Essential Role of Integrin-Linked Kinase in Podocyte Biology: Bridging the Integrin and Slit Diaphragm Signaling
Chunsun Dai*,
Donna B. Stolz,
Sheldon I. Bastacky*,
Rene St.-Arnaud,
Chuanyue Wu*,
Shoukat Dedhar and
Youhua Liu*
Departments of * Pathology and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada; and BC Cancer Research Centre, Vancouver, British Columbia, Canada
Address correspondence to: Dr. Youhua Liu, Department of Pathology, University of Pittsburgh, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; E-mail: liuy{at}upmc.edu
Received for publication January 13, 2006.
Accepted for publication May 24, 2006.
Integrin-linked kinase (ILK) has been implicated in the pathogenesisof proteinuria and congenital nephrotic syndrome. However, thefunction of ILK in glomerular podocyte in a physiologic settingremains unknown. In this study, a mouse model was generatedin which ILK gene was selectively disrupted in podocytes byusing the Cre-LoxP system. Podocyte-specific ablation of ILKresulted in heavy albuminuria, glomerulosclerosis, and kidneyfailure, which led to animal death beginning at 10 wk of age.Podocyte detachment and apoptosis were not observed at 4 wkof age, when albuminuria became prominent, indicating that theyare not the initial cause of proteinuria. Electron microscopyrevealed an early foot process effacement, as well as morphologicabnormality, in ILK-deficient podocytes. ILK deficiency causedan aberrant distribution of nephrin and -actinin-4 in podocytes,whereas the localization of podocin and synaptopodin remainedrelatively intact. Co-immunoprecipitation demonstrated thatILK physically interacted with nephrin to form a ternary complex,and -actinin-4 participated in ILK/nephrin complex formation.Therefore, ILK plays an essential role in specifying nephrinand -actinin-4 distribution and in maintaining the slit diaphragmintegrity and podocyte architecture. These results also illustratethat the integrin and slit diaphragm signals in podocytes areintrinsically coupled through an ILK-dependent mechanism.
Defects in the glomerular filtration barrier, as manifestedby proteinuria, are the principal pathologic feature of a largenumber of primary glomerular diseases that eventually progressto end-stage renal failure (1). Glomerular filter, a structuralapparatus that consists of the fenestrated endothelium, glomerularbasement membrane (GBM), and the epithelial podocyte foot processes,serves as a molecular sieve that selectively restricts the filtrationof different molecules on the basis of their size, shape, andcharge (1–3). Genetic studies have identified severalpodocyte proteins, such as nephrin and podocin, that are ofcritical importance for establishing the size-selective filtrationbarrier of the kidney (4–6). A loss-of-function mutationin the genes that encode these proteins is associated with thedevelopment of massive proteinuria and congenital nephroticsyndrome in both animal models and patients (7–10).
Podocytes are the specialized visceral epithelial cells thatreside on GBM outside the glomerular capillaries. Foot processesof podocytes interdigitate with their neighboring counterpartsand form a filtration slit that is connected by a continuousmembrane-like structure known as the slit diaphragm (SD) (1,2).Increasing evidence suggests that SD is a specialized cell–celladhesion complex that not only provides a structural foundationfor the filter barrier but also participates in cell signalingthat is necessary for the maintenance of podocyte function (2,11,12).Podocytes are anchored to the GBM through the 31-integrin complexthat is present in the sole of the foot processes. Therefore,the cell–matrix integrin signaling also is indispensablefor maintaining the delicate architecture of the glomerularfiltration barrier. Consistent with this, mice that lack 3-integrinare born without podocyte foot processes formation (13). However,whether the integrin and SD signals are connected in some waysin podocytes remains uncertain.
Integrin-linked kinase (ILK) is an intracellular serine/threonineprotein kinase that interacts with the cytoplasmic domains of integrins and mediates the integrin signaling in diverse typesof cell (14,15). In addition to its catalytic kinase activity,ILK is known as an adaptor protein that interacts with numerouscytoskeleton-associated proteins such as PINCH and -parvin (14,16).It seems that ILK works in concert with its interacting partnersto build multicomponent cellular machinery that is essentialfor its function (17,18). Dysregulation of ILK expression hasbeen implicated in the pathogenesis of a wide variety of chronickidney diseases, including nephrotic syndrome and diabetic andobstructive nephropathy (19–23). Overexpression of ILKis observed in patients with congenital nephrotic syndrome andin glomerular podocytes of murine models of proteinuria (19),suggesting a possible connection between aberrant ILK expressionand altered podocyte biology under pathologic conditions (22).However, the function of ILK in glomerular podocyte in a physiologicsetting is completely unknown.
In this study, we demonstrated that selective ablation of ILKin podocytes caused an aberrant distribution of the SD proteinnephrin, as well as an early foot process effacement and morphologicabnormality. These lesions resulted in heavy albuminuria, whichinevitably led to podocyte detachment and apoptosis, glomerulosclerosis,kidney failure, and animal death. Furthermore, we found thatILK physically interacted with nephrin to form a ternary complex.These findings indicate that ILK is essential for podocyte biologythrough bridging the integrin and SD signaling.
