Adriamycin does not damage podocytes of zebrafish larvae.
ABSTRACT: Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.
Project description:Our previous work demonstrated a protective role of protein S in early diabetic kidney disease (DKD). Protein S exerts antiinflammatory and antiapoptotic effects through the activation of TYRO3, AXL, and MER (TAM) receptors. Among the 3 TAM receptors, we showed that the biological effects of protein S were mediated largely by TYRO3 in diabetic kidneys. Our data now show that TYRO3 mRNA expression is highly enriched in human glomeruli and that TYRO3 protein is expressed in podocytes. Interestingly, glomerular TYRO3 mRNA expression increased in mild DKD but was suppressed in progressive DKD, as well as in focal segmental glomerulosclerosis (FSGS). Functionally, morpholino-mediated knockdown of tyro3 altered glomerular filtration barrier development in zebrafish larvae, and genetic ablation of Tyro3 in murine models of DKD and Adriamycin-induced nephropathy (ADRN) worsened albuminuria and glomerular injury. Conversely, the induction of TYRO3 overexpression specifically in podocytes significantly attenuated albuminuria and kidney injury in mice with DKD, ADRN, and HIV-associated nephropathy (HIVAN). Mechanistically, TYRO3 expression was suppressed by activation of TNF-α/NF-κB pathway, which may contribute to decreased TYRO3 expression in progressive DKD and FSGS, and TYRO3 signaling conferred antiapoptotic effects through the activation of AKT in podocytes. In conclusion, TYRO3 plays a critical role in maintaining normal podocyte function and may be a potential new drug target to treat glomerular diseases.
Project description:FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1-associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm-associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.
Project description:Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown. We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids. Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis. These findings establish podocyte cell-autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.
Project description:Idiopathic forms of Focal Segmental Glomerulosclerosis (FSGS) are caused by circulating permeability factors, which can lead to early recurrence of FSGS and kidney failure after kidney transplantation. In the past three decades, many research endeavors were undertaken to identify these unknown factors. Even though some potential candidates have been recently discussed in the literature, "the" actual factor remains elusive. Therefore, there is an increased demand in FSGS research for the use of novel technologies that allow us to study FSGS from a yet unexplored angle. Here, we report the successful treatment of recurrent FSGS in a patient after living-related kidney transplantation by removal of circulating factors with CytoSorb apheresis. Interestingly, the classical published circulating factors were all in normal range in this patient but early disease recurrence in the transplant kidney and immediate response to CytoSorb apheresis were still suggestive for pathogenic circulating factors. To proof the functional effects of the patient's serum on podocytes and the glomerular filtration barrier we used a podocyte cell culture model and a proteinuria model in zebrafish to detect pathogenic effects on the podocytes actin cytoskeleton inducing a functional phenotype and podocyte effacement. We then performed Raman spectroscopy in the < 50 kDa serum fraction, on cultured podocytes treated with the FSGS serum and in kidney biopsies of the same patient at the time of transplantation and at the time of disease recurrence. The analysis revealed changes in podocyte metabolome induced by the FSGS serum as well as in focal glomerular and parietal epithelial cell regions in the FSGS biopsy. Several altered Raman spectra were identified in the fractionated serum and metabolome analysis by mass spectrometry detected lipid profiles in the FSGS serum, which were supported by disturbances in the Raman spectra. Our novel innovative analysis reveals changed lipid metabolome profiles associated with idiopathic FSGS that might reflect a new subtype of the disease.
Project description:Focal segmental glomerular sclerosis (FSGS) is a primary kidney disease that is commonly associated with proteinuria and progressive loss of glomerular function, leading to development of chronic kidney disease (CKD). FSGS is characterized by podocyte injury and depletion and collapse of glomerular capillary segments. Progression of FSGS is associated with TGF-? activation in podocytes; however, it is not clear how TGF-? signaling promotes disease. Here, we determined that podocyte-specific activation of TGF-? signaling in transgenic mice and BALB/c mice with Adriamycin-induced glomerulosclerosis is associated with endothelin-1 (EDN1) release by podocytes, which mediates mitochondrial oxidative stress and dysfunction in adjacent endothelial cells via paracrine EDN1 receptor type A (EDNRA) activation. Endothelial dysfunction promoted podocyte apoptosis, and inhibition of EDNRA or scavenging of mitochondrial-targeted ROS prevented podocyte loss, albuminuria, glomerulosclerosis, and renal failure. We confirmed reciprocal crosstalk between podocytes and endothelial cells in a coculture system. Biopsies from patients with FSGS exhibited increased mitochondrial DNA damage, consistent with EDNRA-mediated glomerular endothelial mitochondrial oxidative stress. Our studies indicate that segmental glomerulosclerosis develops as a result of podocyte-endothelial crosstalk mediated by EDN1/EDNRA-dependent mitochondrial dysfunction and suggest that targeting the reciprocal interaction between podocytes and endothelia may provide opportunities for therapeutic intervention in FSGS.
