Biomedical subjects

Nicole Endlich

Publications and source records attributed to Nicole Endlich.

15 recordsLinked to original sources

PubMed2026

Disease-driven post-transcriptional alterations and alternative splicing in podocytes in focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and progression to end-stage renal disease, yet its molecular pathogenesis remains still incompletely defined. While transcriptional alterations in podocytes have been extensively characterized, the contribution of post-transcriptional regulatory mechanisms is poorly understood. Here, we combined a zebrafish podocyte-specific injury model with glomerulus-resolved transcriptomic profiling to dissect RNA regulatory alterations during FSGS progression. Integrated analyses of bulk RNA sequencing, small RNA profiling, and alternative splicing revealed pronounced, time-dependent remodeling of the glomerular transcriptome. We demonstrate that podocyte injury is associated with loss of key podocyte-specific proteins, activation of inflammatory pathways, remodeling of the extracellular matrix, and altered microRNA expression, such as miR-21 and miR-193. Moreover, we found that alternative splicing influences key podocyte gene expression, affecting genes critical for slit diaphragm integrity, actin cytoskeleton organization, and glomerular basement membrane stability. Isoform analyses identified FSGS-associated isoform switches in SRSF3 and EPB41L5. Importantly, these changes were also evident in glomeruli from FSGS patients, demonstrating that the zebrafish model recapitulates key molecular features of human disease and highlighting alternative splicing as a central regulatory mechanism in FSGS.

Animals↗

PubMed2025

Alternative Splicing in Mechanically Stretched Podocytes as a Model of Glomerular Hypertension.

KEY POINTS: Mechanical stretch induced over 3000 alternative splicing events in podocytes, affecting gene expression and protein abundance. Seventeen genes showed consistent splicing events across multiple analysis tools, with key isoform changes. Shroom3 and Myl6 underwent isoform switches under mechanical stretch, altering the C-terminal sequence and interaction properties of Myl6. BACKGROUND: Alterations in pre-mRNA splicing are crucial to the pathophysiology of various diseases. However, the effects of alternative splicing of mRNA on podocytes in hypertensive nephropathy are still unknown. The Sys_CARE project aimed to identify alternative splicing events involved in the development and progression of glomerular hypertension. METHODS: Murine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA sequencing, and several bioinformatic alternative splicing tools. RESULTS: Using transcriptomic and proteomic analysis, we identified significant changes in gene expression and protein abundance because of mechanical stretch. RNA-Seq identified over 3000 alternative spliced genes after mechanical stretch, including all types of alternative splicing events. Among these, 17 genes exhibited an alternative splicing event across four different splicing analysis tools. From this group, we focused on Myl6, a component of the myosin protein complex, and Shroom3, an actin-binding protein essential for podocyte function. We identified two Shroom3 isoforms with significant expression changes under mechanical stretch, which was validated by quantitative RT-PCR and in situ hybridization. In addition, we observed an expression switch of two Myl6 isoforms after mechanical stretch, accompanied by an alteration in the C-terminal amino acid sequence. CONCLUSIONS: A comprehensive RNA-Seq analysis of mechanically stretched podocytes identified novel potential podocyte-specific biomarkers and highlighted significant alternative splicing events, notably in the mRNA of Shroom3 and Myl6.

podocyte↗

PubMed2006

Podocytes are sensitive to fluid shear stress in vitro.

Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm(2) resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK(1) cells) after 20 h. At 0.015-0.25 dyne/cm(2), lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear zonula occludens-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm(2), the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and alpha-actinin. Because the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors that were all without effect on podocyte number under static conditions. At 0.25 dyne/cm(2), however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces a reorganization of the actin cytoskeleton and activates specific tyrosine kinases that are required to withstand fluid shear stress.

Animals↗

PubMed2005

Stretch, tension and adhesion - adaptive mechanisms of the actin cytoskeleton in podocytes.

