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Integrin-linked kinase as a candidate downstream effector in proteinuria¹

MATTHIAS KRETZLER², VICENTE P. C. TEIXEIRA, PAUL G. UNSCHULD, CLEMENS D. COHEN, RÜDIGER WANKE^*, ILKA EDENHOFER, PETER MUNDEL, DETLEF SCHLÖNDORFF and HARRY HOLTHÖFER

Medical Policlinic and
^* Institute of Veterinary Pathology, University of Munich, Munich, Germany;
Department of Anatomy and Cell Biology, Albert Einstein College of Medicine, New York, New York; and
Department of Immunology, University of Helsinki, Helsinki, Finland

²Correspondence: Medizinische Poliklinik, Schillerstr. 42, D-80336 Munich, Germany. E-mail: kretzler{at}medpoli.med.uni-muenchen.de

SPECIFIC AIM

We sought to identify and characterize novel molecular pathways activated in glomerular filtration barrier failure. An expression screening approach on glomeruli from children with congenital nephrotic syndrome of the Finnish type (CNF) was used. Integrin-linked kinase (ILK) was found to be induced in CNF and its specific role for podocyte function was evaluated in in vivo and in vitro models of proteinuria.

PRINCIPAL FINDINGS

1. ILK mRNA is increased in glomeruli from CNF
To identify novel molecules activated in proteinuria, CNF and control glomeruli were screened with 50 primer pair combinations displaying 5800 PCR products: 37 were found to be differentially expressed between CNF and controls and 12 were further characterized. One clone with increased expression in CNF glomeruli (Fig. 1A ) was found to be identical to human ILK. Changes in ILK expression in CNF glomeruli could be confirmed with quantitative RT-PCR using sequence-specific primers, and a 3.0 ± 0.01-fold mRNA increase was detected in glomerular preparation of CNF kidneys compared with controls (Fig. 1B ).

View larger version (32K): [in this window] [in a new window]   Figure 1. ILK mRNA expression in glomerular damage. A) Representative differential display PCR of glomerular cDNA from two children with CNF (CNF 1 and 2) vs. two controls (CON 1 and 2). Increased PCR product abundance of a 526 bp product in CNF is indicated (arrow). B) RT-PCR of glomerular cDNA from two CNF (CNF) vs. two normal human kidney (CON). A 3.0 ± 0.01-fold increase in ILK mRNA vs. controls is seen. C): RT-PCR of glomerular cDNA from mice with nephrotoxic serum nephritis (NTX) and controls (CON) are shown. ILK mRNA levels increased on day 7 in NTX 3.4 ± 0.9-fold vs. CON, n = 4, P < 0.05. D) RT-PCR of glomerular cDNA from growth hormone transgene mice (GH) and controls (CON) are shown. ILK mRNA levels increase compared with CON 2.2 ± 0.1-fold in GH mice, n = 4, P < 0.05.

2. ILK mRNA is elevated in murine models of proteinuria
To analyze the potential role of ILK in CNF independent proteinuria, ILK mRNA expression was determined in two murine proteinuria models. Accelerated nephrotoxic serum nephritis with nephrotic syndrome, including ascites and albuminuria, was used as an acute inflammatory model. Glomerular ILK mRNA levels determined by RT-PCR showed no alteration on day 2, but increased in parallel with albuminuria with a 3.4 ± 0.9-fold induction on day 7 (Fig. 1C ). Growth hormone (GH) transgene mice develop glomerular hypertrophy, progressive podocyte failure, and a chronic progressive focal segmental glomerulosclerosis. A 2.2 ± 0.1-fold increase in ILK mRNA above control levels was found in glomerular preparations from GH mice vs. wild-type littermates (Fig. 1D ).

3. Podocyte-specific ILK mRNA induction as determined by single podocyte RT-PCR
As antibodies for immunohistological analysis of ILK are unavailable, single podocyte real time RT-PCR was established to quantify ILK mRNA in podocytes in situ. Linearity of the assay was confirmed by generating a standard curve of serial diluted ILK plasmid DNA (10,000 to 10 copies, regression coefficient R²=0.99). In microdissected murine podocytes, a decrease in threshold cycle number from Ct 32.6 ± 3.6 in control podocytes to Ct 29.8 ± 3.2 in GH-transgene single murine podocytes was found. Using the ILK standard curve and the above threshold cycles, an increase in ILK copy number per single podocyte cDNA from 59 copies per cell in controls to 361 copies per cell in GH transgene mice could be calculated, confirming podocyte-specific ILK mRNA induction in proteinuria.

