Parietal Epithelial Cells Research Articles

Association of urinary EGF, FABP3, and VCAM1 levels with the progression of early diabetic kidney disease.

Introduction Diabetic kidney disease (DKD) is a common cause of chronic kidney disease with around 25-40% of patients with diabetes being affected. The course of DKD is variable and estimated glomerular filtration rate (eGFR) and albuminuria, the currently used clinical markers, are not able to accurately predict the individual disease trajectory, in particular in early stages of the disease. The aim of this study was to assess the association of urine levels of selected protein biomarkers with the progression of DKD at an early stage of disease. Methods We measured 22 protein biomarkers using the Mesoscale Discovery platform in 461 urine samples of the PROVALID cohort, an observational study of patients with type 2 diabetes mellitus followed at the primary health care level for a minimum of four years. Odds ratios (OR) were estimated for the effect of marker values above median on fast progression using unadjusted and adjusted logistic regression models. RNA expression at the single cell level in kidney biopsy samples obtained from a cohort of young persons with type 2 diabetes mellitus was in addition determined for markers showing significant associations with disease progression. Results Increased urinary levels of epidermal growth factor (EGF) were linked to lower odds of fast progression (defined as annual eGFR decline greater than 2.58 ml/min per 1.73 m2) with an odds ratio (OR) of 0.60 (95% CI 0.46, 0.78). The association with outcome was even stronger when adjusting for a set of 14 baseline clinical parameters including age, biological sex, eGFR, body mass index, albuminuria, and HbA1c. Elevated urinary levels of fatty acid binding protein 3 (FABP3) and vascular cell adhesion molecule 1 (VCAM1) were each significantly associated with fast progression with an OR of 1.44 (95% CI 1.11, 1.87) and an OR of 1.41 (95% CI 1.08, 1.83), respectively. Enriched expression of EGF and FABP3 was observed in distal convoluted tubular cells and VCAM1 in parietal epithelial cells at single cell level from biopsies of patients with early DKD. Conclusion In summary we show that lower urinary levels of EGF and higher urinary levels of FABP3 and VCAM1 are significantly associated with DKD progression in early-stage disease.

The trans-differentiation promotion of parietal epithelial cells by magnesium isoglycyrrhizinate to improve podocyte injury induced by high fructose consumption

BackgroundPodocytes have limited proliferative capacity, which leads to irreversible glomerular injury in diverse kidney diseases. Magnesium isoglycyrrhizinate (MgIG), a hepatoprotective agent in clinic, has been reported to improve glomerular podocyte injury. However, the underlying mechanism of MgIG in ameliorating podocyte injury remains unclear. PurposeGlomerular parietal epithelial cells (PECs) are recognized as podocyte progenitors and play a pivotal role in the recovery following glomerular injury. This work aims to investigate the protective mechanisms of MgIG in mitigating glomerular injury by promoting PEC trans-differentiation. Study DesignA rat model of progressive glomerular podocyte injury, and in vitro models using the primary podocytes and primary PECs, were established to further explore the pharmacological mechanism of MgIG. MethodsFour-week-old male Sprague-Dawley (SD) rats were fed a 10% fructose solution for 3, 6, 9 and 12 weeks to induce glomerular injury. The effect of MgIG on the progressive changes in podocytes and PECs, and the correlation between PEC density and podocyte loss, were analyzed. The mechanism of MgIG in triggering PEC trans-differentiation was investigated, by examining adenosine secretion in injured podocytes, as well as the expression of cluster of differentiation 44 (CD44), nephrin, adenosine receptor A2B (ARA2B) and glucocorticoid receptor (GR) in PECs were examined both in vivo and in vitro. ResultsRats fed a high fructose diet exhibited progressive changes in glomerular PECs, including increased cell density and a preference for trans-differentiation. A positive correlation was observed between PEC density and podocyte loss. Co-culture experiments demonstrated that extracellular adenosine accumulation in injured podocytes induced by high fructose exposure promoted PEC trans-differentiation via ARA2B. MgIG significantly improved podocyte injury and exhibited effects similar to dexamethasone on nephrin upregulation activation and CD44 inhibition. Moreover, the inhibitory effect of MgIG on PEC ARA2B activation was more effective than that of dexamethasone. The co-expression of paired box 2 (PAX2)+-Nephrin+ in glomeruli indicated that MgIG induced PEC trans-differentiation and podocyte regeneration in model rats. Accordingly, podocyte loss and increased urine albumin-to-creatinine ratio (UACR) were also alleviated. Moreover, MgIG, which acts as a GR agonist to activate GR, reversed the upregulation of CD44 and decreased ARA2B induced by tumor necrosis factor-α (TNF-α) in primary PECs. The siRNA interference experiment manifested that MgIG exhibited a more pronounced enhancement of GR upregulation, in contrast to ARA2B activation, to promote PEC trans-differentiation. ConclusionThis work reports for the first time that PECs respond to the accumulation of extracellular adenosine from injured podocytes via activating ARA2B and focuses on the role of adenosine and adenosine receptors in the trans-differentiation of PECs. Furthermore, this study provides the first evidence that MgIG may promote podocyte regeneration by enhancing PEC trans-differentiation through GR activation, providing a research basis for investigating the glucocorticoid-like activity of MgIG in ameliorating glomerular podocyte injury.

Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFβR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-β1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both invivo and invitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFβR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis invivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFβR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

Mitochondrial-Derived Signaling Mediates Differentiation of Parietal Epithelial Cells into Podocytes.

Aims: Parietal epithelial cells (PECs) are potential stem cells within the glomerulus, migrating into site of podocyte loss to differentiate into podocytes. Little is known about the mechanism mediating differentiation of PECs into podocytes. Results: In vitro differentiation of PECs into podocytes led to upregulation of podocyte markers such as Wilms' tumor gene 1 (WT-1), Forkhead box C1 (FOXC1), synaptopodin and podocin, accompanied by increased mitochondrial abundance. Preincubation with a mitochondrial reactive oxygen species (ROS) inhibitor prevented all these events in PECs. In vivo, adriamycin (ADR)-treated mice exhibited albuminuria, decreased WT1 positive cells, and claudin-1 expressed in glomerular capillary tuft, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) overproduction in PECs. Expression of the ROS-related molecule nuclear factor erythroid 2-related factor 2 (Nrf2) and its target protein Brahma-related gene 1 (Brg1) increased during differentiation of PECs into podocytes. Suppressing Nrf2 or Brg1 reduced the differentiation of PECs, whereas overexpression had the opposite effect. Brg1 directly regulated WT-1 transcription in PECs. Activation of Nrf2 with bardoxolone-methyl (CDDO-Me) resulted in less proteinuria and more WT1 positive cells in ADR mice. PECs conditional human Nrf2 knock-in mice showed increased WT1 cell numbers. Conclusion: It concluded that mitochondria-derived ROS mediated differentiation of PECs into podocytes via Nrf2 and Brg1 signaling.

Artificial Intelligence (AI) Reveals Dynamic Morphological Shifts in Parietal Epithelial Cells after Acute Tubular Injury

Artificial Intelligence (AI) Reveals Dynamic Morphological Shifts in Parietal Epithelial Cells after Acute Tubular Injury

HSP27 Promotes Activation of Parietal Epithelial Cells and Crescent Formation in Crescentic Glomerulonephritis

HSP27 Promotes Activation of Parietal Epithelial Cells and Crescent Formation in Crescentic Glomerulonephritis

Single-cell RNA sequencing data locate ALDH1A2-mediated retinoic acid synthetic pathway to glomerular parietal epithelial cells.