Podocytes form an epithelial layer on the outer aspect of the basement membrane of glomerular capillaries. The interdigitating pattern of podocyte foot processes (PFPs) generates a unique and extremely long cell-cell contact area - the filtration slit. Thus, the interdigitating PFPs are the morphological basis for the high hydraulic conductivity of the glomerular capillaries. Any disturbance in this interdigitating pattern results in a drop of glomerular filtration rate impairing renal function. PFPs are based on the actin cytoskeleton, consisting of a subplasmalemmal network and a central core of filament bundles. Besides giving PFPs their morphology, the actin cytoskeleton anchors cell-cell contact and cell-matrix proteins in podocytes. Several human genetic diseases as well as transgenic mouse models provide evidence for the crucial role of the actin cytoskeleton in podocytes. Varying flow rates of the filtrate, increased glomerular capillary pressure in glomerular hypertension, and varying activation states of contractile proteins in PFPs impose a mechanical load on the actin cytoskeleton, challenging the intricate arrangement of PFPs and podocyte adhesion. Here we review data about the actin cytoskeleton of podocytes and the response of podocytes to mechanical load. From these data possible mechanisms are emerging how the actin cytoskeleton may allow podocytes to adapt to states of increased mechanical load.

Actin Cytoskeleton↗

PubMed2005

Association of CD2AP with dynamic actin on vesicles in podocytes.

The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6-7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of < or =0.5-microm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.

Actins↗

PubMed2004

Characterization of glucose uptake by cultured rat podocytes.

The nonmetabolizable glucose analogue [(3)H]-2-deoxy-D-glucose ((3)H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of (3)H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the (3)H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a V(max) value of approximately 10 mM and S(0.5)of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.

Animals↗

PubMed2004

Functional and structural characterization of a novel member of the natriuretic family of peptides from the venom of Pseudocerastes persicus.

A novel peptide, PNP (Pseudocerastes persicus natriuretic peptide), was isolated from the venom of the Iranian viper P. persicus. Amino acid sequencing revealed that the 37-residue peptide belongs to the family of natriuretic peptides. The physiological effects of intra-venously PNP infused into anesthetized rats on urine flow, sodium excretion and blood pressure were comparable to those of atrial natriuretic peptide (ANP). In PC12 cells that were treated with either PNP, ANP, or C-type natriuretic peptide, PNP induced a similar cGMP response as ANP. Since PC12 cells only express the natriuretic peptide receptor (NPR)-A receptor we conclude that PNP binds to the NPR-A receptor. The solution conformation of PNP was characterized using (1)H nuclear magnetic resonance spectroscopy and indicates a high degree of conformational flexibility.

Amino Acid Sequence↗

PubMed2004

Alteration of podocytes in a murine model of crescentic glomerulonephritis.

Recent observations suggest a central role of podocytes in crescent formation. In experimental glomerulonephritis podocytes disrupt the parietal epithelial layer and attach on its basement membrane, thus forming bridges between the tuft and Bowman's capsule, and they are a major constituent of crescents. In order to explain these findings we hypothesize that inflammation triggers motility in podocytes. In the present study we asked whether podocytes display alterations which suggest a migratory behavior in glomerulonephritis. Glomerulonephritis was induced in mice by injection of a rabbit serum against the glomerular basement membrane. The kidneys were perfusion-fixed 6 days later and examined by light and electron microscopy as well as by immunohistochemistry. In glomerulonephritis the apical cytoplasm of podocytes displayed numerous actin-containing microprotrusions. Cortactin, a protein involved in the regulation of actin polymerization, was predominantly expressed in foot processes of podocytes in control mice. It was redistributed to the cell body in glomerulonephritis. In untreated mice betal-integrin was restricted to the foot processes. In glomerulonephritis it was additionally found in the cytoplasm and in the apical cell membrane. Recycling of integrins is a crucial event in initiation of cell migration. ICAM-1 and CD44, the ligation of which induces migratory behaviors, were absent from healthy podocytes but expressed by some podocytes in glomerulonephritis. Thus, in glomerulonephritis podocytes display some characteristic features of migrating cells. This might explain their ability to break through the parietal epithelium and to become a constituent of early crescents.

Actins↗

PubMed2004

Stable expression of nephrin and localization to cell-cell contacts in novel murine podocyte cell lines.

BACKGROUND: Cell culture of podocytes has become an indispensable tool in the study of podocyte biology. To date, however, podocyte cell lines with stable expression of the crucial slit diaphragm protein nephrin and localization of nephrin to cell-cell contacts are not available. METHODS: Conditionally immortalized cells were grown from isolated glomeruli of mice, harboring the temperature-sensitive SV40 large T antigen. About 60 clonal cell lines were generated by limiting dilution. RESULTS: Among 30 Wilm's tumor (WT)-1- and podocalyxin-positive cell clones, two cell clones stably expressed nephrin as assessed by reverse transcription-polymerase chain reaction (RT-PCR), Northern and Western blotting, and immunofluorescence. In addition, expression of the following podocyte proteins was demonstrated: NEHP1, FAT, P-cadherin, podocin, CD2AP, ZO-1 (alpha(-) isoform), Lmx1b, podoplanin, synaptopodin, cortactin, and vimentin. The nephrin-positive podocyte cell lines formed a monolayer with abundant cell-cell contacts. Transmission electron microscopy revealed formation of primitive foot process-like interdigitations and slit diaphragm-like junctions. Nephrin colocalized with F-actin at cell-cell contacts as demonstrated by immunofluorescence. Intriguingly, nephrin and actin-associated proteins (synaptopodin, CD2AP, and cortactin) were recruited to and accumulated at the entire cell margin only in confluent cells, but not in dispersed cells. CONCLUSION: We present novel murine podocyte cell lines with stable expression of nephrin and abundant formation of cell-cell contacts, possessing several features of in situ podocyte cell-cell contacts. Furthermore, our data suggest that the accumulation of certain proteins in podocyte foot processes is linked to formation of cell-cell contacts.

Animals↗

PubMed2004

C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress.

BACKGROUND: High glucose and mechanical strain resulting from capillary hypertension are relevant risk factors affecting glomerular cells in diabetes. Altered activity of the natriuretic peptide (NP) system acting via vasorelaxing cyclic guanosine 5' monophosphate (cGMP) has been proposed to be one of the reasons for diabetes-dependent impairment of kidney function. Podocytes possess the NP receptors (NPRs) coupled to particular guanylyl cyclase. We investigated whether mechanical stress and high ambient glucose influence cGMP generation in podocytes stimulated with NPs. Additionally, the C-type natriuretic peptide (CNP) system has been characterized in these cells. METHODS: Conditionally immortalized mouse podocytes were stimulated with NP for 15 minutes and cGMP was determined by enzymatic immunoassay. The mRNA expression for CNP and CNP-specific NPR-B was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cGMP synthesis was measured in cells exposed to mechanical stress and to 30 mmol/L glucose for 3 days. RESULTS: Production of cGMP upon stimulation of the NPR-B receptor with CNP and inhibition by an antagonist HS-142-1 was dose-dependent. RT-PCR showed that podocytes express mRNA not only for NPR-B but also for CNP. Mechanical stress reduced the cGMP response by 50%, both to atrial natriuretic peptide (ANP) and to CNP. Conversely, upon high glucose, the CNP-induced production of cGMP was elevated twofold in stretched and in control cells. Furthermore, as compared to ANP, stimulation with CNP caused a larger increase in cGMP levels in stretched as well as in nonstretched cells. CONCLUSION: Expression of CNP together with potent NPR-B receptors suggests that in podocytes, CNP may act in an autocrine and/or paracrine manner. Furthermore, in a diabetic kidney, high glucose and mechanical stress may modulate the CNP-dependent cGMP production in podocytes in an opposite manner.

Animals↗

PubMed2003

Rho-kinase inhibition blunts renal vasoconstriction induced by distinct signaling pathways in vivo.

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure.

Adenosine↗

PubMed2003

Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli.

Drebrins are actin-binding proteins (ABP) initially identified in and thought to be specific for neuronal cells, where they appear to contribute to the formation of cell processes. Recent studies have also detected the isoform drebrin E2 in a wide range of non-neuronal cell types, notably in and near actin-rich lamellipodia and filopodia. The present study demonstrates drebrin enrichment in renal glomeruli. Immunohistochemistry and double-label confocal laser scanning microscopy have shown intense drebrin reactions in the mesangial cells of diverse mammalian species. In adult human and bovine kidneys, drebrin is, in addition, markedly enriched in the foot processes of podocytes, as also demonstrable by immunoelectron microscopy. By contrast, the podocytes of rodent glomeruli appear to contain significant drebrin concentrations only during early developmental stages. In differentiated murine podocytes cultured in vitro, however, drebrin is concentrated in the cell processes, where it partially codistributes with actin and other ABP. In biochemical analyses using protein extracts from renal cortices, large (approximately 20S) complexes ("drebrosomes") were found containing drebrin and actin. These findings confirm and extend our hypothesis that drebrin is involved in the regulation of actin dynamics also outside the nervous system. Clearly, drebrin has to be added to the ensemble of ABP regulating the actomyosin system and the dynamics of mesangial cells and foot processes in podocytes.

Animals↗

PubMed2002

Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress.

