The Pathophysiologic Role of Gelsolin in Chronic Kidney Disease: Focus on Podocytes

Chronic kidney disease (CKD) is normally related to proteinuria, a common finding in a compromised glomerular filtration barrier (GFB). GFB is a structure composed of glomerular endothelial cells, the basement membrane, and the podocytes. CKD with podocyte damage may be associated with actin cytoskeleton reorganization, resulting in podocyte effacement. Gelsolin plays a critical role in several diseases, including cardiovascular diseases and cancer. Our current study aimed to determine the connection between gelsolin and podocyte, and thus the mechanism underlying podocyte injury in CKD. Experiments were carried out on Drosophila to demonstrate whether gelsolin had a physiological role in maintaining podocyte. Furthermore, the survival rate of gelsolin-knocked down Drosophila larvae was extensively reduced after AgNO3 exposure. Secondly, the in vitro podocytes treated with puromycin aminonucleoside (PAN) enhanced the gelsolin protein expression, as well as small GTPase RhoA and Rac1, which also regulated actin dynamic expression incrementally with the PAN concentrations. Thirdly, we further demonstrated in vivo that GSN was highly expressed inside the glomeruli with mitochondrial dysfunction in a CKD mouse model. Our findings suggest that an excess of gelsolin may contribute to podocytes damage in glomeruli.

The Effect of Uncaria gambir Roxb. Extract on Superoxide Dismutase Activity in Proteinuric Wistar Rats Model

Nephrotic syndrome (NS) is a glomerular disease that is most often found in children with proteinuric as clinical manifestation. Gambier extract is a traditional medicine that has antioxidant effect. Its use to treat proteinuric has never been done. Therefore, we conducted an analytical study on the effect of gambier on proteinuric and superoxide dismutase (SOD) activity in proteinuric Wistar rat’s model. This research is an experimental study with a posttest control group design. The aim is analyzing the effect of giving gambier extract in decreasing urinary protein creatinine ratio and increasing SOD activity. Male Wistar rats were given an injection of puromycin aminonucleoside 1.5mg/100g body weight (BW), subcutaneously for 5 consecutive days for being nephrosis. The experimental animals in this study were divided into four groups: K1, the control group, consist of group of rats that were injected with 0.15ml/100 g BW of aquabidest and not given gambier extract. While K2 group consist of rats induced by Puromycin and given 1 ml of aquabidest. K3 consist of groups of rats induced by Puromycin and then given gambier extract 26mg/200g BW. Group K4 consist of rats induced by Puromycin and then given 80mg/200g BW of gambier extract. The four groups were given treatment for 14 days, after which the rats were placed in a metabolic cage for 24 hours to collect urine samples and then knocked down with ketamine for intra-cardiac blood collection. The results of this study showed differences between the four groups of rats in terms of the mean urine protein/creatinine ratio (p=0.015) and SOD activity (p=0.036). Groups of rats that were given gambier extracts 80mg/200g BW had lower urine protein / creatinine ratio and higher SOD activity. Therapy of gambier 80 mg/200g BW is better in managing proteinuric compared to 26mg/200g.

Symmetric Dimethylarginine Is a Sensitive Biomarker of Glomerular Injury in Rats

Glomerular filtration rate is the gold-standard method for assessment of renal function but is rarely performed in routine toxicity studies. Standard serum biomarkers of renal function are insensitive and become elevated only with significant loss of organ function. Symmetric dimethylarginine (SDMA) is a ubiquitous analyte that is freely filtered by the glomerulus and can be detected in serum. It has shown utility for the detection of renal injury in dogs and cats in clinical veterinary practice, but the potential utility of SDMA to detect renal injury in preclinical species or toxicity studies has not been thoroughly investigated. We utilized a well-characterized glomerular toxicant, puromycin aminonucleoside, to induce podocyte injury and subsequent proteinuria in young male Sprague-Dawley rats. At the end of 1 or 2 weeks, blood, urine, and kidney tissue were collected for analysis. One week following a single 50 mg/kg dose, urea nitrogen, creatinine, and albumin mean values were within historical control ranges, while SDMA was increased. Glomerular changes in these animals included periodic acid–Schiff positive globules within podocytes, podocyte hypertrophy by light microscopy, and podocyte degeneration with effacement of foot processes by electron microscopy (EM). Taken together, our data indicate that SDMA may be a useful biomarker for early detection of glomerular toxicities in rats.