Project description:<h4>Background</h4>Podocytes serve as an important constituent of the glomerular filtration barrier. Recently, we and others identified Myo1e as a key molecular component of the podocyte cytoskeleton.<h4>Results</h4>Myo1e mRNA and protein was expressed in human and mouse kidney sections as determined by Northern blot and reverse transcriptase PCR, and its expression was more evident in podocytes by immunofluorescence. By specific knock-down of MYO1E in zebrafish, the injected larvae exhibited pericardial edema and pronephric cysts, consistent with the appearance of protein in condensed incubation supernate. Furthermore, specific inhibition of Myo1e expression in a conditionally immortalized podocyte cell line induced morphological changes, actin cytoskeleton rearrangement, and dysfunction in cell proliferation, migration, endocytosis, and adhesion with the glomerular basement membrane.<h4>Conclusions</h4>Our results revealed that Myo1e is a key component contributing to the functional integrity of podocytes. Its impairment may cause actin cytoskeleton re-organization, alteration of cell shape, and membrane transport, and podocyte drop-out from the glomerular basement membrane, which might eventually lead to an impaired glomerular filtration barrier and proteinuria.
Project description:FSGS is the most common primary glomerular disease underlying ESRD in the United States and is increasing in incidence globally. FSGS results from podocyte injury, yet the mechanistic details of disease pathogenesis remain unclear. This has resulted in an unmet clinical need for cell-specific therapy in the treatment of FSGS and other proteinuric kidney diseases. We previously identified Yes-associated protein (YAP) as a prosurvival signaling molecule, the in vitro silencing of which increases podocyte susceptibility to apoptotic stimulus. YAP is a potent oncogene that is a prominent target for chemotherapeutic drug development. In this study, we tested the hypothesis that podocyte-specific deletion of Yap leads to proteinuric kidney disease through increased podocyte apoptosis. Yap was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Yap silencing in podocytes resulted in podocyte apoptosis, podocyte depletion, proteinuria, and an increase in serum creatinine. Histologically, features characteristic of FSGS, including mesangial sclerosis, podocyte foot process effacement, tubular atrophy, interstitial fibrosis, and casts, were observed. In human primary FSGS, we noted reduced glomerular expression of YAP. Taken together, these results suggest a role for YAP as a physiologic antagonist of podocyte apoptosis, the signaling of which is essential for maintaining the integrity of the glomerular filtration barrier. These data suggest potential nephrotoxicity with strategies directed toward inhibition of YAP function. Further studies should evaluate the role of YAP in proteinuric glomerular disease pathogenesis and its potential utility as a therapeutic target.
Project description:The Centers for Disease Control and Prevention estimates more than 10% of adults in the United States, over 20 million Americans, have chronic kidney disease (CKD). A failure to maintain the glomerular filtration barrier directly contributes to the onset of CKD. The visceral epithelial cells, podocytes, are integral to the maintenance of this renal filtration barrier. Direct podocyte injury contributes to the onset and progression of glomerular diseases such as minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS), diabetic nephropathy, and HIV-associated nephropathy (HIVAN). Since podocytes are terminally differentiated with minimal capacity to self-replicate, they are extremely sensitive to cellular injury. In the past two decades, our understanding of the mechanism(s) by which podocyte injury occurs has greatly expanded. With this newfound knowledge, therapeutic strategies have shifted to identifying targets directed specifically at the podocyte. Although the systemic effects of these agents are important, their direct effect on the podocyte proves to be essential in ameliorating glomerular disease. In this review, we highlight the mechanisms by which these agents directly target the podocyte independent of its systemic effects.
Project description:In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.
Project description:Focal segmental glomerulosclerosis (FSGS) is a chronic glomerular disease with poor clinical outcomes. Podocyte loss via apoptosis is one important mechanism underlying the pathogenesis of FSGS. Recently, Yes-associated-protein (YAP), a key downstream protein in the Hippo pathway, was identified as an activator for multiple gene transcriptional factors in the nucleus to control cell proliferation and apoptosis. To investigate the potential role of YAP in the progression of FSGS, we examined kidney samples from patients with minimal change disease or FSGS and found that increases in podocyte apoptosis is positively correlated with the cytoplasmic distribution of YAP in human FSGS. Utilizing an established mT/mG transgenic mouse model and primary cultured podocytes, we found that YAP was distributed uniformly in nucleus and cytoplasm in the podocytes of control animals. Adriamycin treatment induced gradual nuclear exclusion of YAP with enhanced phospho-YAP/YAP ratio, accompanied by the induction of podocyte apoptosis both in vivo and in vitro. Moreover, we used verteporfin to treat an Adriamycin-induced FSGS mouse model, and found YAP inhibition by verteporfin induced nuclear exclusion of YAP, thus increasing podocyte apoptosis and accelerating disease progression. Therefore, our findings suggest that YAP nuclear distribution and activation in podocytes is an important endogenous anti-apoptotic mechanism during the progression of FSGS.