelial cells, and with podocyte markers (synaptopodin, nephrin). Different Cxs co-expressed with endothelial (CD31) and VSMC (α –SMA) markers in vascular walls. Peak signalling of Cx37, Cx43 and Cx40 accompanied kidney nephrogenesis, while strongest Cx45 signalling paralleled nephron maturation. Spatiotemporal Cxs patterning indicate participation of Cx45 in differentiation of proximal tubules, and Cx43, Cx37 and Cx40 in distal tubules differentiation. CNF characterized disorganized Cx45 expression in proximal tubules, increased Cx43 expression in distal tubules and overall elevation of Cx40 and Cx37, while Cx40 co-localized with increased number of interstitial myofibroblasts.

Inhibition of mTOR delayed but could not prevent experimental collapsing focal segmental glomerulosclerosis

Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation in vitro. However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions.