Lamine Aoudjit

Src-Family Kinase Fyn Phosphorylates the Cytoplasmic Domain of Nephrin and Modulates Its Interaction with Podocin

Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Src-family kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was co-transfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.

Activation of RhoA in Podocytes Induces Focal Segmental Glomerulosclerosis

Proper organization of the actin cytoskeleton is essential for the normal structure and function of podocytes. RhoA modulates actin dynamics but its role in podocyte biology is controversial. Here, we generated transgenic mice that express a constitutively active form of RhoA in a podocyte-specific and doxycycline-inducible manner. Induction of activated RhoA with doxycycline resulted in significant albuminuria. Furthermore, both the degree of albuminuria and the histologic changes in the glomerulus positively correlated with the level of constitutively active RhoA expression: low levels of expression associated with segmental foot-process effacement without changes observable by light microscopy, whereas higher levels of expression associated with both extensive foot-process effacement and histologic features of focal segmental glomerulosclerosis (FSGS). In addition, induction of activated RhoA markedly upregulated glomerular mRNA expression of fibronectin and collagen IA1, and the degree of upregulation positively correlated with the level of albuminuria. Withdrawal of doxycycline led to a decline in albuminuria toward basal levels in most mice, but heavy albuminuria persisted in some mice. Taken together, these data suggest that activation of RhoA in podocytes leads to albuminuria accompanied by a range of histologic changes characteristic of minimal change disease and FSGS in humans. Although most changes are reversible, severe and prolonged activation of RhoA may cause irreversible glomerulosclerosis.

Nck Proteins Maintain the Adult Glomerular Filtration Barrier

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.

Rac1 Contributes to Actin Organization in Glomerular Podocytes

Background/Aims: The function of glomerular podocytes is closely associated with the actin cytoskeleton. In this study, we studied the role of the small Rho-GTPase, Rac1, in actin organization in podocytes. Methods: Conditionally immortalized mouse podocytes (MP) stably expressing nephrin or control plasmid were used. Results: In MP, Rac1 activity increased significantly at 1 week of differentiation. MP stably expressing nephrin showed Rac1 activity significantly higher and more sustained than vector-expressing control cells. Antibody-mediated cross-linking of nephrin also activated Rac1. Differentiated MP showed more distinct lamellipodia/cellular processes, as compared with undifferentiated cells, which was further augmented by nephrin expression. Transient transfection of constitutively active Rac1 markedly increased the number of lamellipodia/cellular processes in undifferentiated MP, while the Rac1 inhibitor caused actin cytoskeleton derangement in differentiating MP. In the rat model of puromycin aminonucleoside nephrosis, RhoA activity was increased at Day 7 (at the peak of proteinuria), while Rac1 activity increased significantly only at Day 14, when the recovery process had started. Conclusion: Rac1 is activated in differentiating MP and nephrin potentiates Rac1 activation. Rac1 likely contributes to lamellipodia formation in differentiating MP and may contribute to process formation in podocytes recovering from injuries.

p21-Activated kinases regulate actin remodeling in glomerular podocytes

The tyrosine phosphorylation of nephrin is reported to regulate podocyte morphology via the Nck adaptor proteins. The Pak family of kinases are regulators of the actin cytoskeleton and are recruited to the plasma membrane via Nck. Here, we investigated the role of Pak in podocyte morphology. Pak1/2 were expressed in cultured podocytes. In mouse podocytes, Pak2 was predominantly phosphorylated, concentrated at the tips of the cellular processes, and its expression and/or phosphorylation were further increased when differentiated. Overexpression of rat nephrin in podocytes increased Pak1/2 phosphorylation, which was abolished when the Nck binding sites were mutated. Furthermore, dominant-negative Nck constructs blocked the Pak1 phosphorylation induced by antibody-mediated cross linking of nephrin. Transient transfection of constitutively kinase-active Pak1 into differentiated mouse podocytes decreased stress fibers, increased cortical F-actin, and extended the cellular processes, whereas kinase-dead mutant, kinase inhibitory construct, and Pak2 knockdown by shRNA had the opposite effect. In a rat model of puromycin aminonucleoside nephrosis, Pak1/2 phosphorylation was decreased in glomeruli, concomitantly with a decrease of nephrin tyrosine phosphorylation. These results suggest that Pak contributes to remodeling of the actin cytoskeleton in podocytes. Disturbed nephrin-Nck-Pak interaction may contribute to abnormal morphology of podocytes and proteinuria.

Planar cell polarity pathway regulates nephrin endocytosis in developing podocytes

Background: The PCP pathway controls many cell processes during development. Results: The PCP pathway induces nephrin endocytosis when cultured podocytes are treated with Wnt5a. Loss of PCP protein Vangl2 decreases nephrin endocytosis. Conclusion: During glomerular development, endocytosis of nephrin is regulated by the PCP pathway. Significance: Implicating the PCP pathway in nephrin endocytosis is important for understanding the complexity of PCP signaling during mammalian development. The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.

Podocyte Protein, Nephrin, Is a Substrate of Protein Tyrosine Phosphatase 1B

Glomerular podocytes are critical for the barrier function of the glomerulus in the kidney and their dysfunction causes protein leakage into the urine (proteinuria). Nephrin is a key podocyte protein, which regulates the actin cytoskeleton via tyrosine phosphorylation of its cytoplasmic domain. Here we report that two protein tyrosine phosphatases, PTP1B and PTP-PEST negatively regulate nephrin tyrosine phosphorylation. PTP1B directly binds to and dephosphorylates nephrin, while the action of PTP-PEST is indirect. The two phosphatases are also upregulated in the glomerulus in the rat model of puromycin aminonucleoside nephrosis. Both overexpression and inhibition of PTP1B deranged the actin cytoskeleton in cultured mouse podocytes. Thus, protein tyrosine phosphatases may affect podocyte function via regulating nephrin tyrosine phosphorylation.