Aldehyde dehydrogenase 1, family member A2, is a retinoic acid-synthesizing enzyme encoded by Aldh1a2 in mice and ALDH1A2 in humans. This enzyme is indispensable for kidney development, but its role in kidney physiology and pathophysiology remains to be fully defined. In this review, we mined single-cell and single-nucleus RNA sequencing databases of mouse and human kidneys and found that glomerular parietal epithelial cells (PECs) express a full set of genes encoding proteins needed for cellular vitamin A uptake, intracellular transport, and metabolism into retinoic acid. In particular, Aldh1a2/ALDH1A2 mRNAs are selectively enriched in mouse and human PECs. Aldh1a2 expression in PECs is greatly increased in a mouse model of anti-glomerular basement membrane glomerulonephritis and moderately induced in a mouse model of ischemia-reperfusion acute kidney injury. Aldh1a2 expression in PECs is substantially repressed in a chronic kidney disease mouse model combining diabetes, hypertension, and partial nephrectomy and is moderately repressed in mouse models of focal segmental glomerulosclerosis and diabetic nephropathy. Single-nucleus RNA sequencing data show that ALDH1A2 mRNA expression in PECs is diminished in patients with chronic kidney disease associated with diabetes, hypertension and polycystic kidney disease. In addition to data mining, we also performed Spearman's rank correlation coefficient analyses and identified gene transcripts correlated with Aldh1a2/ALDH1A2 transcripts in mouse PECs and PEC subtypes, and in human PECs of healthy subjects and patients with AKI or CKD. Furthermore, we conducted Gene Ontology pathway analyses and identified the biological pathways enriched among these Aldh1a2/ALDH1A2-correlated genes. Our data mining and analyses led us to hypothesize that ALDH1A2-mediated retinoic acid synthesis in PECs plays a yet-undefined role in the kidney and that its dysregulation mediates injury. Conditional, PEC-selective Aldh1a2 knockout, RNA silencing and transgenic mouse models will be useful tools to test this hypothesis. Clinical studies on genetics, epigenetics, expression and functions of ALDH1A2 and other genes needed for retinoic acid biosynthesis and signaling are also warranted.

Investigating FSGS-like injury in zebrafish larvae by nifurpirinol: efficacy and molecular insight.

Identifying effective drugs for focal segmental glomerulosclerosis (FSGS) treatment holds significant importance. Our high-content drug screening on zebrafish larvae relies on nitroreductase/metronidazole (NTR/MTZ)-induced podocyte ablation to generate FSGS-like injury. A crucial factor for successful drug screenings is minimizing variability in injury induction. For this, we introduce nifurpirinol (NFP) as a more reliable prodrug for targeted podocyte depletion. NFP showed a 2.3-fold increase in efficiency at concentrations 1,600-fold lower compared with MTZ-mediated injury induction. Integration into the screening workflow validated its suitability for the high-content drug screening. The presence of crucial FSGS hallmarks, such as podocyte foot process effacement, proteinuria, and activation of parietal epithelial cells, was observed. After the isolation of the glomeruli from the larvae, we identified essential pathways by proteomic analysis. This study shows that NFP serves as a highly effective prodrug to induce the FSGS-like disease in zebrafish larvae and is well-suited for a high-content drug screening to identify new candidates for the treatment of FSGS.NEW & NOTEWORTHY This research investigated the use of nifurpirinol in nanomolar amounts as a prodrug to reliably induce focal segmental glomerulosclerosis (FSGS)-like damage in transgenic zebrafish larvae. Through proteomic analysis of isolated zebrafish glomeruli, we were further able to identify proteins that are significantly regulated after the manifestation of FSGS. These results are expected to expand our knowledge of the pathomechanism of FSGS.

Cardiotrophin-1 therapy reduces disease severity in a murine model of glomerular disease.

Cardiotrophin-1 (CT-1), a member of the interleukin (IL)-6 cytokine family, has renoprotective effects in mouse models of acute kidney disease and tubulointerstitial fibrosis, but its role in glomerular disease is unknown. To address this, we used the mouse model of nephrotoxic nephritis to test the hypothesis that CT-1 also has a protective role in immune-mediated glomerular disease. Using immunohistochemistry and analysis of single-cell RNA-sequencing data of isolated glomeruli, we demonstrate that CT-1 is expressed in the glomerulus in male mice, predominantly in parietal epithelial cells and is downregulated in mice with nephrotoxic nephritis. Furthermore, analysis of data from patients revealed that human glomerular disease is also associated with reduced glomerular CT-1 transcript levels. In male mice with nephrotoxic nephritis and established proteinuria, administration of CT-1 resulted in reduced albuminuria, prevented podocyte loss, and sustained plasma creatinine, compared with mice administered saline. CT-1 treatment also reduced fibrosis in the kidney cortex, peri-glomerular macrophage accumulation and the kidney levels of the pro-inflammatory mediator complement component 5a. In conclusion, CT-1 intervention therapy delays the progression of glomerular disease in mice by preserving kidney function and inhibiting renal inflammation and fibrosis.

