Wild-type Podocytes Research Articles

Melanocortin 5 receptor signaling protects against podocyte injury in proteinuric glomerulopathies.

Melanocortin therapeutics, exemplified by adrenocorticotropic hormone, have a proven steroidogenic-independent anti-proteinuric and glomerular protective effect. The biological functions of melanocortins are mediated by melanocortin receptors (MCR), including MC1R, which recent studies have shown to protect against glomerular disease. However, the role of other MCRs like MC5R is unknown. Here, MC5R knockout exacerbated glomerulopathy in mice injured by adriamycin (ADR) or nephrotoxic serum (NTS), as demonstrated by increased albuminuria and podocyte injury. Conversely, selective MC5R agonism using a peptidomimetic agonist improved outcomes of glomerulopathies. Mechanistically, MC5R is expressed in glomerular podocytes. Reconstitution of MC5R in podocytes attenuated glomerular injury and proteinuria in MC5R knockout mouse models of glomerulopathies, indicating a direct podocyte protective effect. In vitro, MC5R agonism in primary wild-type podocytes attenuated ADR-elicited cytoskeleton disruption, hypermotility and apoptosis, associated with restored inhibitory phosphorylation of glycogen synthases kinase 3β (GSK3β), a signaling transducer downstream of MC5R and at the nexus of multiple podocytopathic pathways. In parallel, ADR-induced phosphorylation and activation of GSK3β substrates, such as paxillin and NFκB RelA/p65, were abrogated, leading to improved actin cytoskeleton integrity and diminished expression of mediators of podocyte injury, like MCP-1, B7-1 and Cathepsin L. This protective effect of MC5R agonism was blunted in wild-type podocytes expressing constitutively active GSK3β and was mimicked in MC5R knockout podocytes by ectopic expression of dominant negative GSK3β. Consistently in ADR-injured MC5R knockout mice, worsened podocytopathy was associated with enhanced GSK3β hyperactivity. These findings suggest that MC5R signaling protects against podocyte injury and may serve as a novel therapeutic target for glomerular diseases.

Empagliflozin reduces podocyte lipotoxicity in experimental Alport syndrome.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are anti-hyperglycemic agents that prevent glucose reabsorption in proximal tubular cells. SGLT2i improves renal outcomes in both diabetic and non-diabetic patients, indicating it may have beneficial effects beyond glycemic control. Here, we demonstrate that SGLT2i affects energy metabolism and podocyte lipotoxicity in experimental Alport syndrome (AS). In vitro, we found that the SGLT2 protein was expressed in human and mouse podocytes to a similar extent in tubular cells. Newly established immortalized podocytes from Col4a3 knockout mice (AS podocytes) accumulate lipid droplets along with increased apoptosis when compared to wild-type podocytes. Treatment with SGLT2i empagliflozin reduces lipid droplet accumulation and apoptosis in AS podocytes. Empagliflozin inhibits the utilization of glucose/pyruvate as a metabolic substrate in AS podocytes but not in AS tubular cells. In vivo, we demonstrate that empagliflozin reduces albuminuria and prolongs the survival of AS mice. Empagliflozin-treated AS mice show decreased serum blood urea nitrogen and creatinine levels in association with reduced triglyceride and cholesterol ester content in kidney cortices when compared to AS mice. Lipid accumulation in kidney cortices correlates with a decline in renal function. In summary, empagliflozin reduces podocyte lipotoxicity and improves kidney function in experimental AS in association with the energy substrates switch from glucose to fatty acids in podocytes.

NUP133 Controls Nuclear Pore Assembly, Transcriptome Composition, and Cytoskeleton Regulation in Podocytes.

Steroid-resistant nephrotic syndrome (SRNS) frequently leads to end-stage renal disease, ultimately requiring kidney replacement therapies. SRNS is often caused by hereditary monogenic mutations, specifically affecting specialized epithelial cells (podocytes) of the glomerular filtration barrier. Mutations in several components of the nuclear pore complex, including NUP133 and NUP107, have been recently identified to cause hereditary SRNS. However, underlying pathomechanisms, eliciting podocyte-specific manifestations of these nucleoporopathies, remained largely elusive. Here, we generated an in vitro model of NUP133-linked nucleoporopathies using CRISPR/Cas9-mediated genome editing in human podocytes. Transcriptome, nuclear pore assembly, and cytoskeleton regulation of NUP133 loss-of-function, mutant, and wild-type podocytes were analyzed. Loss of NUP133 translated into a disruption of the nuclear pore, alterations of the podocyte-specific transcriptome, and impaired cellular protrusion generation. Surprisingly, comparative analysis of the described SRNS-related NUP133 mutations revealed only mild defects. Am impaired protein interaction in the Y-complex and decrease of NUP133 protein levels might be the primary and unifying consequence of mutant variants, leading to a partial loss-of-function phenotype and disease manifestation in susceptible cell types, such as podocytes.