Mice and Genotyping
The ILK floxed mice, in which LoxP sites were inserted downstreamfrom exons 4 and 12 at the ILK locus through homologous recombination,was described previously (24). Transgenic mice that expressedCre recombinase under the control of a 2.5-kb fragment of thehuman podocin promoter (2.5P-Cre mice) were reported elsewhere(25). By mating ILK floxed mice with podocin-Cre transgenicmice, mice that were heterozygous for the ILK floxed allelewere generated (genotype ILKfl/+, Cre). These mice were cross-bredto inactivate both ILK alleles by Cre-mediated excision, therebycreating conditional knockout mice in which ILK gene was specificallydisrupted in glomerular podocytes (genotype ILKfl/fl, Cre).The breeding protocol also generated heterozygous littermates(genotype ILKfl/+, Cre), as well as wild-type and several controlgroups with different genotype (ILK+/+; ILKfl/fl; ILKf/+; ILK+/+,Cre, referred to as controls). A routine PCR protocol was usedfor genotyping of tail DNA samples with following primer pairs:Cre transgene, 5'-AGGTGTAGAGAAGGCACTTAGC-3' and 5'-CTAATCGCCATCTTCCAGCAGG-3',which generated a 411-bp fragment; and ILK genotyping, 5'-CAAGGAATAAGGTGAGCTTCAGAA-3'and 5'-AAGGTGCTGAAGGTTCGAGA-3', which yielded 1.3- and 1.1-kbbands for the floxed and wild-type alleles, respectively. Thepresence of excision products was confirmed by PCR analysisof glomerular DNA using the following primer pair: 5'-CCAGGTGGCAGAGGTAAGTA-3'and 5'-CAAGGAATAAGGTGAGCTTCAGAA-3', which gave rise to a 230-bpfragment. All animals were born normally at the expected Mendelianfrequency. All control mice displayed normal phenotype. Animalexperiments were approved by the Institutional Animal Care andUse Committee at the University of Pittsburgh.
Urine Albumin, Total Protein, and Creatinine Assay
Urine total protein levels were determined using a bicinchoninicacid-based protein assay kit (Sigma, St. Louis, MO) with BSAas a standard. Urine albumin was measured by using a mouse AlbuminELISA Quantitation kit, according to the manufacturer's protocol(Bethyl Laboratories, Inc., Montgomery, TX). Serum and urinecreatinine was determined by a routine procedure as describedpreviously (26).
Glomerular Isolation
Glomerular isolation was performed according to the method describedelsewhere (27) with minor modifications. Briefly, mice wereanesthetized by intraperitoneal injection of pentobarbital (50mg/kg) and perfused with 8 x 107 Dynabeads M-450 (Dynal BiotechASA, Oslo, Norway) diluted in 5 ml of PBS by the abdomen artery.After perfusion, kidneys were removed and cut into 1-mm3 piecesand digested in collagenase A (1 mg/ml) at 37°C for 30 minwith gentle shaking. After digestion, the tissue was pressedgently through a 100-µm-cell strainer (BD Falcon, Bedford,MA). Glomeruli that contained Dynabeads then were gathered byusing a magnetic particle concentrator. The isolated glomeruliwere washed three times with cold PBS and used for subsequentstudies.
Western Blot Analysis
The glomeruli that were isolated from eight mice were pooledtogether and lysed with RIPA buffer that contained 1% NP40,0.1% SDS, 100 µg/ml PMSF, 1% protease inhibitor cocktail,and 1% phosphatase I and II inhibitor cocktail (Sigma) in PBSon ice. The supernatants were collected after centrifugationat 13,000 x g at 4°C for 20 min. Protein expression in isolatedglomeruli was analyzed by Western blot analysis as describedpreviously (28). The primary antibodies used were as follows:Anti-ILK (Upstate, Charlottesville, VA), anti–WT-1 (SantaCruz Biotechnology, Santa Cruz, CA), anti–-actinin-4 (AlexisCorp., San Diego, CA), anti-nephrin (Progen, Heidelberg, Germany),and anti–-tubulin (Sigma).
Histology and Immunohistochemical Staining
Kidney sections were prepared at 2 µm thickness by a routineprocedure. Sections were stained with hematoxylin-eosin, periodicacid-Schiff, methenamine silver-trichrome, and Masson-trichromeby standard protocol. The histologic sections were examinedby a renal pathologist. The glomeruli were assessed for segmentalglomerulosclerosis and global glomerulosclerosis, mesangialmatrix expansion, and glomerular size. The tubules were examinedfor dilation by proteinaceous fluid and tubular atrophy. Theinterstitium was assessed for fibrosis and mononuclear inflammation.Immunohistochemical staining for WT-1 was performed using aroutine protocol as described previously (28). For quantitativedetermination of podocyte numbers, the WT-1–positive cellswere counted in at least 10 randomly chosen glomeruli underhigh power (x400) in each mouse. The averages of podocyte numbersper glomerulus were calculated on the basis of individual values,which were determined on four mice per group.
Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick-End Labeling Staining
Apoptotic cell death was determined by using terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL) stainingwith DeadEnd Fluorometric Apoptosis Detection System (Promega,Madison, WI), as previously reported (26). The slide was doublestained with anti–WT-1 antibody (Santa Cruz Biotechnology)for the identification of podocytes. Apoptosis in glomeruliand tubulointerstitial area was counted in all glomeruli andat least 10 randomly chosen nonoverlapping high-power (x400)fields for each mouse, four mice per group (n = 4). Apoptoticpodocytes (both TUNEL and WT-1 positive) were expressed as apoptoticcells per field. For a better view of kidney morphology, anotherset of the paraffin-embedded kidney sections were stained withDeadEnd Colormetric TUNEL System (Promega).
Electron Microscopy
Electron microscopy of kidney samples was carried out by routineprocedures as described previously (29). Briefly, mouse kidneyswere perfusion fixed with 2.5% glutaraldehyde in PBS by leftcardiac ventricular injection and postfixed in aqueous 1% OsO4.Specimens were dehydrated through an ethanol series, infiltratedin a 1:1 mixture of propylene oxide-Polybed 812 epoxy resin(Polysciences, Warrington, PA), and then embedded. Ultrathinsections were stained with 2% uranyl acetate, followed by 1%lead citrate. Sections were observed and photographed usinga JEOL JEM 1210 transmission electron microscope (TEM; JEOL,Peabody, MA). For scanning electron microscopy (SEM), kidneyswere perfusion fixed in 2.5% glutaraldehyde in PBS as for TEM,dissected lengthwise, then postfixed in 1% OsO4 (30). Afterdehydration through an ethanol series, kidney samples were criticalpoint-dried, mounted on aluminum stubs, sputtered with gold-coat,and examined under JEOL 6335F field emission gun scanning electronmicroscope.
Immunofluorescence Staining and Confocal Microscopy
Kidney cryosections were fixed with 3.7% paraformalin for 15min at room temperature, except that methanol was used for sectionsthat were stained with ILK. After blocking with 10% donkey serumfor 30 min, the slides were immunostained with primary antibodiesagainst ILK, nephrin, -actinin-4, podocin (Santa Cruz Biotechnology),and synaptopodin (Progen), respectively. For ILK and -actinin-4staining, the Vector M.O.M. immunodetection kit (Vector Laboratories,Burlingame, CA) was used. As a negative control, the primaryantibody was replaced with nonimmune IgG, and no staining occurred.Slides were viewed under a Leica TCS-SL confocal microscope.For determination of the disruptive nephrin and -actinin-4 distribution,a semiquantitative scoring method was used. Score 0 representsno lesion, whereas 1, 2, 3, and 4 represent the disruptive redistributionof nephrin and -actinin-4, involving <25%, 25 to 50%, 50to 75%, and >75% of the glomerular tuft area, respectively.At least 10 randomly chosen glomeruli were evaluated for eachmouse, and an average composite score was calculated. Four micefrom each group were analyzed.
Immunoprecipitation
Immunoprecipitation was carried out by using an establishedmethod (31). Briefly, isolated glomeruli were lysed on ice in1 ml of nondenaturing lysis buffer that contained 1% TritonX-100, 0.01 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.025% NaN3, 1%protease inhibitors cocktail, and 1% phosphatase inhibitorscocktail I and II (Sigma). After preclearing with normal IgG,glomerular lysates (0.5 mg of protein) were incubated overnightat 4°C with 4 µg of anti-ILK (Upstate) and anti-nephrin(Santa Cruz Biotechnology), respectively, followed by precipitationwith 30 µl of protein A/G Plus-Agarose for 1 h at 4°C.The precipitated complexes were separated on SDS-polyacrylamidegels and blotted with various antibodies as indicated.
Statistical Analyses
Statistical analysis of the data was performed by using SigmaStatsoftware (Jandel Scientific, San Rafael, CA). Comparison betweengroups was made using one-way ANOVA, followed by the Student-Newman-Keulstest. P < 0.05 was considered significant.
Podocyte-Specific Disruption of ILK Gene in Mice
We generated a mouse model in which ILK gene was selectivelydisrupted in glomerular podocytes by mating ILK floxed micewith podocin-Cre transgenic mice (Figure 1). Mice with podocyte-specificdisruption of ILK (podo-ILK–/–), as well as wild-typeand several control groups, were confirmed by PCR analysis oftail genomic DNA (Figure 1B). PCR analysis of the glomerularDNA that was isolated from the podo-ILK–/– micedisplayed a 230-bp band that corresponded to the excised floxedILK allele, indicating the Cre-mediated deletion of the ILKgene (Figure 1C). Figure 1D shows the glomerular ILK proteinlevels in podo-ILK–/– mice and their wild-type littermates.Comparing with the controls, ILK protein level in the isolatedglomeruli in podo-ILK–/– mice was reduced by approximately40% (Figure 1D), which is consistent with the fact that podocytesonly constitute a fraction of total glomerular cell population.Podocyte-specific ablation of ILK in podo-ILK–/–mice also was confirmed by immunofluorescence staining (Figure 8;see the ILK Physically Interacts with Nephrin and -Actinin-4section).
Figure 1. Podocyte-specific ablation of the integrin-linked kinase (ILK) in mice. (A) Diagram illustrates the strategy of generating podocyte-specific ILK knockout mice. Shown is the deletion of exons 5 through 12 in the event of recombination of the ILK gene. (B) Representative picture shows PCR analysis of the genomic DNA from tail clippings. The PCR bands of wild-type (1.1 kb), floxed (1.3 kb), and Cre (411 bp) are indicated. Genotypes of representative litters are indicated. fl, ILK floxed; Cre, Cre recombinase. (C) PCR analysis shows the Cre-excised band (230 bp) in the glomeruli of podo-ILK–/– mice. (D) Western blot analysis of ILK protein expression in whole glomeruli (pooled from eight mice per group) that were isolated from the control and podo-ILK–/– mice. Glomerular lysates were immunoblotted with antibodies against ILK and -tubulin, respectively. (E) Body weights of the podo-ILK–/– mice and their control littermates at different time points after birth. **P < 0.01 versus controls (n = 8). (F) Representative picture shows the phenotypic appearance of podo-ILK–/– mouse and its control littermate at 10 wk after birth.
Figure 8. ILK physically interacts with nephrin and -actinin-4 in vivo. (A through F) Co-localization of ILK and nephrin in glomerular podocyte. Representative micrographs show the staining of ILK (A and D), nephrin (B and E), and co-localization (C and F) in the control (A through C) and podo-ILK–/– mice (D through F). Arrowheads indicate the co-localization of ILK and nephrin in podocyte in normal mice. Arrows denote ILK-deficient podocyte in which nephrin distribution was disturbed in podo-ILK–/– mice. Bar = 12 µm. (G and H) Co-immunoprecipitation demonstrates the ILK/nephrin complex formation. Glomerular lysates (pooled from eight mice) were immunoprecipitated with anti-ILK or normal IgG (G) and anti-ILK or anti-nephrin (H), followed by immunoblotting with anti-nephrin. (I) Co-immunoprecipitation demonstrates the presence of -actinin-4 in the ILK/nephrin complex in vivo. Immunocomplexes that were precipitated with anti-ILK or anti-nephrin were immunoblotted with anti–-actinin-4 antibody. (J and K) Ablation of ILK in podocyte abolished ILK/-actinin/nephrin interaction. Glomerular lysates from either podo-ILK–/– (ko) or control littermates (wt) were immunoprecipitated with anti-ILK and immunoblotted with either anti–-actinin-4 (J) or anti-nephrin (K). (L) Schematic model illustrates that ILK bridges the integrin and slit diaphragm signaling. ILK functions as an adaptor protein that physically and functionally associates with slit diaphragm protein nephrin. -Actinin-4 participates in the ILK/nephrin complex formation. Ablation of ILK disrupts ILK/nephrin complex formation and leads to an aberrant distribution of nephrin and -actinin-4, thereby inducing podocyte foot process effacement and morphologic alterations. That is followed by albuminuria, podocyte detachment and apoptosis, glomerulosclerosis, and kidney failure.
We observed no difference in the body weights in podo-ILK–/–mice at 2 wk of age when compared with the control littermates(Figure 1E). However, significant reduction in body weight wasfound at 10 wk of age in podo-ILK–/– mice (Figure 1,E and F). This growth retardation likely resulted from the severemorbidity characterized by heavy proteinuria that developedin podo-ILK–/– mice (see below). Podo-ILK–/–mice began to die at 10 wk. The majority of podo-ILK–/–mice died of renal failure at approximately 3 to 4 mo, althougha few of them survived beyond 6 mo of age.
Podocyte-Specific Ablation of ILK Leads to Heavy Proteinuria, Glomerulosclerosis, and Kidney Failure
We next determined the urine albumin levels at different timepoints in podo-ILK–/– mice and their control littermates.As shown in Figure 2A, urine albumin levels were dramaticallyelevated at 4 wk in podo-ILK–/– mice and reachedto >7 mg/mg creatinine, whereas no or little urine albuminwas detected at 2 wk. Albuminuria progressively deterioratedat 10 wk in podo-ILK–/– mice. Similar results wereobtained when urine total protein was measured (Figure 2B).Consistent with an elevated albumin, urine protein also wassignificantly increased at 4 and 10 wk in podo-ILK–/–mice, respectively. Separation of urine samples by SDS-PAGErevealed that albumin was the predominant constituent of urineproteins (Figure 2C).
Figure 2. Podocyte-specific ablation of ILK results in heavy albuminuria, proteinuria, and kidney dysfunction. (A) Urinary albumin levels at different time points in podo-ILK–/– mice and their control littermates. Urinary albumin data after correction to creatinine are presented as mean ± SEM. **P < 0.01 versus controls (n = 7 to 10). (B) Urinary total protein levels at different time points in podo-ILK–/– mice and their control littermates. *P < 0.05, **P < 0.01 versus controls (n = 7 to 10). (C) SDS-PAGE demonstrates urine proteins at different time points in podo-ILK–/– mice. Urine samples were separated on 10% SDS-PAGE after correction to creatinine. BSA (1 µg) was loaded onto an adjacent lane as a positive control. Samples from two individual animals per group were used. (D) Serum creatinine levels at different time points in podo-ILK–/– mice and their control littermates. **P < 0.01 versus controls (n = 7 to 10).
