Introduction: X-linked Alport syndrome (XLAS) is a well-known monogenetic kidney disease caused by pathogenic variants in the COL4A5 gene. Routine analysis of exons and direct flanking regions fails to identify a pathogenic variant in 10% to 20% of patients with XLAS. Methods: We evaluated 11 selected patients with clinical features of XLAS, in whom routine analysis failed to identify a pathogenic variant. In 2 patients a variant of unknown significance was detected in the intronic splice site regions. We used mRNA analysis from fibroblasts or urine-derived podocyte-lineage cells to establish a genetic diagnosis. Results: In 2 patients with a variant of unknown significance (VUS), mRNA analysis confirmed the pathogenicity. In 9 patients, mRNA analysis was used to evaluate aberrant splicing and guide genomic DNA sequencing. In 7 patients a novel pathogenic deep-intronic variant was found. Overall, aberrant splicing was complete in 5 patients and partial in 4, whereas kidney disease was less severe in the latter group. Conclusion: This report highlights the importance of mRNA analysis to confirm pathogenicity or facilitate the search for intronic variants to establish a genetic diagnosis in XLAS. This analysis can serve as a diagnostic tool in patients suspected for Alport syndrome (AS) when routine genetic analysis fails to identify a pathogenic variant.

BACKGROUND:Preterm birth disrupts kidney development, resulting in reduced nephron number and structural immaturity of glomeruli and podocytes that increases the risk of hypertension, proteinuria, and subsequently chronic kidney disease (CKD) later in life. Since nephrogenesis ceases around 36 weeks of gestation, preterm infants cannot generate new nephrons after birth, making them vulnerable to long-term renal dysfunction. Unravelling the mechanisms behind this impaired development has been limited by the complexity of human nephrogenesis and the lack of physiologically relevant experimental models. SUMMARY:Recent advances in human-induced pluripotent stem cell-derived kidney organoids have made it possible to model nephrogenesis in vitro. These organoids replicate key processes such as nephron differentiation, ureteric bud branching, and kidney vascularization, allowing detailed study of kidney development and injury. Moreover, molecular interventions - including retinoic acid (RA), glial-cell-line-derived neurotrophic factor (GDNF), and insulin-like growth factor 1 (IGF1) - show the potential to enhance nephron formation and protect against kidney injury. KEY MESSAGES:(1) Kidney organoids provide a powerful, human-relevant system to study nephrogenesis and CKD mechanisms. (2) Integration of the ureteric bud and enhanced vascularization significantly improves organoid maturity towards a more in vivo-like state. (3) Targeted molecular interventions such as RA, GDNF, and IGF1 offer potential strategies to enhance nephron endowment and mitigate CKD risk in preterm-born individuals and may be studied using kidney organoids.

Introduction The disease course of primary nephrotic syndrome (PNS) is highly variable, and is difficult to predict at onset. PNS is characterized by podocyte injury and loss. We hypothesized that measurement of urinary podocyte loss is associated with treatment response in patients with PNS. Methods We included 21 controls, and 59 patients with PNS (minimal change disease [MCD], n =8; focal segmental glomerulosclerosis [FSGS], n = 9; membranous nephropathy [MN]. n = 42). MCD and FSGS were considered manifestations of the same disease entity, and analyzed as one group. Patients’ baseline clinical and follow-up data were recorded. Urinary sediments were collected and stained for podocyte-specific markers, and analyzed using fluorescence-activated cell sorting (FACS). Results In patients with MCD/FSGS, the respective partial and complete remission rates were 24% and 59% during a median follow-up of 12.9 months, and all patients received immunosuppressive treatment. In patients with MN, the respective partial and complete remission rates were 64% and 19% during a median follow-up of 15.5 month, and the majority of patients received immunosuppressive treatment. Patients with PNS had elevated levels of podocyturia when compared with controls, and repeat measurements revealed that podocyturia follows proteinuria course over time, normalizing following complete proteinuria remission. In treatment-responsive immunosuppresive-treated patients with MCD/FSGS, podocyturia at baseline significantly differentiated between early and late treatment responders at 4 weeks, contrary to proteinuria and serum albumin. In symptomatically treated patients with MN, low levels of podocyturia at baseline were associated with spontaneous remission. Conclusion Patients with PNS have increased podocyturia compared with healthy individuals. Quantitative detection of podocyturia may have prognostic relevance in patients with PNS.