4. ILK expression and activity are induced by podocyte damage in vitro
Analysis of the rapidly regulated kinase activity of ILK is problematic in in vivo models of podocyte damage, as kinase activity would change during the procedure and time required to isolate glomeruli. Therefore, we used the in vitro system of conditionally immortalized murine podocytes. The role of ILK for inside-out integrin signaling was assessed by inducing podocyte damage with the aminonucleoside of puromycin, known to cause selective proteinuria in vivo. Differentiated podocytes were incubated for 48 h with 1–50 µg/ml of puromycin, and an increase of ILK mRNA (1.8±0.7-fold with 10 µg/ml puromycin) and protein levels above PBS controls could be demonstrated.

ILK function was analyzed by immunoprecipitation of podocyte cell lysates with an affinity-purified ILK antibody and in vitro kinase assay. An increase in kinase activity 4.1 ± 1.8-fold above basal levels could be seen 48 h after exposure to 10 µg/ml puromycin.

5. Podocyte ILK is found in focal contacts and kinase activity is inhibited by adhesion to matrix
To define the subcellular localization of ILK, podocytes were stained with antibodies specific for ILK and paxillin. A colocalization of the two molecules in a focal contact pattern could be found.

To determine the role of ILK in outside-in signaling of podocytes, differentiated cells were cultured on noncoated tissue culture dishes or collagen I, IV and fibronectin as substrates. Culture on the different matrix components for 48 h resulted in repression of ILK activity vs. the noncoated surface.

6. In vitro podocyte phenotype is altered in response to ILK overexpression
To assess the functional role of ILK for podocyte phenotype in culture, we established stable transfected murine podocytes expressing a wild-type and mutant human ILK construct.

A striking difference could be detected upon examination of the podocyte phenotype. Overexpression of the active kinase led to a highly proliferative, cobblestone phenotype compared with ‘arborized’ cell morphology of controls (Fig. 2I +II ). Kinase mutant clones displayed flattened cell bodies and, as control cells, a growth-arrested phenotype (Fig. 2 III). An ILK-dependent rearrangement of stress fibers could be demonstrated after labeling the actin filaments with TRITC phalloidin (Fig. 2IV , V , VI ). A mouse monoclonal anti--actinin antibody displayed a cytoplasmic signal consistent with a colocalization of -actinin with actin stress fibers in control podocytes (Fig. 2VII ) and kinase-defective mutants (Fig. 2IX ). In ILK overexpressing cells, a relocalization of the actinin staining into cell–cell and cell–matrix contacts was observed (Fig. 2VIII ).

View larger version (107K): [in this window] [in a new window]   Figure 2. ILK alters podocyte phenotype. ILK-dependent podocyte phenotype alteration. Phase contrast microscopy (I–III, 200x). Labeling of the actin filaments with TRIC-phalloidin (IV–VI, 400x). Immunofluorescence with mouse monoclonal -actinin antibody (VII–IX, 400x). I, IV, VII) Control podocytes. II, V, VIII) Cell line overexpressing wild-type ILK. III, VI, IX) ILK kinase-defective mutant cell line. ILK-dependent alterations in cell morphology (cobblestone with ILK overexpression II), stress fiber redistribution (V), and actin association of -actinin with relocalization into focal contacts in ILK overexpression (VIII) are seen.

To analyze the functional consequences of modulating ILK activity, the adhesive properties of the different cell lines were assessed. Overexpression of functional ILK resulted in a significant reduction of cell adhesion to collagen matrix compared with kinase-defective mutants and control podocytes.