A Reference Profile of N-Glycosylation for Human Kidney and the Identification of Cell-Cell Interactions between Parietal Epithelial Cells and Capillary Endothelial Cells by Single-Cell Glycosylation-Sequencing

Background: N-glycosylation is one of the most common posttranslational modifications in humans, and these alterations are associated with kidney diseases. Methods: A novel technological approach, single-cell N-acetyllactosamine sequencing (scLacNAc-seq), was applied to simultaneously detect N-glycosylation expression and the transcriptome at single-cell resolution in three human kidney tissues from zero-time biopsy. Cell clusters, glycation abundance in each cell cluster, functional enrichment analysis, cell-cell crosstalk, and pseudotime analysis were applied. Results: Using scLacNAc-seq, 24,247 cells and 22 cell clusters were identified, and N-glycan abundance in each cell was obtained. Transcriptome analysis revealed a close connection between capillary endothelial cells (CapECs) and parietal epithelial cells (PECs). PECs and CapECs communicate with each other through several pairs of ligand receptors (e.g., TGFB1-EGFR, GRN-EGFR, TIMP1-FGFR2, VEGFB-FLT1, ANGPT2-TEK, and GRN-TNFRSF1A). Finally, a regulatory network of cell-cell crosstalk between PECs and CapECs was constructed, which is involved in cell development. Conclusions: We here, for the first time, constructed the glycosylation profile of 22 cell clusters in the human kidney from zero-time biopsy. Moreover, cell-cell communication between PECs and CapECs through the ligand-receptor system may play a crucial regulatory role in cell proliferation.

#417 Targeting parietal epithelial cell activation with mineralocorticoid receptor antagonism controls FSGS and crescentic glomerulonephritis

Abstract Background and Aims We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury [1]. Accumulating evidence has indicated the potential contributions of the mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is expressed in endothelial cells, glomerular mesangial cells, podocytes, and distal tubular cells. Previous studies have shown that administering mineralocorticoid receptor antagonists has beneficial effects in various renal injury animal models, the role of the MR in extracapillary glomerulopathies is still elusive and mechanistically unclear. Methods To investigate the cell-specific role of the MR in PEC in the course of focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (GN), we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Maladaptive FSGS was induced by combining a high-salt diet and deoxycorticosterone acetate (DOCA) with unilateral nephrectomy (DOCA-UniN). Crescentic glomerulonephritis (GN) was induced using the anti-glomerular basement membrane nephrotoxic serum (NTS) model. Results At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged by DOCA-UniN, PEC-selective Nr3c2 targeting significantly inhibited the expression of PEC activation markers Ki67, CD44, and FSGS lesions. Importantly, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function compared to Pec Cre Nr3c2 wt/wt counterparts. Likewise, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function in the NTS-induced CGN model. Crescents were also more numerous and organised in Pec Cre Nr3c2wt/wt mice than in Pec Cre Nr3c2lox/lox animals. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less expression of the CD44 activation marker on PECs. Thus, genetic disruption of MR in PEC and pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC within FSGS and crescents. We next evaluated PEC migration under various conditions (DMSO, HB-EGF, Eplerenone, or HB-EGF+Eplerenone) for 24 hours. Eplerenone fully abrogated HB-EGF-induced PEC migration and Pcna, Snai1, and Vim mRNA expressions. Conclusion Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis, along with the recently discovered CD9/EGFR/PDGFR [1] and CLDN1 pathways. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in FGSS and, more surprisingly, in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

#2837 Novel therapeutic for crescentic glomerulonephritis through targeting CLDN1 in parietal epithelial cells