BACKGROUND:Primary focal segmental glomerulosclerosis (FSGS) is characterized by podocyte injury and treatment-resistant nephrotic syndrome. Recurrence of the original disease after kidney transplantation (rFSGS) occurs in 10%-50% of patients. Unidentified circulating permeability factors (CPF) are likely involved in FSGS pathogenesis. We hypothesized that donor podocyte susceptibility to CPF is also relevant. We developed a personalized model for (r)FSGS using induced pluripotent stem cell (iPSC)-derived podocytes from patients and kidney donors. METHODS:Five patients and their respective living kidney donors were included. Three patients had developed rFSGS, and two patients manifested no symptoms of rFSGS. One patient (P5) had heterozygous mutations in NPHS2. Peripheral blood mononuclear cells were reprogrammed to iPSC, and differentiated to podocytes. iPSC-derived podocytes from either patients or donors were exposed to presumed CPF-containing plasma/serum of corresponding patients. Three assays to detect podocyte injury were performed: (i) reactive oxygen species formation, (ii) cellular granularity induction, and (iii) quantitative assessment of F-actin redistribution (FAR), a new quantitative method. Crossmatch experiments with donor iPSC-derived podocytes and patients samples assessed individual susceptibility to CPF-induced injury. RESULTS:Successful podocyte differentiation was confirmed by morphology and protein expression. Only FAR differentiated consistently between patient and healthy donor samples. All pre-transplant patient samples except P5 caused significant FAR in corresponding patient podocytes. Significant FAR was observed in donor podocytes exposed to corresponding patient samples in the setting of rFSGS, and not in donor podocytes exposed to samples of patients who did not develop rFSGS. Effects of FSGS patient samples on non-corresponding donor podocytes were variable. CONCLUSIONS:In vitro assays using iPSC-derived donor podocytes may allow individualized assessment of rFSGS. Prospective studies in a larger cohort are required to validate our findings.

Rationale & Objective:Clinical outcome of primary nephrotic syndrome (PNS) is highly variable, and predicting an individual patient's treatment response remains difficult. PNS is characterized by means of podocyte injury and loss. We hypothesized that histologic parameters related to podocyte depletion predict treatment response. Study Design:Retrospective cohort study. Setting & Participants:We analyzed biopsy tissue of 106 patients with PNS (minimal change disease, N = 26; focal segmental glomerulosclerosis, N = 21; and membranous nephropathy [MN], N = 59) and 9 controls. Minimal change disease and focal segmental glomerulosclerosis were considered manifestations of the same entity, defined as idiopathic nephrotic syndrome (iNS), and analyzed as one group. Patients' baseline clinical and follow-up data were recorded. Kidney biopsies, stained for podocyte-specific and fibrosis markers, were quantitatively analyzed. Predictors:Glomerular density, glomerulosclerosis, podocyte number, podocyte density, and cortical fibrosis. Outcomes:Complete remission (CR) and delayed treatment response. Analytical Approach:Odds ratios and receiver operating characteristic-the area under the curve (ROC-AUC) values identified predictors. Results:In patients with iNS, the respective partial remission and CR rates were 29% and 60% during a median follow-up of 40 months. The majority of patients received high-dose corticosteroid treatment. Quantitation of cortical fibrosis had the highest discriminative power (ROC-AUC value, 0.79; 95% CI, 0.655-0.923) to predict CR. Other significant predictors included podocyte density, nonsclerotic glomerular density, and percentage of nonsclerotic glomeruli.In patients with MN, respective partial remission and CR rates were 41% and 54% during a median follow-up of 50 months. The percentage of nonsclerotic glomeruli and nonsclerotic glomerular density were predictors for CR (patients receiving immunosuppressive treatment [ROC-AUC value, 0.71; 95% CI, 0.535-0.893]; patients receiving nonimmunosuppressive treatment alone [ROC-AUC value, 0.80; 95% CI, 0.584-1.000]). Limitations:Relatively small cohorts prevented the use of covariates. Conclusions:In patients with iNS, higher podocyte density and nonsclerotic glomerular density, and lower glomerulosclerosis and cortical fibrosis predicted CR. In patients with MN, lower glomerulosclerosis and higher nonsclerotic glomerular density predicted CR. Biopsy parameters may thus be useful for estimating proteinuria outcome.