CONCLUSIONS AND SIGNIFICANCE

Kidney function depends on an intact glomerular filtration unit, allowing the excretion of potentially hazardous small molecular substances but retaining essential macromolecules. The permselectivity of the glomerular filter is defined by a fenestrated endothelial cell layer, the glomerular basement membrane (GBM), and podocytes. The podocyte forms the filtration slit, an ultrastructural membrane bridging the delicate web of interdigitating podocyte foot processes.

By various approaches we have identified ILK as a candidate signaling molecule linking cell–cell and cell–matrix interaction to podocyte function and, if disturbed, to proteinuria. An increase of ILK mRNA was found by differential display screening of CNF glomeruli. This led to the examination of ILK expression in two murine models of proteinuria. Surprisingly, ILK induction was found to be a common theme in progressive podocyte damage. ILK was originally identified via a yeast two-hybrid screen using the cytoplasmic tail of the ß₁-integrin as ‘bait,’ and has been shown to be a key player in integrin-mediated cell adhesion and signaling. ILK activity is inhibited by ligation of the corresponding integrins to matrix, confirming its relevance for outside-in signaling.

Examining outside-in signaling of ILK, a repression of kinase activity by matrix attachment of podocytes was found that is consistent with an involvement of this kinase in cell matrix interaction. Podocytes respond to matrix alteration of the GBM, a hallmark in many glomerular diseases, with foot process effacement and cytoskeletal changes. The above data implicate ILK as a candidate signaling molecule, mediating the cellular response of podocytes to GBM modification.

In inside-out integrin signaling, ILK kinase activity increased after in vitro challenge of podocytes with puromycin. The aminonucleoside of puromycin induces podocyte foot processes effacement, actin filament alteration, and podocyte detachment from the GBM, leading to severe proteinuria in vivo. An increase in ILK activity is consistent with this kinase being a downstream effector in in vitro podocyte damage and modulating podocyte matrix attachment.

ILK not only modulates integrin avidity and affinity, but also appears to be involved in the cross talk between podocyte matrix anchorage, cytoskeleton, and cell phenotype. In addition to interfering with podocyte matrix interaction, ILK overexpression severely changed podocyte phenotype from the arborized cell morphology to a proliferative cobblestone pattern. These alterations were paralleled by significant rearrangements of the cytoskeleton. We could show a redistribution of -actinin, the causative gene of an autosomal dominant focal segmental glomerulosclerosis, from a stress fiber association into a focal contact pattern. ILK could directly phosphorylate -actinin and thereby alter its binding specificity to actin. Alternatively, the phenotype changes could be mediated by activation of the Wnt pathway with nuclear translocation of ß-catenin and induction of LEF-1. Members of this pathway are indeed expressed in podocytes (ß-catenin and cadherins) and located at the slit diaphragm. An activation of the Wnt signaling pathway or a change of the highly restricted podocyte cell cycle regulators, as seen in collapsing glomerulonephritis, could induce the phenotype changes seen in vitro.

In summary, using an unbiased mRNA expression screening approach, we identified the induction of ILK in proteinuric disease in vivo and after podocyte challenge in vitro. The functional and phenotype alterations found in ILK-overexpressing podocytes paralleled those seen with in vivo podocyte damage. These data are consistent with a positioning of ILK at the intersection between podocyte cytoskeleton and cell–matrix contacts. ILK appears to be an interesting candidate kinase for the orchestration of signal cross talk between those two critical components of the glomerular filtration barrier. As kinase cascades have proved to be viable targets for therapeutic intervention, the detection of ILK as a potential mediator of glomerular disease has identified a new focus for future drug discovery targets.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic diagram. Working model of integrin mediated podocyte-GBM interaction. Intact filtration barrier: 3ß1-Integrins are in a high-affinity binding state anchored in the GBM. The cytoplasmic domain of ß1-integrins is coupled to the actin cytoskeleton via focal contacts consisting of -actinin, talin, paxillin, and vinculin. -Actinin cross-links actin filaments to stress fibers. ILK is in its inactive state. Altered filtration barrier: podocyte damage or GBM matrix alterations activate ILK. Active ILK phosphorylates the cytoplasmic domain of ß1-integrins, resulting in a low-affinity binding state and detachment of podocytes from the GBM.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0832fje ; to cite this article, use FASEB J. (June 18, 2001) 10.1096/fj.00-0832fje

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