Loss of sphingosine kinase 2 enhances Wilm's tumor suppressor gene 1 and nephrin expression in podocytes and protects from streptozotocin-induced podocytopathy and albuminuria in mice

The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2-/- mice compared to STZ-treated wildtype mice.To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation.The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation.Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

Podocyte-specific knockout of the neonatal Fc receptor (FcRn) results in differential protection depending on the model of glomerulonephritis.

Podocytes have been proposed to be antigen presenting cells (APCs). In traditional APCs, the neonatal Fc receptor (FcRn) is required for antigen presentation and global knockout of FcRn protects against glomerulonephritis. Since podocytes express FcRn, we sought to determine whether the absence of podocyte FcRn ameliorates immune-mediated disease. We examined MHCII and costimulatory markers expression in cultured wild type (WT) and FcRn knockout (KO) podocytes. Interferon gamma (IFNγ) induced MHCII expression in both WT and KO podocytes but did not change CD80 expression. Neither WT nor KO expressed CD86 or inducible costimulatory ligand (ICOSL) at baseline or with IFNγ. Using an antigen presentation assay, WT podocytes but not KO treated with immune complexes induced a modest increase in IL-2. Induction of the anti-glomerular basement membrane (anti-GBM) model resulted in a significant decrease in glomerular crescents in podocyte-specific FcRn knockout mouse (podFcRn KO) versus controls but the overall percentage of crescents was low. To examine the effects of the podocyte-specific FcRn knockout in a model with a longer autologous phase, we used the nephrotoxic serum nephritis (NTS) model. We found that the podFcRn KO mice had significantly reduced crescent formation and glomerulosclerosis compared to control mice. This study demonstrates that lack of podocyte FcRn is protective in immune mediated kidney disease that is dependent on an autologous phase. This study also highlights the difference between the anti-GBM model and NTS model of disease.

Knockout of the neonatal Fc receptor in cultured podocytes alters IL-6 signaling and the actin cytoskeleton.

The neonatal Fc receptor (FcRn) has been shown to be required for antigen presentation in dendritic cells, and global knockout of FcRn attenuates immune-mediated kidney disease. Podocytes express interleukin-6 (IL-6) receptor and produce IL-6 under proinflammatory conditions. Here we examined the role of FcRn in the IL-6-mediated inflammatory response in podocytes. We examined IL-6 production by ELISA and expression by qPCR in wild type (WT) and FcRn knockout (KO) podocytes after treatment with proinflammatory stimuli as well as IL-6-mediated signaling via the JAK/STAT pathway. We also examined podocyte motility in cultured WT and KO podocytes after a proinflammatory challenge. We found that FcRn KO podocytes produced minimal amount of IL-6 after treatment with albumin, IgG, or immune complexes whereas WT podocytes had a robust response. FcRn KO podocytes also had minimal expression of IL-6 compared with WT. By Western blotting, there was significantly less phosphorylated STAT3 in KO podocytes after treatment with IFNγ or immune complexes. In a scratch assay, FcRn KO podocytes showed increased motility comparted KO, suggesting a defect in actin dynamics. Cultured FcRn KO podocytes also demonstrated abnormal stress fibers compared with WT and the defect could be rescued by IL-6 treatment. This study shows that in podocytes, FcRn modulates the IL-6 mediated response to proinflammatory stimuli and regulates podocytes actin structure, motility and synaptopodin expression.