Abstract Background and Aims Crescentic glomerulonephritis (CrGN) is the final mode of kidney injury common to several immune-mediated kidney diseases. CrGN is characterized by extensive glomerular parietal epithelial cells (PECs) proliferation forming crescents. The destructive crescent is then progressively replaced by fibrosis. Currents therapies for CrGN do not directly target the deleterious response of resident kidney cells. In a pathological context, claudin-1 (CLDN1), a transmembrane protein involved in epithelial tight junctions, can be exposed outside the tight junctions and mediate pro-fibrotic pathways and extracellular matrix (ECM) remodelling. Lixudebart (formerly ALE.F02) is a first-in-class monoclonal antibody that specifically targets and blocks exposed CLDN1 in injured epithelial cells. A phase II clinical trial is under way for patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) with rapidly progressive glomerulonephritis which induces kidney fibrosis (RENAL-F02, NCT06047171). CLDN1 is highly expressed by glomerular PECs. This study investigated the functional role of CLDN1 in CrGN and the potential benefit to target CLDN1 in CrGN. Method CLDN1 expression in renal tissues of CrGN patients was analyzed using kidney multicolor immunofluorescence staining and spatial transcriptomics. Correlation between CLDN1 expression and clinical endpoints (eGFR, proteinuria), disease biomarkers and crescent evolution were studied. A spatially resolved molecular roadmap from CLDN1+ crescentic glomeruli was conducted. Proof-of-concept studies using anti-CLDN1 mAb were performed in preclinical models of CrGN. Results In tissues (n = 150) of patients with AAV and IgAN, multicolor immunofluorescence revealed up-regulated CLDN1 expression by cellular and fibrocellular crescents. At the time of diagnostic kidney biopsy, glomerular CLDN-1 expression was correlated with podocyte loss (as measured with P57 staining) and fibronectin area. The extent of dual expression of CLDN1 and CD44 at the surface of active PECs was associated with poor renal outcome (eGFR < 30 ml/min) in AAV and IgAN patients with a median follow-up of 2.5 and 3.7 years respectively. Spatial transcriptomics analysis highlighted the association between CLDN1+ crescentic glomeruli and ECM genes. Treatment with anti-CLDN1 mAb reduced albuminuria, improved kidney function and decreased fibrosis biomarkers in nephrotoxic serum-induced CrGN in mice. Conclusion Our results suggest a functional role of CLDN1 in the pathogenesis of CrGN, providing preclinical proof-of-concept for anti-CLDN1 antibodies as a novel therapeutic approach in patients with CrGN.

#1603 A potent protein inhibitor of HB-EGF protects kidneys from glomerular lesions in a pig model of crescentic glomerulonephritis

