Signaling In Podocytes Research Articles

Letter to the Editor

LETTER TO THE EDITORLetter to the EditorKevin D. Burns and Ningjun LiKevin D. Burns and Ningjun LiPublished Online:01 Mar 2009https://doi.org/10.1152/ajprenal.90544.2008MoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat to the editor: We are writing in response to the corrigendum by Velez et al. (2) to their original manuscript, in which they have determined that the peptide residing at mass 1,181 Da corresponds to angiotensin-(2-10), and not angiotensin-(2-9). In their letter, the authors have incorrectly suggested that our previous publication using isolated rat proximal tubules assigned a peptide with mass 1,181 Da as angiotensin-(1-9) (1). In our manuscript, angiotensin-(1-9) was assigned a mass of 1,183 Da, as clearly indicated in Figs. 4, 5C, and 7 (1). This assignment was based on sequence analysis. The small peaks in the mass spectra at 1,181 Da, depicted in Fig. 5, A and B, were not assigned to angiotensin-(1-9). Indeed, these peaks were not sequenced, but may correspond to angiotensin-(2-10), as Velez et al. have determined. We would like to emphasize therefore that the corrigendum by Velez et al. (2) does not alter in any way the results or conclusions of our study.REFERENCES1 Li N, Zimpelmann J, Cheng K, Wilkins JA, Burns KD. The role of angiotensin converting enzyme 2 in the generation of angiotensin 1-7 by rat proximal tubules. Am J Physiol Renal Physiol 288: F353–F362, 2005.Link | ISI | Google Scholar2 Velez JC, Bland AM, Arthur JM, Raymond JR, Janech MG. Characterization of renin-angiotensin system activities in cultured mouse podocytes. Am J Physiol Renal Physiol 293: F398–F407, 2007. Erratum in: Am J Physiol Renal Physiol 295: F857, 2008.Link | ISI | Google Scholar Download PDF Previous Back to Top FiguresReferencesRelatedInformationCited ByActivation of NFAT Signaling in Podocytes Causes Glomerulosclerosis22 July 2010 | Journal of the American Society of Nephrology, Vol. 21, No. 10 More from this issue > Volume 296Issue 3March 2009Pages F666-F666 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/ajprenal.90544.2008PubMed19234095History Published online 1 March 2009 Published in print 1 March 2009 Metrics

The Role of Osteopontin in the Development of Albuminuria

Several gene array studies have suggested that osteopontin (Opn) expression strongly correlates with albuminuria and glomerular disease. Urinary Opn concentration and kidney Opn immunoreactivity were found to be increased in patients with steroid-sensitive nephrotic syndrome. In addition, renal Opn mRNA was increased in the Ins2(Akita) mouse model of type 1 diabetic nephropathy, in the LPS-induced albuminuria model, and in glomeruli of puromycin aminonucleotide-induced nephrotic rats. Opn knockout mice did not develop albuminuria in response to LPS injection, and Opn knockout mice were protected from diabetes-induced albuminuria and mesangial expansion. In the glomerulus, Opn immunostaining was increased specifically in podocytes. Treatment of podocytes with recombinant Opn activated the NF-kappaB pathway, increased expression of urokinase plasminogen activator and matrix metalloproteinases 2 and 9, and increased podocyte motility. Taken together, these results indicate that Opn plays an important role in the development of albuminuria, possibly by modulating podocyte signaling and motility.

Erythropoietin Prevents Diabetes-Induced Podocyte Damage

Objective: Erythropoietin (EPO) has cytoprotective effects apart from its hematopoietic effects. We studied the effects of different EPO molecules on podocyte signaling in vitro and on podocyte survival in an experimental model of diabetic kidney injury (db/db mouse). Methods: We elucidated intracellular signaling by epoetin-β, darbepoetin-α, and the continuous erythropoietin receptor activator (CERA) in immortalized murine podocyte cultures. Moreover, we treated db/db micewith placebo or with CERA in a chronic (14-week) randomized controlled study. We also studied non-diabetic db/m mice as controls. Results: We could clearly demonstrate phosphorylation of the JAK/PI3K pathway and Akt signaling in podocytes by epoetin-β, darbepoetin-α and CERA. In the long-term animal study we found significantly reduced podocyte numbers in placebo-treated db/db mice compared to db/m control mice (7.4 ± 0.2 vs. 10.2 ± 0.9 per glomerular field; p < 0.05). Chronic CERA treatment ameliorated podocyte loss in kidneys of diabetic animals (8.5 ± 0.5 per glomerular field; p < 0.05 vs. placebo-treated db/db mice). Conclusion: EPO activates pro-survival intracellular pathways in podocytes in vitro, and ameliorates diabetes-induced podocyte loss in vivo. Chronic EPO administration may be a feasible way to protect podocyte from diabetic injury.