New CRISPR/Cas9 mouse model confirms association of phosphorylation of ACTN4 at serine 159 with proteinuria and focal segmental glomerulosclerosis

Mutations in α‐actinin‐4 (ACTN4)—an important cytoskeleton protein that binds actin—cause a form of kidney injury in humans called familial focal segmental glomerulosclerosis (FSGS). FSGS is characterized by proteinuria and eventual progression to renal failure. All of the known disease‐causing mutations result in increased ACTN4 binding affinity to actin. We previously showed that this increased actin binding was associated with biomechanical changes that left podocytes vulnerable to mechanical stress and eventual detachment. We also previously detected with mass spectrometry that ACTN4 is phosphorylated at the serine 159 site (S159) in cultured human podocytes. Moreover, phosphomimetic S159D ACTN4 protein (which mimics the effect of phosphorylation at S159) confers similar increased actin binding affinity as disease‐causing mutant ACTN4. In the current study, we hypothesized that the changes in binding affinity associated with phosphorylation of S159 may also render the podocyte vulnerable to mechanical stress and lead to eventual proteinuria and FSGS in vivo. Using CRISPR/Cas9 technology, we developed a new phosphomimetic mouse model Actn4 S160D (analogous to S159D mutation in humans). We first isolated primary podocytes from wild‐type (WT) and homozygous Actn4S160D/S160D mice. Similar to podocytes carrying disease‐causing mutant Actn4, homozygous Actn4S160D/S160D podocytes demonstrated more aligned actin filaments and increased number of focal adhesions (quantified using vinculin staining) in comparison with WT podocytes. We subjected both WT and homozygous Actn4S160D/S160D mice to subtotal nephrectomy to induce glomerular hyperfiltration and increase glomerular mechanical stress upon podocytes. We found that in comparison with WT, homozygous Actn4S160D/S/160D mice demonstrated significantly increased albuminuria at 2, 3, and 7 weeks after nephrectomy. Additionally, kidney sections were used for PAS staining and examined by a renal pathologist to assess for glomerulosclerosis. Whereas only 1% of glomeruli were found to be sclerosed in WT, 12% were sclerosed in homozygous Actn4S160D/S/160D mice (p<0.001). Our findings confirm that increased phosphorylation at S159 is pathologic, in showing that a phosphomimetic mouse model develops albuminuria and FSGS. Moreover, we show that phosphomimetic podocytes demonstrate changes in actin alignment and focal adhesion, markers of podocyte dysfunction that are shared by disease‐causing mutant ACTN4 podocytes. Altogether, these findings add to the building evidence suggesting that abnormal phosphorylation at S159 leads to changes on a protein, cellular, and whole animal level that are similar to mutant ACTN4‐mediated disease. This evidence provides new insights into the importance of a post‐translational modification affecting the podocyte cytoskeleton.Support or Funding InformationK01DK114329 and R37DK059588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutant Actn4 alters the distribution of intracellular stress within podocytes and contributes to their mechanical failure in response to stretch

Alpha‐actinin‐4 (ACTN4 in humans, Actn4 in mice) crosslinks actin filaments in podocytes, providing them mechanical resilience as they help maintain the glomerular filtration barrier. Although human disease‐causing mutant ACTN4 leads to brittle reconstituted actin networks in vitro, how this affects the ability of podocytes to withstand the external mechanical challenges within the glomerulus remains unknown. We studied primary podocytes isolated from wild‐type (WT) and disease‐causing mutant Actn4 knock‐in mice. We previously reported that, after transient stretch, WT cells recovered their baseline contraction and actin cytoskeleton breakages, whereas mutant podocytes developed irrecoverable reductions in their contraction and irreparable disruptions in their cytoskeletons. We now report that after stretch, when challenged in a centrifugal detachment assay, mutant podocytes (11/23) had a higher probability of detachment than WT (2/19) (odds ratio =7.42, 95% CI 1.27, 80.75; p=0.017). Through an analysis of baseline biophysical properties, we found that the actin filaments of mutant Actn4 podocytes displayed a more uniform distribution of orientation (aligned more parallel to each other) compared with the filaments of WT podocytes (which displayed more random orientation). We then computed baseline intracellular stress in terms of two constituent components: the magnitude of mean principal stress as tension (σ), and the orientation of first principal stress as principal orientation (θ). The spatial distribution of intracellular stress within the podocytes was measured using the autocorrelation function C(r). The distance over which the C(r) decays to a value of 0.5 was extrapolated as a singular value from the autocorrelation curves and is termed correlation length, or r1/2. The greater the r1/2 value, the greater the distance over which intracellular stress maintains uniformity. We found that mutant podocytes displayed a more uniform distribution of intracellular stress across the cell, both in terms of magnitude (r1/2 σ mean ± SE: mutant 18.0 ± 0.8 μm vs. WT 13.4 ± 0.8 μm) and orientation (r1/2 θ mean ± SE: mutant 34.7 ± 2.2 μm vs. WT 25.9 ± 2.3 μm). Moreover, the more uniform the intracellular stress was distributed at baseline, the less likely mutant podocytes recovered their baseline contraction after periodic stretch. Taken together, our findings provide novel insight into the mechanism by which ACTN4 mutation alters the alignment of the cytoskeleton and impairs the resiliency of the podocyte, leaving it vulnerable to mechanical stretch and more prone to detachment in its glomerular environment.Support or Funding InformationR37DK059588 and T32DK007199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fyn-binding protein ADAP supports actin organization in podocytes.