Abstract Background and Aims Crescentic glomerulonephritis (CGN) is a severe disease that rapidly destroys the glomerular architecture. CGN is due to immuno-inflammatory aggressions triggered by anti-neutrophil cytoplasmic autoantibody-associated vasculitides, anti-glomerular basement membrane (GBM) autoantibodies, or immune complexes deposition as occurs in systemic lupus erythematosus, IgA nephropathy or infections. Treatments are based on aggressive immunosuppression. Although required, immunosuppression doesn't directly target the deleterious glomerular response to inflammation and leads to significant morbidity. Moreover, most patients still develop chronic or end-stage kidney failure, indicating that complementary strategies are needed. We showed previously that local over-expression of the growth factor HB-EGF (Heparin-binding EGF-like growth factor) and subsequent activation of EGFR lead to dedifferentiation and proliferation of parietal epithelial cells and podocytes, leading to crescentic lesions that irreversibly obliterate the glomeruli. We aimed to evaluate the therapeutic potential of a protein inhibitor of HB-EGF in a pig model of CGN. Method We engineered a protein inhibitor of HB-EGF, DTR8, from the receptor-binding domain of diphtheria toxin, a highly selective natural ligand of HB-EGF. Twelve mutations improved solubility, led to pM affinity, and reduced immunogenicity and antigenicity. Pigs received decomplemented nephrotoxic serum (NTS) from sheep immunized against pig GBM to mimic anti-GBM antibodies disease responsible for one form of CGN. Ten pigs were treated with 0.3 mg/kg of DTR8 twice a day by intramuscular route for 20 days, and 12 pigs received vehicle only starting 6 hours after NTS. Animals were sacrificed on day 21, and blood, urine and kidneys were analyzed. Five animals that had received pre-immune sheep serum served as controls. Results Pigs developed a nephritic syndrome with high proteinuria, hypoalbuminemia, kidney failure with increased BUN and blood creatinine levels, anasarca and crescentic lesions typical of CGN along with de novo HB-EGF glomerular overexpression. The vehicle-treated group had only 24.8% of histologically normal glomeruli, while the DTR8-treated group had 57.3% of healthy glomeruli. DTR8 treatment significantly reduced the percentage of crescentic lesions (P = .0365) and preserved podocyte differentiation as assessed by the expression of nephrin that was markedly reduced in vehicle-treated animals but maintained close to normal in DTR8-treated animals (P = .0028). DTR8 administration reduced EGFR phosphorylation in glomerular cells (P = .0161), suggesting local inhibition of the HB-EGF/EGFR cascade. Lymphocyte infiltration was reduced (P = .2543). Notably, the treatment group also displayed a global reduction in interstitial fibrosis (P = .0313). Conclusion The therapeutic protein candidate DTR8 protects kidney glomeruli in a CGN model in pigs.

#2145 Sexual dimorphism in kidney regenerative capacity decline and in progressive tubular cell polyploidization during aging

Abstract Background and Aims Aging is characterized by a progressive kidney function decline, resulting in chronic kidney disease (CKD), which is steeper in men than in women. However, sex-related differences in mechanisms underpinning CKD development during aging still remain to be clarified. In most organs regenerative capacity declines overtime from a pool of resident stem cells leading to tissue aging. In the kidney, we have revealed that two adaptive responses co-exist to recover kidney function after injury. On one hand a population of renal progenitors (RPC), which is interspersed among parietal epithelial cells of Bowman's capsule and tubular cells (TC) of nephron segments, proliferate and generate new podocytes and TC to recover structural integrity during the regenerative processes occurring in glomerular and tubular diseases. On the other, TC enter the alternative cell cycles to become polyploid in order to increase their functional capacity and maintain the residual kidney function after injury. However, a continuous TC polyploidization comes with the long term trade-offs (fibrosis and senescence), driving CKD. Here, we aimed to investigate the role of sexual dimorphism in these two different adaptive responses during aging. Method To trace RPC (Pax2+cells) we used the inducible Pax2/Confetti or Pax2/mTmG mice (RPC are randomly labelled by one of the four fluorescent reporter genes or with GFP respectively). To study polyploid TC, we used the inducible Pax8/FUCCI2aR mice (polyploid TC are identified by expression of cell cycle fluorescent proteins in combination with DNA content analysis). Mice were analyzed at 2, 5, 12 and 20 months of age. scRNA sequencing was performed at 2 and 20 months of age. Results Pax2/Confetti mice revealed that the number of Pax2+RPC either of the Bowman's capsule or in tubules was higher in female than in male mice at all ages. Specifically, tracing analysis during aging showed that in females Pax2+RPC of the Bowman's capsule generated new podocytes, resulting in glomeruli with a better integrity of the slit-diaphragm compared with aged males. Moreover, tubular Pax2+RPC in females clonally expanded, generating a higher number of clones, which were longer than those in males. scRNAseq analysis on kidneys showed that females were endowed with a greater number of Pax2+RPC with a higher expression of Pax2 than males, suggesting that RPC in females kept a higher regenerative capacity than males during aging. Simultaneously, analysis of tubular polyploidy demonstrated that, whereas in females the polyploid fraction progressively increased, it was drastically reduced in males during aging. These results suggested that whereas in females the increase of polyploid TC provided a better maintenance of kidney function overtime, loss of polyploid response in males made them more susceptible to develop CKD. Indeed, aged male mice showed a higher percentage of polyploid TC undergoing further cycles of polyploidization, which were associated with fibrosis and senescence. In agreement with these findings, we observed a progressive kidney function decline along with an increase of glomerulosclerosis, interstitial fibrosis and blood pressure, which were significantly greater in males than in females. Conclusion Female mice showed a higher regenerative and polyploid response compared to male mice, aimed likely to cope kidney function decline during aging. These results reveal the intriguing role of a new kidney adaptive response, which may explain the higher susceptibility of males to develop a more premature CKD during aging.