Modification of kidney barrier function by the urokinase receptor

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin. Mice lacking uPAR (Plaur-/-) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active beta3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate alphavbeta3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of alphavbeta3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes

Kidney podocytes and their slit diaphragms (SDs) form the final barrier to urinary protein loss. There is mounting evidence that SD proteins also participate in intracellular signaling pathways. The SD protein nephrin serves as a component of a signaling complex that directly links podocyte junctional integrity to actin cytoskeletal dynamics. Another SD protein, CD2-associated protein (CD2AP), is an adaptor molecule involved in podocyte homeostasis that can repress proapoptotic TGF-beta signaling in podocytes. Here we show that dendrin, a protein originally identified in telencephalic dendrites, is a constituent of the SD complex, where it directly binds to nephrin and CD2AP. In experimental glomerulonephritis, dendrin relocates from the SD to the nucleus of injured podocytes. High-dose, proapoptotic TGF-beta1 directly promotes the nuclear import of dendrin, and nuclear dendrin enhances both staurosporine- and TGF-beta1-mediated apoptosis. In summary, our results identify dendrin as an SD protein with proapoptotic signaling properties that accumulates in the podocyte nucleus in response to glomerular injury and provides a molecular target to tackle proteinuric kidney diseases. Nuclear relocation of dendrin may provide a mechanism whereby changes in SD integrity could translate into alterations of podocyte survival under pathological conditions.

CD2AP/CIN85 Balance Determines Receptor Tyrosine Kinase Signaling Response in Podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.

TGF-β Concentration Specifies Differential Signaling Profiles of Growth Arrest/Differentiation and Apoptosis in Podocytes

Podocyte depletion occurs in most progressive glomerular diseases and is thought to result from podocyte loss while the remaining podocytes are unable to proliferate. The underlying mechanisms for podocyte growth arrest/differentiation and depletion remain poorly understood but may involve TGF-beta, which is typically upregulated in injured glomeruli. The TGF-beta are multifunctional cytokines that regulate growth, differentiation, and apoptosis in most cells. Determinants of functional specificity of TGF-beta signaling in cell-cycle control and apoptosis remain poorly understood. Using a unique system of conditionally immortalized podocytes, it is demonstrated that autocrine TGF-beta2 induces G0/G1 arrest and differentiation under nonpermissive culture through Smad3-dependent induction of the cyclin-dependent kinase inhibitor p15(Ink4b) (Cdkn2b). When exposed to recombinant TGF-beta1 (or TGF-beta2), nonpermissive culture podocytes switch to G2/M arrest and apoptosis, selectively at advanced TGF-beta concentrations and specifically in association with suppression of Cdkn2b and activation of proapoptotic p38 mitogen-activated protein kinase. Thus, distinct signaling profiles activated in a concentration-dependent manner by TGF-beta were identified. Autocrine TGF-beta2/Smad3/Cdkn2b signaling in podocytes specifies G0/G1 arrest associated with podocyte differentiation, whereas increasing TGF-beta concentrations beyond a critical threshold induces G2/M block and apoptosis associated with selective p38 mitogen-activated protein kinase activation and with suppression of Cdkn2b. In summary, the results suggest a new functional requirement of TGF-beta2 in growth arrest and differentiation of murine podocytes in vitro and demonstrate that a critical TGF-beta concentration threshold may specify a molecular switch to proapoptotic signaling profiles and apoptosis.

Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats.

Angiotensin II (AngII) is a critical determinant of glomerular function involving both hemodynamic and pressure-independent effects that are insufficiently understood. A novel transgenic rat (TGR) model with overexpression of the human AngII type 1 receptor (hAT1) in podocytes was developed to study the consequences of an increased AT1 signaling on the structure and function of the glomerular filter. Use of the nephrin promoter to target the podocytes resulted in an expression of the hAT1 at a level roughly two times higher than the endogenous AT1 throughout life. All male TGR developed significant albuminuria starting at 8 to 15 wk of age; systolic BP was not elevated. More or less concurrently, structural changes at the glomerulus were encountered, starting with ubiquitous formation of pseudocysts at podocytes, followed by foot process effacement and local detachments. This damage progressed to nephron loss via the well known pathway typical for classic focal segmental glomerulosclerosis. The structural changes significantly correlated with age (r(2) = 0.76) and urinary albumin excretion (r(2) = 0.70). The data provide direct evidence that increased AT1 signaling in podocytes leads to protein leakage and structural podocyte damage progressing to focal segmental glomerulosclerosis.

