Oxysterol-binding protein like 7 deficiency leads to ER stress mediated apoptosis in podocytes and proteinuria.

Chronic kidney disease (CKD) is associated with renal lipid dysmetabolism among a variety of other pathways. We recently demonstrated that oxysterol-binding protein like 7 (OSBPL7) modulates the expression and function of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in podocytes, a specialized type of cell essential for kidney filtration. Drugs that target OSBPL7 lead to improved renal outcomes in several experimental models of CKD. However, the role of OSBPL7 in podocyte injury remains unclear. Employing mouse models and cellular assays, we investigated the influence of OSBPL7 deficiency on podocytes. We demonstrated that reduced renal OSBPL7 levels as observed in two different models of experimental CKD are linked to increased podocyte apoptosis, primarily mediated by heightened endoplasmic reticulum (ER) stress. While as expected the absence of OSBPL7 also resulted in lipid dysregulation (increased lipid droplets and triglycerides content), OSBPL7-deficiency related lipid dysmetabolism did not contribute to podocyte injury. Similarly, we demonstrated that the decreased autophagic flux we observed in OSBPL7-deficient podocytes was not the mechanistic link between OSBPL7-deficiency and apoptosis. In a complementary zebrafish model, osbpl7 knockdown was sufficient to induce proteinuria and morphological damage to the glomerulus, underscoring its physiological relevance. Our study shed new light on the mechanistic link between OSBPL7 deficiency and podocyte injury in glomerular diseases associated with CKD, and it strengthen the role of OSBPL7 as a novel therapeutic target.

Partner, Neighbor, Housekeeper and Dimension: 3D versus 2D Glomerular Co-Cultures Reveal Drawbacks of Currently Used Cell Culture Models.

Signaling-pathway analyses and the investigation of gene responses to different stimuli are usually performed in 2D monocultures. However, within the glomerulus, cells grow in 3D and are involved in direct and paracrine interactions with different glomerular cell types. Thus, the results from 2D monoculture experiments must be taken with caution. We cultured glomerular endothelial cells, podocytes and mesangial cells in 2D/3D monocultures and 2D/3D co-cultures and analyzed cell survival, self-assembly, gene expression, cell-cell interaction, and gene pathways using live/dead assay, time-lapse analysis, bulk-RNA sequencing, qPCR, and immunofluorescence staining. Without any need for scaffolds, 3D glomerular co-cultures self-organized into spheroids. Podocyte- and glomerular endothelial cell-specific markers and the extracellular matrix were increased in 3D co-cultures compared to 2D co-cultures. Housekeeping genes must be chosen wisely, as many genes used for the normalization of gene expression were themselves affected in 3D culture conditions. The transport of podocyte-derived VEGFA to glomerular endothelial cells confirmed intercellular crosstalk in the 3D co-culture models. The enhanced expression of genes important for glomerular function in 3D, compared to 2D, questions the reliability of currently used 2D monocultures. Hence, glomerular 3D co-cultures might be more suitable in the study of intercellular communication, disease modelling and drug screening ex vivo.

Glomerular Endothelial Cell-Derived microRNA-192 Regulates Nephronectin Expression in Idiopathic Membranous Glomerulonephritis.

BACKGROUND: Autoantibodies binding to podocyte antigens cause idiopathic membranous glomerulonephritis (iMGN). However, it remains elusive how autoantibodies reach the subepithelial space because the glomerular filtration barrier (GFB) is size selective and almost impermeable for antibodies. METHODS: Kidney biopsies from patients with iMGN, cell culture, zebrafish, and mouse models were used to investigate the role of nephronectin (NPNT) regulating microRNAs (miRs) for the GFB. RESULTS: Glomerular endothelial cell (GEC)-derived miR-192-5p and podocyte-derived miR-378a-3p are upregulated in urine and glomeruli of patients with iMGN, whereas glomerular NPNT is reduced. Overexpression of miR-192-5p and morpholino-mediated npnt knockdown induced edema, proteinuria, and podocyte effacement similar to podocyte-derived miR-378a-3p in zebrafish. Structural changes of the glomerular basement membrane (GBM) with increased lucidity, splitting, and lamellation, especially of the lamina rara interna, similar to ultrastructural findings seen in advanced stages of iMGN, were found. IgG-size nanoparticles accumulated in lucidity areas of the lamina rara interna and lamina densa of the GBM in npnt-knockdown zebrafish models. Loss of slit diaphragm proteins and severe structural impairment of the GBM were further confirmed in podocyte-specific Npnt knockout mice. GECs downregulate podocyte NPNT by transfer of miR-192-5p-containing exosomes in a paracrine manner. CONCLUSIONS: Podocyte NPNT is important for proper glomerular filter function and GBM structure and is regulated by GEC-derived miR-192-5p and podocyte-derived miR-378a-3p. We hypothesize that loss of NPNT in the GBM is an important part of the initial pathophysiology of iMGN and enables autoantigenicity of podocyte antigens and subepithelial immune complex deposition in iMGN.