Adhesion G Protein-Coupled Receptor Gpr126 (Adgrg6) Expression Profiling in Diseased Mouse, Rat, and Human Kidneys.

Uncovering the function of understudied G protein-coupled receptors (GPCRs) provides a wealth of untapped therapeutic potential. The poorly understood adhesion GPCR Gpr126 (Adgrg6) is widely expressed in developing kidneys. In adulthood, Gpr126 expression is enriched in parietal epithelial cells (PECs) and epithelial cells of the collecting duct and urothelium. Whether Gpr126 plays a role in kidney disease remains unclear. Here, we characterized Gpr126 expression in diseased kidneys in mice, rats, and humans. RT-PCR data show that Gpr126 expression is altered in kidney disease. A quantitative RNAscope® analysis utilizing cell type-specific markers revealed that Gpr126 expression upon tubular damage is mainly increased in cell types expressing Gpr126 under healthy conditions as well as in cells of the distal and proximal tubules. Upon glomerular damage, an increase was mainly detected in PECs. Notably, Gpr126 expression was upregulated in an ischemia/reperfusion model within hours, while upregulation in a glomerular damage model was only detected after weeks. An analysis of kidney microarray data from patients with lupus nephritis, IgA nephropathy, focal segmental glomerulosclerosis (FSGS), hypertension, and diabetes as well as single-cell RNA-seq data from kidneys of patients with acute kidney injury and chronic kidney disease indicates that GPR126 expression is also altered in human kidney disease. In patients with FSGS, an RNAscope® analysis showed that GPR126 mRNA is upregulated in PECs belonging to FSGS lesions and proximal tubules. Collectively, we provide detailed insights into Gpr126 expression in kidney disease, indicating that GPR126 is a potential therapeutic target.

Renin-Angiotensin System Signaling in Parietal Epithelial Cells

INTRODUCTION. Bowman's capsule surrounds the kidney glomerular tuft and consists of a monolayer of parietal epithelial cells (PECs) creating a physiologically functioning barrier between the urinary space and the renal interstitium. Pathophysiological PEC activation describes the phenotypic switch from their normal quiescent state to one of proliferation and migration to the glomerular tuft, leading to glomerular injury. The development of several proteinuric kidney diseases is closely associated with PEC activation, however, the mechanisms of functional receptor signaling in these cells are not well described. This study investigates the functional presence of renin-angiotensin system (RAS) components and corresponding intracellular mechanisms in PECs. Methods. The vibrodissociation method was utilized to obtain freshly isolated Bowman’s capsules from discarded adult male human transplant tissues and 12-week old Wistar male and female rats. Confocal fluorescent microscopy of freshly isolated Bowman’s capsule PECs was performed using Fluo-8 or DAF-FM fluorescent dyes to detect changes in intracellular Ca2+ concentrations and nitric oxide (NO) response to the variety of RAS peptides. RESULTS. In separate experiments on freshly isolated rodent and human Bowman’s capsule PECs, we demonstrate the changes in redox response to the stimulation of RAS components. In particular, the acute application of Ang II peptide caused a rapid increase in NO production. Interestingly, we did not indicate significant changes in intracellular Ca2+ influx. To find out which receptor is responsible for the NO release we preincubated PECs with AT1R and AT2R blockers (Losartan and PD123319 respectively). Our data indicate that RAS mediates NO production through the AT2R activation. There is no sex difference was observed in these experiments. Further experiments with the variety of nitric oxide synthase (NOS) pharmacology reveal the functional distribution of NOS in rodent PECs. CONCLUSION. Our study provides an overview of the RAS signaling within PECs. For the first time, we present evidence demonstrating a direct connection between Ang II and redox homeostasis in PECs. Further investigation using the developed approach will be essential for understanding the role of Bowman's capsule cells in the glomerulus and the development of potential treatment against the hyperplasia of activated PECs and corresponding chronic kidney pathologies. Funding Sources: R01 DK129227, R01 DK126720, VA CDA-2 1IK2BX005605-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obesity-related glomerulopathy is associated with elevated WT1 expression in podocytes.