TRPC5 Channel Inhibition Protects Podocytes in Puromycin-Aminonucleoside Induced Nephrosis Models

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction

<b><i>Introduction:</i></b> Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. <b><i>Methods:</i></b> The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. <b><i>Results:</i></b> Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. <b><i>Conclusion:</i></b> Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

Title Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes

Background Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes.Objectives We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury.Methods A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods.Results Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8 and − 3, suggesting that rituximab may inhibit apoptosis.Conclusions In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.

Human relevance of blocking the Rac1-TRPC5 pathway as a podocyte-protective strategy for progressive kidney diseases

Podocyte injury and the appearance of proteinuria are key features of progressive kidney diseases including focal segmental glomerulosclerosis (FSGS). Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of disease; however, less is known about the human relevance and translatability of TRPC5 inhibition into effective drug development programs. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated human iPSC-derived podocytes and kidney organoids.A single i.p. injection of PAN (50mg/kg) was given to wild-type Sprague-Dawley rats (Male, 4-5 weeks, Charles River). AC1903 was administered twice a day for 7 days after PAN injection. 24-hour urine albumin levels were measured on day 7. Human iPS cells were used to generate podocyte and kidney organoid. PAN treatment was used to induce human podocyte injury in these in vitro model systems. PAN treatment triggered the Rac1-TRPC5 injury pathway in human iPSC-derived podocyte cultures and kidney organoids. The TRPC5 inhibitor AC1903 reversed the effects of PAN-induced injury providing the first evidence for therapeutic applicability of TRPC5 inhibition to human podocytes in both 2D and 3D culture systems. Cross-validation in rats with PAN-induced nephrosis, an established model of podocyte injury and progressive kidney disease, confirmed that inhibition of TRPC5 by AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria. Taken together, our results confirmed the relevance of the TRPC5-Rac1 pathway in human kidney tissue thus highlighting the potential of this therapeutic strategy for patients.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments

BackgroundMaintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood.MethodsCells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside–induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes.ResultsNebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation.ConclusionsLIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model

Urinary exosomes, small extracellular vesicles present in urine, are secreted from all types of renal epithelial cells. Aquaporin-2 (AQP2), a vasopressin-regulated water channel protein, is known to be selectively excreted into the urine through exosomes (UE-AQP2), and its renal expression is decreased in nephrotic syndrome. However, it is still unclear whether excretion of UE-AQP2 is altered in nephrotic syndrome. In this study, we examined the excretion of UE-AQP2 in an experimental rat model of nephrotic syndrome induced by the administration of puromycin aminonucleoside (PAN). Rats were assigned to two groups: a control group administered saline and a PAN group given a single intraperitoneal injection of PAN (125 mg/kg) at day 0. The experiment was continued for 8 days, and samples of urine, blood, and tissue were collected on days 2, 5, and 8. The blood and urine parameters revealed that PAN induced nephrotic syndrome on days 5 and 8, and decreases in the excretion of UE-AQP2 were detected on days 2 through 8 in the PAN group. Immunohistochemistry showed that the renal expression of AQP2 was decreased on days 5 and 8. The release of exosomal marker proteins into the urine through UEs was decreased on day 5 and increased on day 8. These data suggest that UE-AQP2 is decreased in PAN-induced nephrotic syndrome and that this reflects its renal expression in the marked proteinuria phase after PAN treatment.