Novel role for podocin as a genetic cause of hypertension

OR-42

Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling.

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes.

Inhibitory smads and tgf-Beta signaling in glomerular cells.

Smad6 and Smad7 are inhibitory SMADs with putative functional roles at the intersection of major intracellular signaling networks, including TGF-beta, receptor tyrosine kinase (RTK), JAK/STAT, and NF-kappaB pathways. This study reports differential functional roles and regulation of Smad6 and Smad7 in TGF-beta signaling in renal cells, in murine models of renal disease and in human glomerular diseases. Smad7 is upregulated in podocytes in all examined glomerular diseases (focal segmental glomerulosclerosis [FSGS], minimal-change disease [MCD], membranous nephropathy [MNP], lupus nephritis [LN], and diabetic nephropathy [DN]) with a statistically significant upregulation in "classical" podocyte-diseases such as FSGS and MCD. TGF-beta induces Smad7 synthesis in cultured podocytes and Smad6 synthesis in cultured mesangial cells. Although Smad7 expression inhibited both Smad2- and Smad3-mediated TGF-beta signaling in podocytes, it inhibited only Smad3 but not Smad2 signaling in mesangial cells. In contrast, Smad6 had no effect on TGF-beta/Smad signaling in podocytes and enhanced Smad3 signaling in mesangial cells. These data suggest that Smad7 is activated in injured podocytes in vitro and in human glomerular disease and participates in negative control of TGF-beta/Smad signaling in addition to its pro-apoptotic activity, whereas Smad6 has no role in TGF-beta response and injury in podocytes. In contrast, Smad6 is upregulated in the mesangium in human glomerular diseases and may be involved in functions independent of TGF-beta/Smad signaling. These data indicate an important role for Smad6 and Smad7 in glomerular cells in vivo that could be important for the cell homeostasis in physiologic and pathologic conditions.

Cyclic GMP signaling in podocytes

Natriuretic peptides (NP), together with nitric oxide (NO) are powerful relaxing factors acting via a common second messenger, cyclic GMP (cGMP). Together with other vasoactive modulators, these vasorelaxing factors play an essential role in regulating the function of kidney glomeruli. The presence of NP receptors in podocytes has been well documented. Recently, also mRNA for soluble guanylate cyclase, the NO receptor, has been shown in these cells. Stimulation of podocytes with atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), and NO donors results in considerable upregulation of cellular cGMP synthesis. The podocyte foot processes contain a highly organized network of microfilaments adhering to the glomerular basement membrane (GBM). Changes in podocyte cytoskeleton accompanied by detachment of the cells from the GBM are closely associated with many glomerulopathies. The contractile apparatus in the podocyte foot processes seems to be an obvious target for the cyclic GMP signaling cascade. However, little is known about implications of the cGMP synthesis in these cells. We briefly review the current art regarding generation and modulation of cyclic GMP levels in podocytes. We discuss also the possible targets for this secondary messenger as well as its functional role in podocytes.

Regulation of adhesive interaction between podocytes and glomerular basement membrane.

Glomerular filtration depends on well-orchestrated cell-cell and cell-matrix contacts of glomerular podocytes. Over the last years critical constituents of these contacts have been identified via molecular approaches. Podocyte cell-matrix interactions have been shown to be mediated in part by alpha(3)beta(1)-integrin heterodimers. Disturbances of integrin matrix interaction lead to detachment of podocytes in vitro, corresponding to the critical event of foot process retraction and glomerular basement membrane (GBM) denudation in vivo. Further, dystroglycan-mediated matrix attachment appears to play a critical role for podocyte foot process architecture. Downstream signaling events are currently elucidated concentrating mainly on integrin-dependent cascades and their consequences for podocyte adhesion and proliferation. An activation of the integrin-linked kinase in podocyte damage in vivo and in vitro makes this molecule a particularly interesting candidate for integrin-mediated inside-out and outside-in signaling in podocytes. Podocyte cell-cell interaction has been characterized in a few studies in vitro, indicating the slit diaphragm to be a modified adherens junction. The structural link between the cell-matrix and cell-cell contacts is maintained by the actin cytoskeleton, which may also enable cross-talk between these two cell contact sites. Examining podocyte function in tissue culture, animal models and human expression studies should allow further detailed dissection of the molecular pathways responsible for maintenance and failure of the glomerular filtration barrier.