The prevalence of obesity is increasing worldwide at an alarming rate. In addition to the increased incidence of cardiovascular and metabolic diseases, obesity is the most potent risk factor for developing chronic kidney disease (CKD). Although systemic events such as hemodynamic factors, metabolic effects, and lipotoxicity were implicated in the pathophysiology of obesity-related glomerulopathy (ORG) and kidney dysfunction, the precise mechanisms underlying the association between obesity and CKD remain unexplored. In this study, we employed spontaneous WNIN/Ob rats to investigate the molecular events that promote ORG. Further, we fed a high-fat diet to mice and analyzed the incidence of ORG. Kidney functional parameters, micro-anatomical manifestations, and podocyte morphology were investigated in both experimental animal models. Gene expression analysis in the rodents was compared with human subjects by data mining using Nephroseq and Kidney Precision Medicine Project database. WNIN/Ob rats were presented with proteinuria and several glomerular deformities, such as adaptive glomerulosclerosis, decreased expression of podocyte-specific markers, and effacement of podocyte foot process. Similarly, high-fat-fed mice also showed glomerular injury and proteinuria. Both experimental animal models showed increased expression of podocyte-specific transcription factor WT1. The altered expression of putative targets of WT1 such as E-cadherin, podocin (reduced), and α-SMA (increased) suggests elevated expression of WT1 in podocytes elicits mesenchymal phenotype. Curated data from CKD patients revealed increased expression of WT1 in the podocytes and its precursors, parietal epithelial cells. WT1 is crucial during nephron development and has minimal expression in adult podocytes. Our study discovered elevated expression of WT1 in podocytes in obesity settings. Our analysis suggests a novel function for WT1 in the pathogenesis of ORG; however, the precise mechanism of WT1 induction and its involvement in podocyte pathobiology needs further investigation.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Comparative analysis of effects of conditioned mediums obtained from 2D or 3D cultured mesenchymal stem cells on kidney functions of diabetic rats: Early intervention could potentiate transdifferentiation of parietal epithelial cell into podocyte precursors

AimThe secretome of mesenchymal stem cells (MSCs) could be a potential therapeutic intervention for diabetes and associated complications like nephropathy. This study aims to evaluate the effects of conditioned mediums (CMs) collected from umbilical cord-derived MSCs incubated under 2-dimensional (2D) or 3D culture conditions on kidney functions of rats with type-I diabetes (T1D). Main methodsSprague-Dawley rats were treated with 20 mg/kg streptozocin for 5 consecutive days to induce T1D, and 12 doses of CMs were applied intraperitoneally for 4 weeks. The therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological, and immunohistochemical analysis. Key findings3D-CM had significantly higher total protein concentration than the 2D-CM Albumin/creatinine ratios of both treatment groups were significantly improved in comparison to diabetes. Light microscopic evaluations showed that glomerular and cortical tubular damages were significantly ameliorated in only the 3D-CM applied group compared to the diabetes group, which were correlated with transmission electron microscopic observations. The nephrin and synaptopodin expressions increased in both treatment groups compared to diabetes. The WT1, Ki-67, and active caspase-3 expressions in glomeruli and parietal layers of the treatment groups suggest that both types of CMs suppress apoptosis and promote possible parietal epithelial cells' (PECs') transdifferentiation towards podocyte precursor cells by switching on WT1 expression in parietal layer rather than inducing new cell proliferation. Significance3D-CM was found to be more effective in improving kidney functions than 2D-CM by ameliorating glomerular damage through the possible mechanism of transdifferentiation of PECs into podocyte precursors and suppressing glomerular apoptosis.