In accordance with heavy albuminuria and proteinuria, kidneyfunction in podo-ILK–/– mice was severely impairedin an age-dependent manner. An elevation of serum creatininelevels was observed at 10 wk in podo-ILK–/– mice,compared with their control littermates (Figure 2D). However,little difference in serum creatinine levels was found at 4wk between podo-ILK–/– mice and controls (Figure 2D),suggesting that the development of albuminuria and proteinuriaprecedes kidney dysfunction in podo-ILK–/– mice.
Figure 3 shows the representative micrographs of kidney sectionsat different time points. Kidney morphology was normal at 2wk after birth in podo-ILK–/– mice (data not shown).At 4 wk, kidney histology remained essentially normal, whencompared with the control littermates (Figure 3, A and B). However,dramatic morphologic abnormality was observed at 10 wk in thepodo-ILK–/– kidney, which was characterized by primaryglomerular lesions, including an enlargement of glomeruli insize, moderate to marked mesangial expansion, and segmentalglomerulosclerosis to global sclerosis (Figure 3, C, F, andI). There was widespread tubular dilation distended by proteinaceousfluid and cellular debris. The interstitium contained a mild,patchy mononuclear inflammatory infiltrate, and interstitialfibrosis generally was mild (Figure 3).
Figure 3. Mice that lacked ILK in podocyte developed primary glomerulosclerotic lesions in a time-dependent manner. Representative micrographs show the morphologic lesions in the podo-ILK–/– mice at different time points after birth. Kidney sections from the control littermate at 4 wk (A, D, and G) and podo-ILK–/– mice at 4 (B, E, and H) and 10 wk (C, F, and I) were subjected to periodic acid-Schiff (PAS) staining (A through C), methenamine silver-trichrome staining (D through F), and Masson's staining (G through I). Bar = 30 µm.
Podocyte Detachment and Apoptosis Are Not the Initial Causes of Albuminuria
Because ILK plays an important role in mediating cell–matrixadhesion and cell survival (32), we reasoned that ILK deficiencymay lead to podocyte detachment and apoptosis, thereby causingproteinuria and kidney dysfunction. To test this hypothesis,we first examined the podocyte abundance in podo-ILK–/–kidney at different time points. As shown in Figure 4, podocytesin the glomeruli were readily identifiable by the nuclear stainingfor transcription factor WT-1. Quantitative determination revealedthat there was no difference in podocyte number per glomerulibetween podo-ILK–/– mice and control littermatesat 2 and 4 wk, respectively (Figure 4E), suggesting a lack ofpodocyte depletion at the time point when albuminuria becameprominent (Figure 2). This finding was supported further bymeasuring WT-1 abundance from the isolated glomeruli using Westernblot analysis (Figure 4F). However, podocyte depletion was predominantat 10 wk in podo-ILK–/– kidney (Figure 4D). Thenumber of podocytes per glomeruli was decreased by approximately70%, compared with the controls. WT-1–positive podocyteswere obviously detached from the GBM and easily found in thelumens of the dilated tubules. Some tubular epithelial cellsoccasionally were stained positively for WT-1, possibly becausethey endocytosed the debris or apoptotic bodies of the deadpodocytes (Figure 4D).
Figure 4. Podocyte depletion is a late event secondary to the development of albuminuria in mice that lack ILK in podocyte. (A through D) Representative micrographs show the podocyte abundance at 2 (B), 4 (C), and 10 wk (D) in the glomeruli of podo-ILK–/– mice and their control littermates at 4 wk (A). Kidney sections were stained with antibody against WT-1. Arrow denotes the glomerulus with a reduced podocyte number at 10 wk in podo-ILK–/– mice (D). Arrowhead indicates the WT-1–positive podocytes in the lumen of renal tubules. Bar = 25 µm. (E) Quantitative determination of the glomerular podocyte numbers at different time points in podo-ILK–/– mice and their control littermates. **P < 0.01 versus controls (n = 4). (F) Western blot analysis demonstrates a comparable glomerular WT-1 protein level at 4 wk in podo-ILK–/– mice and their control littermates. Whole glomerular lysates (pooled from eight mice) were immunoblotted with antibodies against WT-1 and -tubulin, respectively.
We also assessed the podocyte apoptosis in podo-ILK–/–kidney. As illustrated in Figure 5, no apoptosis was found at2 and 4 wk, respectively, in the kidney with podocyte-specificdeficiency of ILK. At 10 wk, little or no podocyte apoptosiswas found in situ in the glomeruli of podo-ILK–/–kidney (Figure 5, G through I), even though podocyte depletionwas eminent at this time point (Figure 4E). Substantial apoptosiswas observed in the tubulointerstitial area of the kidney at10 wk in podo-ILK–/– mice (Figure 5, J through M).Co-staining with TUNEL and WT-1 displayed that significant podocyteswere positive for TUNEL staining in the lumens of tubules atthis stage, suggesting that detached podocytes underwent apoptoticdeath. Intriguingly, there were the WT-1–positive podocytesthat were negative for TUNEL staining, indicating that thesedetached podocytes were still alive in the tubular lumens. Someapoptotic cells lacked WT-1 protein, indicating their originof tubular epithelium (Figure 5L). Similar results were obtainedwhen kidney tissues were stained by using a colormetric apoptosisdetection kit (Figure 5, N through P). Together, it became clearthat podocyte detachment and apoptosis are not the initial causesof proteinuria that takes place in podo-ILK–/– miceat 4 wk of age.
Figure 5. Podocyte apoptosis is secondary to cell detachment in mice with podocyte-specific ablation of ILK. (A through L) Representative micrographs demonstrate apoptotic podocytes in podo-ILK–/– mice and their control littermates. Kidney sections were subjected to terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining to identify apoptotic cells (A, D, G, and J) and WT-1 staining to recognize podocytes (B, E, H, and K). Merging of the TUNEL and WT-1 staining images is presented (C, F, I, and L). (A through C) Control littermates at 4 wk. (D through F) Podo-ILK–/– mice at 4 wk. (G through I) Podo-ILK–/– mice at 10 wk. (J through L) Tubulointerstitial area of podo-ILK–/– mice at 10 wk. Arrow indicates an apoptotic tubular cell with negative staining for WT-1, whereas arrowhead denotes WT-1–positive podocyte with negative TUNEL staining. Bar = 30 µm. (M) Quantitative determination of apoptotic podocytes in tubulointerstitial area at 4 and 10 wk in podo-ILK–/– mice and their control littermates. **P < 0.01 versus controls (n = 4). (N through P) Representative micrographs show apoptotic cells in the tubular lumens detected by colorimetric TUNEL staining at 2 (N), 4 (O), and 10 wk (P) in podo-ILK–/– mice. Arrows indicate glomeruli. Bar = 30 µm.
ILK Deficiency Causes an Early Podocyte Foot Process Effacement and Morphologic Abnormality
We next examined the ultrastructural alterations of the podocytefoot processes. TEM revealed an intact filtration apparatusthat consisted of the fenestrated endothelial cytoplasm, GBM,and the foot processes of podocytes (Figure 6A). However, amarked alteration that was characterized by podocyte foot processeffacement was observed at 4 wk in podo-ILK–/– mice(Figure 6C). The foot processes were adhered to GBM as continuouscytoplasmic processes, which led to the disappearance of theSD. It is interesting that slight foot process effacement withnarrower SD occasionally was found in some areas of podocytesat 2 wk (Figure 6B), a time point when no albuminuria had beendeveloped. This suggests that podocyte foot process effacementis an early event that precedes and/or parallels proteinuria.As expected, the foot process effacement of podocytes deterioratedat 10 wk in podo-ILK–/– mice (Figure 6D). In addition,podocyte foot processes sometimes detached from the GBM, leadingto the appearance of naked GBM on the side of the Bowman spacein some areas at this stage (Figure 6D, arrowhead). GBM lesionsthat were characterized by an increased thickness and irregularshape also were observed in some areas of the glomeruli at 4wk in podo-ILK–/– mice.
Figure 6. ILK deficiency causes podocyte foot process effacement and induces surface morphologic changes. (A through D) Representative electron micrographs demonstrate the ultrastructural alterations in the glomerular filtration barrier at different time points after selective ablation of ILK in podocyte. (A) Control littermates at 4 wk. (B) Podo-ILK–/– mice at 2 wk. (C) Podo-ILK–/– mice at 4 wk. (D) Podo-ILK–/– mice at 10 wk. Arrow indicates fused foot processes, whereas arrowhead denotes naked GBM. CL, capillary lumen; BS, Bowman space. Bar = 0.5 µm. (E and F) Scanning electron microscopy shows the surface morphology of glomeruli at 4 wk in podo-ILK–/– mice (F) and their control littermates (E). Massive microvilli were developed in the surface of podocytes that lacked ILK. FP, foot processes. Bar = 5 µm. Microvilli-like structures also were revealed by transmission electron microscopy (arrowhead; G). Bar = 1 µm.
We also examined the three-dimensional structure and topographyof the glomerular podocytes in podo-ILK–/– miceby SEM. As shown in Figure 6, ablation of ILK caused a significantchange in podocyte surface morphology. Compared with controls,the podocytes that lacked ILK developed massive microvilli-likestructures (Figure 6F), which were confirmed by TEM (Figure 6G).
Podocyte-Specific Ablation Results in Aberrant Distribution of Nephrin and -Actinin-4
In light of a possible connection between ILK and the SD integrity,we further examined the potential alterations in the abundanceand distribution of the SD proteins in podo-ILK–/–kidney. As shown in Figure 7, an aberrant distribution of nephrinwas observed at 4 wk in the glomeruli of the mice that lackedILK in podocytes, compared with their control littermates. Innormal glomeruli, nephrin staining revealed a linear patternalong the GBM, corresponding to its localization in the SD betweenthe foot processes of the podocytes (Figure 7A). However, ILKdeficiency caused an altered distribution of nephrin from theSD to the entire body of podocytes, resulting in a granularstaining pattern (Figure 7B), as illustrated by confocal immunofluorescencemicroscopy. Quantitative determination exhibited that such lesionsin nephrin distribution took place in almost all glomeruli ofpodo-ILK–/– mice at 4 wk with different degreesof severity, although little redistribution of nephrin was observedat 2 wk (Figure 7C). The abundance of nephrin in podo-ILK–/–mice remained comparable to that in control littermates (Figure 7D).Therefore, it seems that the specific subcellular localizationof nephrin depends on the presence of ILK in podocytes.
Figure 7. ILK deficiency results in aberrant distribution of nephrin and -actinin-4 in podocyte. Confocal immunofluorescence microscopy demonstrates the distribution of nephrin (A and B) or -actinin-4 (E and F) in the glomeruli of podo-ILK–/– mice at 4 wk (B and F) and their control littermates (A and E). Boxed areas were enlarged and presented. Arrowheads indicate the staining pattern. Quantitative data on the disruptive nephrin and -actinin-4 redistribution in podo-ILK–/– mice at 2 and 4 wk are presented in C and G, respectively. **P < 0.01 (n = 4). (D and H) Western blot analysis shows a comparable protein abundance of nephrin (D) or -actinin-4 (H) in podo-ILK–/– mice and their control littermates. Glomerular lysates (pooled from eight mice) from mice at 4 wk were immunoblotted with specific antibodies against nephrin, -actinin-4, and -tubulin, respectively. Little difference was observed in the localization of podocin (I and J) and synaptopodin (K and L) in podo-ILK–/– mice (J and L) and their control littermates (I and K) at 4 wk. Bar = 10 µm.
We also investigated the abundance and distribution of -actinin-4,an actin-associated protein that links the SD to actin cytoskeleton.Although there was no difference in its cellular abundance (Figure 7H),-actinin-4 distribution underwent drastic changes in the podocytesthat lacked ILK (Figure 7, E and F). The -actinin-4 also displayeda linear staining along the GBM in normal podocytes, a patternsimilar to nephrin, podocin, and other SD proteins. However,ILK deficiency caused an aberrant distribution of -actinin-4characterized by forming large, aggregated dots (Figure 7F).This disturbed localization of -actinin-4 began to occur at4 wk in podo-ILK–/– mice (Figure 7G). Under theidentical conditions, the localization of podocin and synaptopodinwas largely unaffected by ablation of ILK in podocytes (Figure 7,I through L), suggesting the specificity of the interplay amongILK, nephrin, and -actinin-4.
ILK Physically Interacts with Nephrin and -Actinin-4
The finding that ILK deficiency resulted in nephrin redistributionprompted us to explore their intrinsic connection. Double immunofluorescencestaining exhibited a co-localization of ILK and nephrin in glomerularpodocytes in vivo. In normal glomeruli, ILK localization withinthe podocytes displayed a linear pattern along the GBM (Figure 8A),similar to that of nephrin (Figure 8B). Merging of ILK and nephrinimages revealed a clear co-localization in podocytes (Figure 8C).In the glomeruli of podo-ILK–/– mice, ILK was ablatedspecifically in podocytes (Figure 8D, arrow), whereas its expressionin the mesangium was intact (Figure 8D). Deficiency of ILK inpodocytes led to a diffused distribution of nephrin from theSD to the entire cell body (Figure 8E, arrow).
We examined the potential interaction between ILK and nephrinin the glomeruli by co-immunoprecipitation. As shown in Figure 8,G and H, nephrin was readily detected in the complex that wasprecipitated by anti-ILK antibody. As a negative control, nonephrin was detected when the glomerular lysates were precipitatedwith normal IgG (Figure 8G, lane 2). Likewise, podocyte-specificablation of ILK abolished the interaction between ILK and nephrin(Figure 8K). These results suggest that ILK physically interactswith the SD protein nephrin in vivo.
We next investigated the potential involvement of -actinin-4in the formation of ILK/nephrin complex, because ILK ablationalso disturbs its localization. As shown in Figure 8, I andJ, -actinin-4 also was detected easily in the complex that wasprecipitated by either anti-ILK or anti-nephrin antibodies.Ablation of ILK in podocytes largely abolished the interactionbetween ILK and -actinin-4 (Figure 8J). The weak band of -actinin-4that was detected in the immunocomplexes likely was caused bythe contamination of other cell types such as tubular cellsin the glomerular preparation from the podo-ILK–/–mice (Figure 8J). Double immunofluorescence staining also revealedthat -actinin-4 co-localized with nephrin and ILK in glomerularpodocytes in vivo (data not shown). Therefore, -actinin-4 participatesin the ILK/nephrin complex formation and perhaps functions asan adaptor protein bridging ILK and nephrin (Figure 8L).