The mechanisms driving the development of extracapillary lesions in crescentic glomerulonephritis (CGN) and focal segmental glomerulosclerosis (FSGS) remain poorly understood. This study investigates the role of serotonin (5-HT) and its receptors, specifically HTR2A and HTR2B, in murine models of these diseases. This study employed a combination of genetic, pharmacological, and in vitro approaches to elucidate the role of serotonin receptors in CGN and FSGS. We found that both receptors were upregulated in glomerular parietal epithelial cells in both human and murine CGN. We found that both genetic deletion and pharmacological inhibition of the 5-HT2A receptor effectively reduced glomerular crescents and sclerosis, while the compensatory upregulation of 5-HT2A receptors in response to 5-HT2B receptor deficiency or antagonism underscores a complex interplay between these receptor subtypes. Bone marrow-specific deletion and repletion of HTR2A-competent cells did not alter the course of experimental CGN, suggesting that the 2A signals driving CGN originate from the kidney. The Alport model, which is primarily degenerative, also benefitted in a similar way from 2A deletion, further corroborating the role of kidney resident 2A signaling in these diseases. Additionally, platelets were identified as a source of 5-HT and a crucial upstream factor in promoting PEC activation and exacerbating the disease, with their depletion leading to significant mitigation of disease severity. Finally, in vitro studies using murine PECs indicated the pivotal role of 5-HT signaling in PEC activation. Our findings highlight a critical role of 5-HTR2A in PEC activation, proposing it as a new potential target for the treatment of glomerular kidney diseases.

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3(-/-) mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3(-/-) mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3(-/-) mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3(-/-) Pdgfd(-/-) mice compared with Col4a3(-/-) Pdgfd(+/+) littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3(-/-) mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

Renal proximal tubular reabsorption of proteins and polypeptides is tightly regulated by a concerted action of the multi-ligand receptors with subsequent processing from the clathrin-coated pits to early/recycling and late endosomes and towards lysosomes. We performed whole exome-sequencing in a male patient from a consanguineous family, who presented with low- and intermediate molecular weight proteinuria, nephrocalcinosis and oligospermia. We identified a new potential player in tubular endocytosis, coiled-coil domain containing 158 (CCDC158). The variant in CCDC158 segregated with the phenotype and was also detected in a female sibling with a similar clinical kidney phenotype. We demonstrated the expression of this protein in kidney tubules and modeled its structure in silico. We hypothesized that the protein played a role in the tubular endocytosis by interacting with other endocytosis regulators, and used mass spectrometry to identify potential interactors. The role of CCDC158 in receptor-mediated endocytosis was further confirmed by transferrin and GST-RAP trafficking analyses in patient-derived proximal tubular epithelial cells. Finally, as CCDC158 is known to be expressed in the testis, the presence of oligospermia in the male sibling further substantiated the pathogenic role of the detected missense variant in the observed phenotype. In this study, we provide data that demonstrate the potential role of CCDC158 in receptor-mediated endocytosis, most likely by interaction with other endocytosis-related proteins that strongly correlate with the proximal tubular dysfunction phenotype as observed in the patients. However, more studies are needed to fully unravel the molecular mechanism(s) in which CCDC158 is involved.

Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13(+)CD24(-)CD133(-) proximal tubule epithelial cells (PTECs) and CD13(+)CD24+ and CD13(+)CD133(+) STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor kappa B, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. (c) 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting ‘iPSC organoid-derived (iPSCod)’ tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