Protein Tyrosine Phosphatase 1B Inhibition Protects against Podocyte Injury and Proteinuria

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed nonreceptor protein-tyrosine phosphatase that regulates various cellular functions, including migration. Recent studies suggest that an increased migratory phenotype of podocytes may be responsible for proteinuria and foot process effacement. The current study addresses the role of PTP1B in podocyte injury and proteinuria. PTP1B was markedly up-regulated in the glomerulus, notably in podocytes, in three rodent models of podocyte injury. Podocyte-specific ablation of the PTP1B gene ameliorated proteinuria induced by lipopolysaccharide and Adriamycin (doxorubicin). The use of a specific PTP1B inhibitor also protected against lipopolysaccharide-induced proteinuria. In contrast, podocyte-specific PTP1B transgenic male mice developed spontaneous proteinuria and foot process effacement. In cultured mouse podocytes, PTP1B knockdown and/or pretreatment with the PTP1B inhibitor blunted lipopolysaccharide-induced cell migration, activation of Src-family kinases (SFKs), and phosphorylation of focal adhesion kinase at Y397 (pFAK(Y397)), the latter being crucial for cell migration. Lipopolysaccharide-injected mice showed increased glomerular expression of active SFKs and pFAK(Y397), both of which were inhibited by podocyte-specific PTP1B knockout and the PTP1B inhibitor. Moreover, podocyte-specific PTP1B transgenic mice showed increased glomerular expression of active SFKs and pFAK(Y397). In summary, PTP1B up-regulation in podocytes induces a migratory response by activating SFKs and FAK, leading to foot process effacement and proteinuria. Pharmacological inhibition of PTP1B may have therapeutic potential in the treatment of proteinuric diseases.

Role of calcium-independent phospholipase A2 in complement-mediated glomerular epithelial cell injury

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase A(2) (PLA(2)) activation. The present study addresses the role of calcium-independent PLA(2) (iPLA(2)) in GEC injury. iPLA(2)beta short and iPLA(2)gamma were expressed in cultured rat GEC and normal rat glomeruli. To determine whether iPLA(2) is involved in complement-mediated arachidonic acid (AA) release, GEC were stably transfected with iPLA(2)gamma or iPLA(2)beta cDNAs (GEC-iPLA(2)gamma; GEC-iPLA(2)beta). Compared with control cells (GEC-Neo), GEC-iPLA(2)gamma and GEC-iPLA(2)beta demonstrated greater expression of iPLA(2) proteins and activities. Complement-mediated release of [(3)H]AA was augmented significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the augmented [(3)H]AA release was inhibited by the iPLA(2)-directed inhibitor bromoenol lactone (BEL). For comparison, overexpression of iPLA(2)gamma also amplified [(3)H]AA release after incubation of GEC with H(2)O(2), or chemical anoxia followed by reexposure to glucose (in vitro ischemia-reperfusion injury). In parallel with release of [(3)H]AA, complement-mediated production of prostaglandin E(2) was amplified in GEC-iPLA(2)gamma. Complement-mediated cytotoxicity was attenuated significantly in GEC-iPLA(2)gamma compared with GEC-Neo, and the cytoprotective effect of iPLA(2)gamma was reversed by BEL, and in part by indomethacin. Overexpression of iPLA(2)beta did not amplify complement-dependent [(3)H]AA release, but nonetheless attenuated complement-mediated cytotoxicity. Thus iPLA(2)gamma may be involved in complement-mediated release of AA. Expression of iPLA(2)gamma or iPLA(2)beta induces cytoprotection against complement-dependent GEC injury. Modulation of iPLA(2) activity may prove to be a novel approach to reducing GEC injury.

Disease-causing mutations of RhoGDIα induce Rac1 hyperactivation in podocytes

ABSTRACT Nephrotic syndrome (NS) describes a group of kidney disorders in which there is injury to podocyte cells, specialized cells within the kidneys glomerular filtration barrier, allowing proteins to leak into the urine. Three mutations in ARHGDIA, which encodes Rho GDP dissociation inhibitor α (GDIα), have been reported in patients with heritable NS and encode the following amino acid changes: ΔD185, R120X, and G173V. To investigate the impact of these mutations on podocyte function, endogenous GDIα was knocked-down in cultured podocytes by shRNA and then the cells were re-transfected with wild-type or mutant GDIα constructs. Among the 3 prototypical Rho-GTPases, Rac1 was markedly hyperactivated in podocytes with any of the 3 mutant forms of GDIα while the activation of RhoA and Cdc42 was modest and variable. All three mutant GDIα proteins resulted in slow podocyte motility, suggesting that podocytes are sensitive to the relative balance of Rho-GTPase activity. In ΔD185 podocytes, both random and directional movements were impaired and kymograph analysis of the leading edge showed increased protrusion and retraction of leading edge (phase switching). The mutant podocytes also showed impaired actin polymerization, smaller cell size, and increased cellular projections. In the developing kidney, GDIα expression increased as podocytes matured. Conversely, active Rac1 was detected only in immature, but not in mature, podocytes. The results indicate that GDIα has a critical role in suppressing Rac1 activity in mature podocytes, to prevent podocyte injury and nephrotic syndrome.