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-ß) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.

Role of endothelial microRNA 155 on capillary leakage in systemic inflammation.

BACKGROUND: Capillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage. METHODS: SmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance. RESULTS: Endothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects. CONCLUSIONS: We found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.

The Zebrafish Model to Understand Epigenetics in Renal Diseases.

Epigenetic modifications are able to alter gene expression and include DNA methylation, different histone variants, and post-transcriptional modifications (PTMs), such as acetylation or phosphorylation, and through short/long RNAs, respectively. In this review, we focus on current knowledge concerning epigenetic modifications in gene regulation. We describe different forms of epigenetic modifications and explain how epigenetic changes can be detected. The relevance of epigenetics in renal diseases is highlighted with multiple examples and the use of the zebrafish model to study glomerular diseases in general and epigenetics in renal diseases in particular is discussed. We end with an outlook on how to use epigenetic modifications as a therapeutic target for different diseases. Here, the zebrafish model can be employed as a high-throughput screening tool not only to discover epigenetic alterations contributing to disease, but also to test novel substances that change epigenetic signatures in vivo. Therefore, the zebrafish model harbors the opportunity to find novel pathogenic pathways allowing a pre-selection of potential targets and compounds to be tested for renal diseases.

IPNA clinical practice recommendations for the diagnosis and management of children with steroid-resistant nephrotic syndrome.

Idiopathic nephrotic syndrome newly affects 1-3 per 100,000 children per year. Approximately 85% of cases show complete remission of proteinuria following glucocorticoid treatment. Patients who do not achieve complete remission within 4-6 weeks of glucocorticoid treatment have steroid-resistant nephrotic syndrome (SRNS). In 10-30% of steroid-resistant patients, mutations in podocyte-associated genes can be detected, whereas an undefined circulating factor of immune origin is assumed in the remaining ones. Diagnosis and management of SRNS is a great challenge due to its heterogeneous etiology, frequent lack of remission by further immunosuppressive treatment, and severe complications including the development of end-stage kidney disease and recurrence after renal transplantation. A team of experts including pediatric nephrologists and renal geneticists from the International Pediatric Nephrology Association (IPNA), a renal pathologist, and an adult nephrologist have now developed comprehensive clinical practice recommendations on the diagnosis and management of SRNS in children. The team performed a systematic literature review on 9 clinically relevant PICO (Patient or Population covered, Intervention, Comparator, Outcome) questions, formulated recommendations and formally graded them at a consensus meeting, with input from patient representatives and a dietician acting as external advisors and a voting panel of pediatric nephrologists. Research recommendations are also given.

Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex.

BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.

Identification of cell and disease specific microRNAs in glomerular pathologies.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR-screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR-screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR-screening in renal cells was done in untreated conditions and after stimulation with TGF-ß. A merge of the detected regulated miRs led us to identify disease-specific, cell type-specific and cell stress-induced miRs. Most miRs were down-regulated following the stimulation with TGF-ß in all cell types. Up-regulation of miRs after TGF-ß was cell type-specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR-155 was up-regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR-screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.

Podocytes from the diagnostic and therapeutic point of view.

The central role of podocytes in glomerular diseases makes this cell type an interesting diagnostic tool as well as a therapeutic target. In this review, we discuss the current literature on the use of podocytes and podocyte-specific markers as non-invasive diagnostic tools in different glomerulopathies. Furthermore, we highlight the direct effects of drugs currently used to treat primary glomerular diseases and describe their direct cellular effects on podocytes. A new therapeutic potential is seen in drugs targeting the podocytic actin cytoskeleton which is essential for podocyte foot process structure and function. Incubation of cultured human podocyte cell lines with sera from patients with active glomerular diseases is currently also used to identify novel circulating factors with pathophysiological relevance for the glomerular filtration barrier. In addition, treatment of detached urinary podocytes from patients with substances that restore their cytoskeleton might serve as a novel personalized tool to estimate their potential for podocyte recovery ex vivo.

Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases.

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Disease modeling in genetic kidney diseases: zebrafish.

Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs. Several options are available to determine the functional role of a specific gene or gene sets. Permanent genome editing can be induced via complete gene knockout by using the CRISPR/Cas-system, among others, or via transient modification by using various morpholino techniques. Cross-species rescues succeeding knockdown techniques are employed to determine the functional significance of a target gene or a specific mutation. This article summarizes the current techniques and discusses their perspectives.

Removal of focal segmental glomerulosclerosis (FSGS) factor suPAR using CytoSorb.

Treatment of primary focal segmental glomerulosclerosis (FSGS) and its recurrence after kidney transplantation associated with rapid deterioration of kidney function remains to be challenging despite advances in immunosuppressive therapy. The presence of circulating factors has been postulated to be a pivotal player in the pathogenesis of FSGS, although suPAR and CLCF-1 have been identified as the most promising causative factors. The potential therapeutic effect of suPAR elimination in an FSGS patient using CytoSorb, a hemoadsorption device that gained attention in the cytokine elimination in septic patients, was studied. Efficiency of total plasma exchange to remove suPAR was determined. CytoSorb hemoadsorption caused a 27.33% reduction of the suPAR level in a single treatment, whereas total plasma exchange showed a suPAR level reduction of 25.12% (n = 3; 95% confidence interval, 0.2777-0.8090; P < 0.01), which may indicate therapeutic potential in the treatment of primary FSGS and its recurrence in a kidney transplant.

Overexpression of TGF-ß Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans.

BACKGROUND: TGF-ß is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-ß. Since miR-143-3p (miR-143) is described as a TGF-ß inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. METHODS: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-ß. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. RESULTS: We detected a time dependent, TGF-ß inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. CONCLUSION: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

Podocyte directed therapy of nephrotic syndrome-can we bring the inside out?

Several of the drugs currently used for the treatment of glomerular diseases are prescribed for their immunotherapeutic or anti-inflammatory properties, based on the current understanding that glomerular diseases are mediated by immune responses. In recent years our understanding of podocytic signalling pathways and the crucial role of genetic predispositions in the pathology of glomerular diseases has broadened. Delineation of those signalling pathways supports the hypothesis that several of the medications and immunosuppressive agents used to treat glomerular diseases directly target glomerular podocytes. Several central downstream signalling pathways merge into regulatory pathways of the podocytic actin cytoskeleton and its connection to the slit diaphragm. The slit diaphragm and the cytoskeleton of the foot process represent a functional unit. A breakdown of the cytoskeletal backbone of the foot processes leads to internalization of slit diaphragm molecules, and internalization of slit diaphragm components in turn negatively affects cytoskeletal signalling pathways. Podocytes display a remarkable ability to recover from complete effacement and to re-form interdigitating foot processes and intact slit diaphragms after pharmacological intervention. This ability indicates an active inside-out signalling machinery which stabilizes integrin complex formations and triggers the recycling of slit diaphragm molecules from intracellular compartments to the cell surface. In this review we summarize current evidence from patient studies and model organisms on the direct impact of immunosuppressive and supportive drugs on podocyte signalling pathways. We highlight new therapeutic targets that may open novel opportunities to enhance and stabilize inside-out pathways in podocytes.

A new rescue regimen with plasma exchange and rituximab in high-risk membranous glomerulonephritis.

BACKGROUND: Even though current treatment guidelines for idiopathic membranous glomerulonephritis (iMGN) exist, many questions regarding an optimal therapy remain unanswered. Complete remission cannot be achieved in all patients; relapses occur, in some cases frequently, and side effects from the immunosuppressive therapy are common. Therapeutic options in high-risk patients not responding to standard immunosuppressive therapies are limited. Recent research reveals that the human M-type phospholipase A2 receptor (PLA2 R) is a causative factor in iMGN that parallels clinical disease activity. However, in some patients, this correlation is not evident and additional undetermined factors seem to play a role. DESIGN: We evaluated a new rescue protocol including plasma exchanges (PE) against albumin, intravenous immunoglobulins (IVIGs) and rituximab for 10 patients with a biopsy-proven diagnosis of iMGN who were therapy-resistant to all conventional regimens and had a urinary protein to creatinine ratio of more than 10 000 mg/g Crea. We compared this protocol with standard immunosuppressive protocols including monthly alternating prednisolone plus cyclophosphamide (18 patients), cyclosporine plus prednisolone (23 patients) and rituximab alone (eight patients) in a retrospective design. RESULTS: Our rescue regimen with PE, IVIGs and rituximab achieved partial remission in 90% of patients who had been otherwise refractory to therapy. The mean time to partial remission was 2·1 months. Furthermore, two anti-PLA2 R-antibody negative patients were also treated with this rescue regimen, achieving partial remission after 1 and 4 months. CONCLUSION: A combination of PE, IVIGs and rituximab is a treatment option to consider for high-risk patients with iMGN who are refractory to conventional therapy.