The renal podocyte is central to the filtration function of the kidney that is dependent on maintaining both highly organized, branched cell structures forming foot processes, and a unique cell‐cell junction, the slit diaphragm. Our recent studies investigating the developmental formation of the slit diaphragm identified a novel claudin family tetraspannin, TM4SF10, which is a binding partner for ADAP (also known as Fyn binding protein Fyb). To investigate the role of ADAP in podocyte function in relation to Fyn and TM4SF10, we examined ADAP knockout (KO) mice and podocytes. ADAP KO mice developed glomerular pathology that began as hyalinosis and progressed to glomerulosclerosis, with aged male animals developing low levels of albuminuria. Podocyte cell lines established from the KO mice had slower attachment kinetics compared to wild‐type cells, although this did not affect the total number of attached cells nor the ability to form focal contacts. After attachment, the ADAP KO cells did not attain typical podocyte morphology, lacking the elaborate cell protrusions typical of wild‐type podocytes, with the actin cytoskeleton forming circumferential stress fibers. The absence of ADAP did not alter Fyn levels nor were there differences between KO and wild‐type podocytes in the reduction of Fyn activating phosphorylation events with puromycin aminonucleoside treatment. In the setting of endogenous TM4SF10 overexpression, the absence of ADAP altered the formation of cell‐cell contacts containing TM4SF10. These studies suggest ADAP does not alter Fyn activity in podocytes, but appears to mediate downstream effects of Fyn controlled by TM4SF10 involving actin cytoskeleton organization.

A small molecule screening to detect potential therapeutic targets in human podocytes.

A small molecule screening to detect potential therapeutic targets in human podocytes. Am J Physiol Renal Physiol 312: F157-F171, 2017. First published October 19, 2016; doi:10.1152/ajprenal.00386.2016. Steroid-resistant nephrotic syndrome (SRNS) inevitably progresses to end-stage kidney disease, requiring dialysis or transplantation for survival. However, treatment modalities and drug discovery remain limited. Mutations in over 30 genes have been discovered as monogenic causes of SRNS. Most of these genes are predominantly expressed in the glomerular epithelial cell, the podocyte, placing it at the center of the pathogenesis of SRNS. Podocyte migration rate (PMR) represents a relevant intermediate phenotype of disease in monogenic causes of SRNS. We therefore adapted PMR in a high-throughput manner to screen small molecules as potential therapeutic targets for SRNS. We performed a high-throughput drug screening of a National Institutes of Health Clinical Collection (NCC) library (n = 725 compounds) measuring PMR by videomicroscopy. We used the Woundmaker to perform individual 96-well scratch wounds and screened compounds using a quantitative kinetic live cell imaging migration assay using IncuCyte ZOOM technology. Using a normal distribution for the average PMR in wild-type podocytes with a vehicle control (DMSO), we applied a 90% confidence interval to define "distinct" compounds (5% faster/slower PMR) and found that 12 of 725 compounds (at 10 μM) reduced PMR. Clusters of drugs that alter PMR included actin/tubulin modulators such as the azole class of antifungals and antineoplastic vinca-alkaloids. We hereby identify compounds that alter PMR. The PMR assay provides a new avenue to test therapeutics for nephrotic syndrome. Positive results may reveal novel pathways in the study of glomerular diseases such as SRNS.

Dynamin-Related Protein 1 Deficiency Improves Mitochondrial Fitness and Protects against Progression of Diabetic Nephropathy.

