Diabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide. Podocytes are important for glomerular filtration barrier function and maintenance of size selectivity in protein filtration in the kidney. Podocyte damage is the basis of many glomerular diseases characterized by loss of interdigitating foot processes and decreased expression of components of the slit diaphragm. Nephrin, a podocyte-specific protein, is the main component of the slit diaphragm. Loss of nephrin is observed in human and rodent models of diabetic kidney disease. The long isoform of CIN85 (RukL) is a binding partner of nephrin that mediates nephrin endocytosis via ubiquitination in podocytes. Here we demonstrate that the loss of nephrin expression and the onset of proteinuria in diabetic mice correlate with an increased accumulation of ubiquitinated proteins and expression of CIN85/RukL in podocytes. CIN85/RukL deficiency preserved nephrin surface expression on the slit diaphragm and reduced proteinuria in diabetic mice, whereas overexpression of CIN85 in zebrafish induced severe edema and disruption of the filtration barrier. Thus, CIN85/RukL is involved in endocytosis of nephrin in podocytes under diabetic conditions, causing podocyte depletion and promoting proteinuria. CIN85/RukL expression therefore shows potential to be a novel target for antiproteinuric therapy in diabetes.

Background: TGF-β 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-β. Since miR-143-3p (miR-143) is described as a TGF-β 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-β. 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-β 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.

Keywords: Adoptive cell transfer r Dendritic cells r Experimental allergic asthma r Histamine H 4 -receptor (H 4 R) r Inflammation r Ovalbumin r T cells Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe biogenic amine histamine is involved in a series of physiological processes, especially inflammatory and allergic reactions, gastric acid secretion, and neuronal transmission [1][2][3]. The effects of histamine are mediated by four different G-protein-coupled receptors, the histamine H 1 -, H 2 -, H 3 -, and H 4 -receptors (H 1-4 R), the Correspondence: Dr. Detlef Neumann e-mail: neumann.detlef@mh-hannover.de first two of which are already of broad pharmacological relevance [4][5][6][7][8][9]. The H 4 R is expressed mainly on cells of hematopoietic origin, including immune cells such as DCs, T cells, mast cells, splenocytes, BM cells, and peripheral leukocytes [10][11][12][13][14]. Therefore, the H 4 R is a promising drug target for the treatment of inflammatory diseases and allergic reactions [10,[15][16][17].In the airways, histamine is detected in high concentrations during an asthmatic response and promotes typical symptoms of allergic inflammation [18][19][20] Eur. J. Immunol. 2015Immunol. . 45: 1129Immunol. -1140 induction of migration of eosinophils [24][25][26], of DCs [27], and of mast cells [14] as well as Th2-directed polarization of CD4 + T cells [28]. However, although the function of histamine as mediator of inflammation and allergy has been well described [4,29], its precise cellular function in the pathogenesis of allergic asthma remains to be elucidated. Many symptoms of an allergic reaction are mediated by histamine via the H 1 R [30], including rhinitis or conjunctivitis. In the therapy of allergic asthma, however, H 1 R antagonists lack effectiveness. In contrast, using specific H 4 R antagonists, the functional relevance of the H 4 R in both the sensitization phase and effector phase of an acute allergic reaction in the lungs of mice was demonstrated [22,29,31,32]. In vitro data point to a potential role of the H 4 R on T-cell priming by DCs [22]. However, whether this effect can be transferred into the in vivo situation of experimental asthma is still unknown. In addition, direct effects of the H 4 R on T cells cannot be excluded [4,10,33]. Nevertheless, such information particularly with regard to future therapies using H 4 R antagonists, which have recently been demonstrated to be of clinical relevance [34,35], and the amplification of chronic allergic diseases, would be of fundamental relevance [36]. Results Lack of H 4 R on DCs during sensitization leads to reduced BAL-f eosinophilia and airway inflammationIn the present study, we took advantage of the adoptive transfer model of asthma, in which mice are sensitized by injection of OVA-specific CD4 + T cells, that had been polarized in vitro under (control), massively enhanced numbers of cells in the BAL-f were found in comparison to mice, which had been sham...

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.

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.

Background Disruption of the endothelial glycocalyx (eGC) is observed in septic patients and its injury is associated with multiple-organ failure and inferior outcomes. Besides this biomarker function, increased blood concentrations of shedded eGC constituents might play a mechanistic role in septic organ failure. We hypothesized that therapeutic plasma exchange (TPE) using fresh frozen plasma might influence eGC-related pathology by removing injurious mediators of eGC breakdown while at the time replacing eGC protective factors. Methods We enrolled 20 norepinephrine-dependent (NE > 0.4 μg/kg/min) patients with early septic shock (onset < 12 h). Sublingual assessment of the eGC via sublingual sidestream darkfield (SDF) imaging was performed. Plasma eGC degradation products, such as heparan sulfate (HS) and the eGC-regulating enzymes, heparanase (Hpa)-1 and Hpa-2, were obtained before and after TPE. A 3D microfluidic flow assay was performed to examine the effect of TPE on eGC ex vivo. Results were compared to healthy controls. Results SDF demonstrated a decrease in eGC thickness in septic patients compared to healthy individuals (p = 0.001). Circulating HS levels were increased more than sixfold compared to controls and decreased significantly following TPE [controls: 16.9 (8–18.6) vs. septic patients before TPE: 105.8 (30.8–143.4) μg/ml, p < 0.001; vs. after TPE: 70.7 (36.9–109.5) μg/ml, p < 0.001]. The Hpa-2 /Hpa-1 ratio was reduced in septic patients before TPE but normalized after TPE [controls: 13.6 (6.2–21.2) vs. septic patients at inclusion: 2.9 (2.1–5.7), p = 0.001; vs. septic patients after TPE: 13.2 (11.2–31.8), p < 0.001]. Ex vivo stimulation of endothelial cells with serum from a septic patient induced eGC damage that could be attenuated with serum from the same patient following TPE. Conclusions Septic shock results in profound degradation of the eGC and an acquired deficiency of the protective regulator Hpa-2. TPE removed potentially injurious eGC degradation products and partially attenuated Hpa-2 deficiency. Trial registration clinicaltrials.gov NCT04231994, retrospectively registered 18 January 2020

Proteinuria can be induced by impairment of any component of the glomerular filtration barrier (GFB). To determine the role of circulating permeability factors on glomerular damage, we developed a parabiosis-based zebrafish model to generate a common circulation between zebrafish larvae. A morpholino-mediated knockdown of a podocyte specific gene (nephronectin) was induced in one zebrafish larva which was then fused to an un-manipulated fish. Notably, proteinuria and glomerular damage were present in the manipulated fish and in the parabiotically-fused partner. Thus, circulating permeability factors may be induced by proteinuria even when an induced podocyte gene dysregulation is the initiating cause. Kidney International (2019) 96, 342-349; https://doi.NTR/MTZ podocyte injury model The parabionts ("ET" fused with "Cherry") were treated with 80 mmol/l or 1 mmol/l MTZ (in 0.1% dimethylsulfoxide) at 96 hpf. 17 J Müller-Deile et al.: Proteinuria in parabiotic zebrafish b a s i c r e s e a r c h Kidney International (2019) 96, 342-349 J Müller-Deile et al.: Proteinuria in parabiotic zebrafish b a s i c r e s e a r c h Kidney International (2019) 96, 342-349

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