Online Kt/V monitoring in haemodialysis by UV absorbance: variations during intra-dialytic meals.

BACKGROUND/AIMS: The aim of this study was to analyse if intra-dialytic food intake may lead to changes in online Kt/V. METHODS: Forty patients were analysed using haemodialysis machines with integrated online Kt/V monitoring based on UV absorbance or ionic dialysance. Kt/V was monitored before, during and after food intake. Samples of dialysate were analysed for urea, uric acid, creatinine and glucose levels. RESULTS: Food intake was associated with a significant decrease in relative Kt/V when determined by UV absorbance. The drop in Kt/V was independent of changes in blood pressure or body posture. No significant changes in online Kt/V were found using the ionic dialysance method. Dialysate samples showed a continuous clearance of urea, uric acid and creatinine during meals but a rapid increase in glucose. CONCLUSION: Our data suggest that the short meal-associated decrease in online Kt/V as measured by UV absorbance does not represent a true decline in Kt/V but might reflect an intermittent increase in UV-absorbing dialysed solutes.

Distinct Changes in Gut Microbiota of Patients With Kidney Graft Rejection.

Background: Kidney graft rejection still represents the major cause of graft loss in kidney transplant recipients. Of growing interest is the bidirectional relationship between gut microbiome and immune system suggesting that gut microbiota can affect allograft outcome. Methods: In this cross-sectional case-control study, we characterized the gut microbial profile of adult renal transplant recipients with and without graft rejection to define a cohort-specific microbial fingerprint through 16S ribosomal RNA gene sequencing. We used very strict inclusion and exclusion criteria to address confounder of microbiota composition. Results: Different relative abundances in several gut microbial taxa were detectable in control patients compared with patients with kidney allograft rejection. Alpha diversity was lower in the rejection group and beta diversity revealed dissimilarity between patients with and without kidney graft rejection (P < 0.01). When the rejection group was stratified according to different types of allograft rejection, major changes were identified between patients with chronic T-cellular-mediated rejection and controls. Changes in alpha diversity within the gut microbiome were related to the probability of chronic T-cellular-mediated rejection (P < 0.05). Kidney transplant patients without rejection showed significant enrichment of rather anti-inflammatory taxa whereas in the rejection group bacteria well known for their role in chronic inflammation were increased. For example, amplicon sequence variant (ASV) 362 belonging to the genus Bacteroides and ASV 312 belonging to Tannerellaceae were enriched in no rejection (P < 0.001 and P < 0.01), whereas ASV 365 was enriched in patients with allograft rejection (P = 0.04). Looking at metagenomic functions, a higher abundance of genes coding for enzymes involved in bacterial multidrug resistance and processing of short-chain fatty acids was found in patients without rejection but an increase in enzymes involved in nicotinamide adenine dinucleotide phosphate production was seen in patients with allograft rejection. Conclusions: A distinct microbial fingerprint of patients with allograft rejection might serve as noninvasive biomarker in the future.

Zebrafish Model to Study Podocyte Function Within the Glomerular Filtration Barrier.

The zebrafish model has been used in many different fields of research because of its high homology to the human genome, its easy genetic manipulation, its high fecundity, and its rapid development. For glomerular diseases, zebrafish larvae have proven to be a versatile tool to study the contribution of different genes, because the zebrafish pronephros is very comparable to the human kidney in function and ultrastructure. Here we describe the principle and use of a simple screening assay based on the measurement of the fluorescence in the retinal vessel plexus of the Tg(l-fabp:DBP:eGFP) zebrafish line ("eye assay") to indirectly determine proteinuria as a hallmark of podocyte dysfunction. Furthermore, we illustrate how to analyze the obtained data and outline methods to attribute the findings to podocyte impairment.

Generation of Patient-Derived Podocytes from Skin Biopsies.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes. Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients' mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.