Mitochondrial fission has been linked to the pathogenesis of diabetic nephropathy (DN). However, how mitochondrial fission affects progression of DN in vivo is unknown. Here, we report the effect of conditional podocyte-specific deletion of dynamin-related protein 1 (Drp1), an essential component of mitochondrial fission, on the pathogenesis and progression of DN. Inducible podocyte-specific deletion of Drp1 in diabetic mice decreased albuminuria and improved mesangial matrix expansion and podocyte morphology. Ultrastructure analysis revealed a significant increase in fragmented mitochondria in the podocytes of wild-type diabetic mice but a marked improvement in mitochondrial structure in Drp1-null podocytes of diabetic mice. When isolated from diabetic mice and cultured in high glucose, Drp1-null podocytes had more elongated mitochondria and better mitochondrial fitness associated with enhanced oxygen consumption and ATP production than wild-type podocytes. Furthermore, administration of a pharmacologic inhibitor of Drp1, Mdivi1, significantly blunted mitochondrial fission and rescued key pathologic features of DN in mice. Taken together, these results provide novel correlations between mitochondrial morphology and the progression of DN and point to Drp1 as a potential therapeutic target in DN.

Lack of CD2AP disrupts Glut4 trafficking and attenuates glucose uptake in podocytes.

The adapter protein CD2-associated protein (CD2AP) functions in various signaling and vesicle trafficking pathways, including endosomal sorting and/or trafficking and degradation pathways. Here, we investigated the role of CD2AP in insulin-dependent glucose transporter 4 (Glut4, also known as SLC2A4) trafficking and glucose uptake. Glucose uptake was attenuated in CD2AP(-/-) podocytes compared with wild-type podocytes in the basal state, and CD2AP(-/-) podocytes failed to increase glucose uptake in response to insulin. Live-cell imaging revealed dynamic trafficking of HA-Glut4-GFP in wild-type podocytes, whereas in CD2AP(-/-) podocytes, HA-Glut4-GFP clustered perinuclearly. In subcellular membrane fractionations, CD2AP co-fractionated with Glut4, IRAP (also known as LNPEP) and sortilin, constituents of Glut4 storage vesicles (GSVs). We further found that CD2AP forms a complex with GGA2, a clathrin adaptor, which sorts Glut4 to GSVs, suggesting a role for CD2AP in this process. We also found that CD2AP forms a complex with clathrin and connects clathrin to actin in the perinuclear region. Furthermore, clathrin recycling back to trans-Golgi membranes from the vesicular fraction containing GSVs was defective in the absence of CD2AP. This leads to reduced insulin-stimulated trafficking of GSVs and attenuated glucose uptake into CD2AP(-/-) podocytes.

Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli

A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis. Here we examined the effects of angiotensin II on intracellular calcium ion levels and endogenous channels in intact podocytes of freshly isolated decapsulated mouse glomeruli. An ion channel with distinct TRPC6 properties was identified in wild type, but was absent in TRPC6 knockout mice. Single channel electrophysiological analysis found that angiotensin II acutely activated native TRPC-like channels in both podocytes of freshly isolated glomeruli and TRPC6 channels transiently overexpressed in CHO cells; the effect was mediated by changes in the channel open probability. Angiotensin II evoked intracellular calcium transients in the wild type podocytes, which was blunted in TRPC6 knockout glomeruli. Pan-TRPC inhibitors gadolinium and SKF 96365 reduced the response in wild type glomerular epithelial cells, whereas the transient in TRPC6 knockout animals was not affected. Thus, angiotensin II-dependent activation of TRPC6 channels in podocytes may have a significant role in the development of kidney diseases.

CD2AP Regulates SUMOylation of CIN85 in Podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.

The Prorenin Receptor

The discovery of the prorenin receptor, now nearly a decade ago, altered our view of the renin-angiotensin system (RAS).[1][1],[2][2] Binding of prorenin, the inactive precursor of renin, to the prorenin receptor results in full catalytic activity of prorenin through a nonproteolytic mechanism that

Glomerular podocytes: A study of mechanical properties and mechano-chemical signaling

Kidney glomeruli function as filters, allowing the passage of small solutes and waste products into the urinary tract, while retaining essential proteins and macromolecules in the blood stream. These structures are under constant mechanical stress due to fluid pressure, driving filtration across the barrier. We mechanically stimulated adherent wildtype podocytes using the methods of magnetic tweezer and twisting as well as cell stretching. Attaching collagen IV-coated or poly-l-lysine-coated magnetic beads to cell receptors allowed for the determination of cellular stiffness. Angiotensin II-treated podocytes showed slightly higher stiffness than untreated cells, the cell fluidity (i.e. internal dynamics) remained similar, and showed an increase with force. The bead detachment (a measure of the binding strength) was higher in angiotensin II-treated compared to untreated podocytes. Magnetic twisting confirmed that angiotensin II treatment of podocytes increases and CDTA treatment decreases cell stiffness. However, treatment with both angiotensin II and CDTA increased the cell stiffness only slightly compared to solely CDTA-treated cells. Exposing podocytes to cyclic, uniaxial stretch showed an earlier onset of ERK1/2 phosphorylation compared to MEF (control) cells. These results indicate that angiotensin II might free intracellularly stored calcium and affects actomyosin contraction, and that mechanical stimulation influences cell signaling.

Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

355 A Novel Role for Cd2Ap in Normal Podocyte Differentiation

Background: CD2AP is a multifunctional adaptor molecule, binding other key podocyte proteins within the slit-diaphragm complex. Methods: The cellular phenotype of conditionally immortalized wild-type (WT) and CD2AP mutant podocytes was compared by light microscopy. Immunoflourescence and western blot was then used to examine the expression of characteristic podocyte markers nephrin, podocin, CD2AP, PAX2 WT1 as well as WTIP and mesenchymal markers fibronectin and α-SMA in CD2AP mutant podocytes. To investigate differences in localisation of WTIP further, nuclear and cytoplasmic fractions were isolated from each cell line and nuclear/cytoplasmic expression ratios compared. Results: In contrast to WT podocytes, CD2AP mutant demonstrated spindle shaped fibroblast like morphology similar to WT1 mutatant podocytes and were unable to form recognizable cell-cell contacts. Although nephrin, CD2AP podocin and WT1 were expressed at comparable levels in both cell lines, mesenchymal markers fibronectin and α-SMA were significantly overexpressed in CD2AP mutants compared with WT. Western blot and immunoflourescence data showed that PAX-2 was also upregulated in CD2AP mutant cells in keeping with a WT1-related defect. Immunofluorescence staining confirmed that WTIP co-localized at the cell membrane in WT podocytes, whereas in mutant cells WTIP expression was primarily nuclear. This observation was confirmed by a significant increase. (p=0.03) in the nuclear/cytoplasmic ratio of WTIP expression in CD2AP mutants. Conclusion: a role for CD2AP in maintainance of the normal differentiated podocyte phenotype. This may be partly mediated through WT1. Our findings have clear implications for a novel role for CD2AP in development and progression of glomerular disease.

Inhibition of Podocyte FAK Protects against Proteinuria and Foot Process Effacement

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.

Angiotensin type 2 receptor actions contribute to angiotensin type 1 receptor blocker effects on kidney fibrosis

Angiotensin type 1 (AT1) receptor blocker (ARB) ameliorates progression of chronic kidney disease. Whether this protection is due solely to blockade of AT1, or whether diversion of angiotensin II from the AT1 to the available AT2 receptor, thus potentially enhancing AT2 receptor effects, is not known. We therefore investigated the role of AT2 receptor in ARB-induced treatment effects in chronic kidney disease. Adult rats underwent 5/6 nephrectomy. Glomerulosclerosis was assessed by renal biopsy 8 wk later, and rats were divided into four groups with equivalent glomerulosclerosis: no further treatment, ARB, AT2 receptor antagonist, or combination. By week 12 after nephrectomy, systolic blood pressure was decreased in all treatment groups, but proteinuria was decreased only with ARB. Glomerulosclerosis increased significantly in AT2 receptor antagonist vs. ARB. Kidney cortical collagen content was decreased in ARB, but increased in untreated 5/6 nephrectomy, AT2 receptor antagonist, and combined groups. Glomerular cell proliferation increased in both untreated 5/6 nephrectomy and AT2 receptor antagonist vs. ARB, and phospho-Erk2 was increased by AT2 receptor antagonist. Plasminogen activator inhibitor-1 mRNA and protein were increased at 12 wk by AT2 receptor antagonist in contrast to decrease with ARB. Podocyte injury is a key component of glomerulosclerosis. We therefore assessed effects of AT1 vs. AT2 blockade on podocytes and interaction with plasminogen activator inhibitor-1. Cultured wild-type podocytes, but not plasminogen activator inhibitor-1 knockout, responded to angiotensin II with increased collagen, an effect that was completely blocked by ARB with lesser effect of AT2 receptor antagonist. We conclude that the benefical effects on glomerular injury achieved with ARB are contributed to not only by blockade of the AT1 receptor, but also by increasing angiotensin effects transduced through the AT2 receptor.