Proliferative forms of glomerulonephritis are characterized by the influx of leukocytes, albuminuria, and loss of kidney function. The glomerular endothelial glycocalyx is a thick carbohydrate layer that covers the endothelium and is comprised of heparan sulfate (HS), which plays a pivotal role in glomerular inflammation by facilitating endothelial-leukocyte trafficking. We hypothesize that the exogenous glomerular glycocalyx may reduce the glomerular influx of inflammatory cells during glomerulonephritis. Indeed, administration of mouse glomerular endothelial cell (mGEnC)-derived glycocalyx constituents, or the low-molecular-weight heparin enoxaparin, reduced proteinuria in mice with experimental glomerulonephritis. Glomerular influx of granulocytes and macrophages, as well as glomerular fibrin deposition, was reduced by the administration of mGEnC-derived glycocalyx constituents, thereby explaining the improved clinical outcome. HSglx also inhibited granulocyte adhesion to human glomerular endothelial cells in vitro. Notably, a specific HSglx fraction inhibited both CD11b and L-selectin binding to activated mGEnCs. Mass spectrometry analysis of this specific fraction revealed six HS oligosaccharides, ranging from tetra- to hexasaccharides with 2-7 sulfates. In summary, we demonstrate that exogenous HSglx reduces albuminuria during glomerulonephritis, which is possibly mediated via multiple mechanisms. Our results justify the further development of structurally defined HS-based therapeutics for patients with (acute) inflammatory glomerular diseases, which may be applicable to non-renal inflammatory diseases as well.

ABSTRACT In the glomerulus, Bowman's space is formed by a continuum of glomerular epithelial cells. In focal segmental glomerulosclerosis (FSGS), glomeruli show segmental scarring, a result of activated parietal epithelial cells (PECs) invading the glomerular tuft. The segmental scars interrupt the epithelial continuum. However, non-sclerotic segments seem to be preserved even in glomeruli with advanced lesions. We studied the histology of the segmental pattern in Munich Wistar Frömter rats, a model for secondary FSGS. Our results showed that matrix layers lined with PECs cover the sclerotic lesions. These PECs formed contacts with podocytes of the uninvolved tuft segments, restoring the epithelial continuum. Formed Bowman's spaces were still connected to the tubular system. In biopsies of patients with secondary FSGS, we also detected matrix layers formed by PECs, separating the uninvolved from the sclerotic glomerular segments. PECs have a major role in the formation of glomerulosclerosis; we show here that in FSGS they also restore the glomerular epithelial cell continuum that surrounds Bowman's space. This process may be beneficial and indispensable for glomerular filtration in the uninvolved segments of sclerotic glomeruli.

1Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud university medical center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 2Department of Pathology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 3Department of Pediatric Nephrology, Amsterdam University Medical Center, Amsterdam, The Netherlands, 4Department of Laboratory Medicine, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences Nijmegen, The Netherlands and 5Department of Development and Regeneration, University Hospital Leuven, Leuven, Belgium

Significance Statement We investigated the role of the profibrotic PDGF in the development and progression of FSGS in a murine model resembling human FSGS. Injured podocytes expressed PDGF-B, inducing parietal epithelial cell activation, proliferation, and a profibrotic switch–driving FSGS. Therapeutic inhibition of PDGF-B significantly reduced proteinuria and FSGS, suggesting that inhibition of the PDGF signaling pathway might be a potential novel treatment for patients with FSGS. Background FSGS is the final common pathway to nephron loss in most forms of severe or progressive glomerular injury. Although podocyte injury initiates FSGS, parietal epithelial cells (PECs) are the main effectors. Because PDGF takes part in fibrotic processes, we hypothesized that the ligand PDGF-B and its receptor PDGFR-β participate in the origin and progression of FSGS. Methods We challenged Thy1.1 transgenic mice, which express Thy1.1 in the podocytes, with anti-Thy1.1 antibody to study the progression of FSGS. We investigated the role of PDGF in FSGS using challenged Thy1.1 mice, 5/6 nephrectomized mice, Col4−/− (Alport) mice, patient kidney biopsies, and primary murine PECs, and challenged Thy1.1 mice treated with neutralizing anti–PDGF-B antibody therapy. Results The unchallenged Thy1.1 mice developed only mild spontaneous FSGS, whereas challenged mice developed progressive FSGS accompanied by a decline in kidney function. PEC activation, proliferation, and profibrotic phenotypic switch drove the FSGS. During disease, PDGF-B was upregulated in podocytes, whereas PDGFR-β was upregulated in PECs from both mice and patients with FSGS. Short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation, and profibrotic activation. In vitro, stimulation of primary murine PECs with PDGF-B recapitulated in vivo findings with PEC activation and proliferation, which was inhibited by PDGF-B antibody or imatinib. Conclusion PDGF-B–PDGFR-β molecular crosstalk between podocytes and PECs drives glomerulosclerosis and the progression of FSGS.