Recurrence of proteinuria after kidney transplantation in primary focal segmental glomerulosclerosis (FSGS) is unpredictable. Several putative circulating permeability factors (CPFs) have been ...suggested, but none have been validated. A clinically relevant experimental model is required that demonstrates the presence of CPF(s) in patient material, to study CPF(s) and possibly predict recurrence in patients. We aimed to develop a FSGS-prone Thy-1.1 transgenic mouse model with accelerated proteinuria after injection of samples from patients with FSGS. The Thy-1.1 transgene was backcrossed into 5 mouse strains. The age of onset and severity of spontaneous proteinuria varied between the different genetic backgrounds. 129X1/Sv
Thy-1.1
and 129S2/SvPas
Thy-1.1
mice displayed proteinuria at 4 weeks, whereas Balb/c
Thy-1.1
and C57BL/6J
Thy-1.1
mice developed proteinuria from 6 weeks, and were used further. We determined the maximum protein dose that could be injected without causing protein overload in each background. Balb/c
Thy-1.1
and C57BL/6J
Thy-1.1
males and females were injected with presumably CPF-containing plasmapheresis effluent from 6 FSGS patients, which induced albuminuria particularly in Balb/c
Thy-1.1
males. Unfortunately, no response could be detected when using sera instead of plasmapheresis effluent, serum being more clinically relevant in the context of predicting FSGS recurrence. Considering the differences between responses elicited by serum and plasmapheresis effluent, simultaneously collected serum, plasma, and plasmapheresis effluent were tested. Whereas we could detect responses using a validated
in vitro
model, none of these presumably CPF-containing samples induced proteinuria in Balb/c
Thy-1.1
males. Thus, we have extensively tested the Thy-1.1 mouse model on different genetic backgrounds with proteinuria after injection of FSGS patient material as clinically relevant readout. The Balb/c
Thy-1.1
male mouse strain demonstrated the most promising results, but to detect CPF activity in FSGS serum e.g. prior to kidney transplantation, this strain clearly lacks sensitivity and is therefore not yet clinically applicable. It could, however, still be used as research tool to study CPFs in patient samples that did induce proteinuria.
Bartter syndrome is a rare inherited salt-losing renal tubular disorder characterized by secondary hyperaldosteronism with hypokalemic and hypochloremic metabolic alkalosis and low to normal blood ...pressure. The primary pathogenic mechanism is defective salt reabsorption predominantly in the thick ascending limb of the loop of Henle. There is significant variability in the clinical expression of the disease, which is genetically heterogenous with 5 different genes described to date. Despite considerable phenotypic overlap, correlations of specific clinical characteristics with the underlying molecular defects have been demonstrated, generating gene-specific phenotypes. As with many other rare disease conditions, there is a paucity of clinical studies that could guide diagnosis and therapeutic interventions. In this expert consensus document, the authors have summarized the currently available knowledge and propose clinical indicators to assess and improve quality of care.
Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular ...microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.
Excessive activation of the immune system is the cause of a wide variety of renal diseases. However, the pathogenic mechanisms underlying the aberrant activation of the immune system in the kidneys ...often remain unknown. TRPC6, a member of the Ca2+-permeant family of TRPC channels, is important in glomerular epithelial cells or podocytes for the process of glomerular filtration. In addition, TRPC6 plays a crucial role in the development of kidney injuries by inducing podocyte injury. However, an increasing number of studies suggest that TRPC6 is also responsible for tightly regulating the immune cell functions. It remains elusive whether the role of TRPC6 in the immune system and the pathogenesis of renal inflammation are intertwined. In this review, we present an overview of the current knowledge of how TRPC6 coordinates the immune cell functions and propose the hypothesis that TRPC6 might play a pivotal role in the development of kidney injury via its role in the immune system.
The hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit ...diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive.
We tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes.
Compared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects.
We elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.
Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes ...to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.
Increased expression and activity of the Ca
channel transient receptor potential channel 6 (TRPC6) is associated with focal segmental glomerulosclerosis, but therapeutic strategies to target TRPC6 ...are currently lacking. Nitric oxide (NO) is crucial for normal glomerular function and plays a protective role in preventing glomerular diseases. We investigated if NO prevents podocyte injury by inhibiting injurious TRPC6-mediated signaling in a soluble guanylate cyclase (sGC)-dependent manner and studied the therapeutic potential of the sGC stimulator Riociguat. Experiments were performed using human glomerular endothelial cells and podocytes. Podocyte injury was induced by Adriamycin incubation for 24 h, with or without the NO-donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP), the sGC stimulator Riociguat or the TRPC6 inhibitor Larixyl Acetate (LA). NO and Riociguat stimulated cGMP synthesis in podocytes, decreased Adriamycin-induced TRPC6 expression, inhibited the Adriamycin-induced TRPC6-mediated Ca
influx and reduced podocyte injury. The protective effects of Riociguat and NO were blocked when sGC activity was inhibited with 1H-1,2,4Oxadiazolo4,3-aquinoxalin-1-one (ODQ) or when TRPC6 activity was inhibited by LA. Our data demonstrate a glomerular (e)NOS-NO-sGC-cGMP-TRPC6 pathway that prevents podocyte injury, which can be translated to future clinical use by, e.g., repurposing the market-approved drug Riociguat.
Thiazide diuretics enhance renal Na+ excretion by blocking the Na+-Cl- cotransporter (NCC), and mutations in NCC result in Gitelman syndrome. The mechanisms underlying the accompanying hypocalciuria ...and hypomagnesemia remain debated. Here, we show that enhanced passive Ca2+ transport in the proximal tubule rather than active Ca2+ transport in distal convolution explains thiazide-induced hypocalciuria. First, micropuncture experiments in mice demonstrated increased reabsorption of Na+ and Ca2+ in the proximal tubule during chronic hydrochlorothiazide (HCTZ) treatment, whereas Ca2+ reabsorption in distal convolution appeared unaffected. Second, HCTZ administration still induced hypocalciuria in transient receptor potential channel subfamily V, member 5-knockout (Trpv5-knockout) mice, in which active distal Ca2+ reabsorption is abolished due to inactivation of the epithelial Ca2+ channel Trpv5. Third, HCTZ upregulated the Na+/H+ exchanger, responsible for the majority of Na+ and, consequently, Ca2+ reabsorption in the proximal tubule, while the expression of proteins involved in active Ca2+ transport was unaltered. Fourth, experiments addressing the time-dependent effect of a single dose of HCTZ showed that the development of hypocalciuria parallels a compensatory increase in Na+ reabsorption secondary to an initial natriuresis. Hypomagnesemia developed during chronic HCTZ administration and in NCC-knockout mice, an animal model of Gitelman syndrome, accompanied by downregulation of the epithelial Mg2+ channel transient receptor potential channel subfamily M, member 6 (Trpm6). Thus, Trpm6 downregulation may represent a general mechanism involved in the pathogenesis of hypomagnesemia accompanying NCC inhibition or inactivation.
The transient receptor potential channel C6 (TRPC6) is a slit diaphragm–associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause ...hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo . In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca2+ influx and the activation of the Ca2+ -dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.
Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a ...prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.
PDF