Glomerular hypertension is a major determinant advancing progression to end-stage renal failure. Podocytes, which are thought to counteract pressure-mediated capillary expansion, are increasingly challenged in glomerular hypertension. Studies in animal models of glomerular hypertension indicate that glomerulosclerosis develops from adhesions of the glomerular tuft to Bowman's capsule due to progressive podocyte loss. However, the molecular alterations of podocytes in glomerular hypertension are unknown. In this study, we determined the changes in gene expression in podocytes induced by mechanical stress in vitro (cyclic biaxial stretch, 0.5 Hz, 5% linear strain, 3 days) using cDNA arrays (6144 clones). Sixteen differentially regulated genes were identified, suggesting alterations of cell-matrix interaction, mitochondrial/metabolic function, and protein synthesis/degradation in stretched podocytes. The transcript for the matricellular protein osteopontin (OPN) was most strongly up-regulated by stretch (approximately threefold). By reverse transcriptase-polymer chain reaction, up-regulation of OPN mRNA was also detected in glomeruli of rats treated for 2.5 wk with desoxycorticosterone acetate-salt, an animal model of glomerular hypertension. In cultured podocytes, OPN coating induced a motile phenotype increasing actin nucleation proteins at cell margins and reducing stress fibers and focal adhesions. Intriguingly, additional OPN coating of collagen IV-coated membranes accelerated stretch-induced actin reorganization and markedly diminished podocyte loss at higher strain. This study delineates the molecular response of podocytes to mechanical stress and identifies OPN as a stretch-adapting molecule in podocytes.

Actins↗

PubMed2002

cAMP pathway in podocytes.

Podocytes possess receptors for a variety of hormones. The following receptors whose stimulation results in increased cAMP levels have been detected in podocytes: adrenergic beta(2) receptor, dopamine D(1) receptor, prostaglandin IP and EP(4) receptors, and parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Besides activating protein kinase A, increased levels of cAMP depolarize podocytes via opening of chloride channels. Relatively little is known about the impact of the cAMP pathway on podocyte function. Results obtained in a limited number of studies indicate that cAMP in podocytes may regulate cell morphology, actin assembly, and matrix production. In addition, cAMP seems to attenuate the action of hormones, which activate the Ca(2+)/protein kinase C pathway. Effects of the cAMP pathway on further aspects of podocyte biology, such as contractility, phosphorylation state of slit membrane-associated proteins, glomerular permeability, cell cycle control, and synthesis of reactive oxygen species can be anticipated from studies on other cell types and from studies on isolated glomeruli. In summary, the data available indicate that the cAMP pathway affects several aspects of podocyte biology in an overall glomerulo-protective manner.

Animals↗

PubMed2001

Podocytes respond to mechanical stress in vitro.

Glomerular capillary pressure is thought to affect the structure and function of glomerular cells. However, it is unknown whether podocytes are intrinsically sensitive to mechanical forces. In the present study, differentiated mouse podocytes were cultured on flexible silicone membranes. Biaxial cyclic stress (0.5 Hz and 5% linear strain) was applied to the membranes for up to 3 d. Mechanical stress reduced the size of podocyte cell bodies, and processes became thin and elongated. Podocytes did not align in the inhomogeneous force field. Whereas the network of microtubules and that of the intermediate filament vimentin exhibited no major changes, mechanical stress induced a reversible reorganization of the actin cytoskeleton: transversal stress fibers (SF) disappeared and radial SF that were connected to an actin-rich center (ARC) formed. Epithelial and fibroblast cell lines did not exhibit a comparable stress-induced reorganization of the F-actin. Confocal and electron microscopy revealed an ellipsoidal and dense filamentous structure of the ARC. Myosin II, alpha-actinin, and the podocyte-specific protein synaptopodin were present in radial SF, but, opposite to F-actin, they were not enriched in the ARC. The formation of the ARC and of radial SF in response to mechanical stress was inhibited by nonspecific blockade of Ca(2+) influx with Ni(2+) (1 mM), by Rho kinase inhibition with Y-27632 (10 microM), but not by inhibition of stretch-activated cation channels with Gd(3+) (50 microM). In summary, mechanical stress induces a unique reorganization of the actin cytoskeleton in podocytes, featuring radial SF and an ARC, which differ in protein composition. The F-actin reorganization in response to mechanical stress depends on Ca(2+) influx and Rho kinase. The present study provides the first direct evidence that podocytes are mechanosensitive.

Actins↗