The SD of podocyte foot processes is a pivotal component ofthe glomerular filtration barrier, and disruption of its integrityrepresents a key event in the development of proteinuria andnephrotic syndrome in a variety of inherited and acquired glomerulardiseases (1,2,33). Previous studies largely focused on elucidatingthe role of various SD proteins such as nephrin and podocinin the SD function and glomerular filter integrity (5,6,8,10).Not surprising, mutations or deletion of these SD proteins leadto the development of congenital nephrotic syndrome. In thisstudy, we found that selective ablation of ILK in podocyte causesheavy proteinuria, podocyte detachment and apoptosis, glomerulosclerosis,and kidney failure. The underlying mechanism of these pathologicmanifestations seems to be attributable to a disturbance ofnephrin localization and foot process effacement. More important,we have uncovered that ILK can physically interact with nephrin,which provides direct evidence that the integrin signaling isintrinsically linked to SD function in podocytes. These findingsnot only underscore an essential role of ILK in podocyte biologyin normal physiologic setting but also suggest that a dysregulationof ILK abundance and/or activity potentially may play a criticalrole in the development of proteinuria and podocyte dysfunctions.Because the integrin/ILK signaling is regulated by many extracellularcues, such as matrix components, high glucose, growth factors,and cytokines, the intrinsic connection of ILK with nephrinthat was unraveled in this study offers important insights intounderstanding the pathogenesis of proteinuria in many commonglomerular diseases, such as diabetic nephropathy. Therefore,our study may have wide implications in comprehending how alterationsin the extracellular environment lead to defects in SD functionand impairment of glomerular filtration.
The finding that ILK binds to nephrin defines a novel, physiologicallyimportant interacting partner for ILK. By interacting with thecytoplasmic domains of -integrins and nephrin, ILK virtuallyacts as an adaptor or scaffolding protein that bridges and integratesthe integrin and the SD signals. Physical interaction betweenILK and nephrin is clearly evident in the glomeruli of normalkidney in vivo, as demonstrated by co-immunoprecipitation (Figure 8).Whether ILK binds to nephrin directly or via additional adaptorproteins, however, remains uncertain. In this aspect, it isinteresting to note that -actinin-4 was involved in the ILK/nephrincomplex formation, suggesting the presence of a multicomponentternary complex in which ILK likely is a central element. Wepropose that the coupling of ILK to nephrin likely is mediatedby additional proteins such as -actinin-4 (Figure 8L). Furtherstudies are warranted in this area to characterize fully themolecular details of the ILK/nephrin interaction in podocyte.
Given the physical interaction between ILK and nephrin, it isnot difficult to understand that ablation of ILK causes a redistributionof nephrin, resulting in a diffused pattern throughout the podocytesin podo-ILK–/– mice. In the absence of ILK, nephrinwould lose its anchoring sites for proper trafficking and localizationto the SD. This underscores that ILK may be essential for specifyingnephrin distribution in podocytes, possibly by acting as ananchoring apparatus. It is worthwhile to point out that in manyglomerular diseases, such as diabetic nephropathy, redistributionand reduction of nephrin are predominant pathologic featuresin podocytes (34–36). Although the exact mechanism remainselusive, dysregulation of ILK abundance and/or activity, whichis common in chronic kidney diseases, may play a significantrole in causing nephrin redistribution in these conditions.
Another striking feature in ILK-deficient podocyte is the aberrantdistribution of -actinin-4, an actin cross-linking protein thatbinds to actin filaments (37,38). Genetic mutations in -actinin-4gene have been shown to cause an autosomal dominant focal segmentalglomerulosclerosis in patients (39). In normal glomeruli, thepattern of -actinin-4 distribution essentially resembles thatof SD proteins such as nephrin and podocin, consistent withits close association with the SD. Similar to nephrin, selectiveablation of ILK in podocyte causes -actinin-4 redistribution,indicating that ILK also directs -actinin-4 localization. Thisnotion is corroborated further by -actinin-4's binding to ILKand nephrin (Figure 8). The complex interactions among ILK,nephrin, and -actinin-4 virtually connect the integrin and theSD with the actin cytoskeleton and integrate both extracellularand intracellular signals from multiple directions.
Our study also strengthens the notion that the disorganizationof actin cytoskeleton may play a critical role in the developmentof podocyte foot process effacement and structural abnormality.In ILK-deficient podocyte, -actinin-4 forms aggregated dots(Figure 7F). Such mislocalization of -actinin-4 certainly wouldchange the actin cytoskeleton dynamics (40) and may cause thecollapse of the actin meshwork underneath or near the SD ofthe foot processes. Because actin cytoskeleton defines cellshape and morphology, altered cytoskeletal structure of thefoot processes will lead to disappearance of the SD structuresand development of an "effaced" phenotype, as demonstrated byelectron microscopy studies (Figure 6). This speculation alsois supported by the observations that mutations or deletionof -actinin-4 gene cause proteinuria and focal segmental glomerulosclerosisin both animal models and patients (39,41,42). Because no alterationin -actinin-4 level was seen in podocytes that lacked ILK, thissuggests that not only the quality (mutations) and the quantity(deletion or induction) of -actinin-4 but also its locationare of extreme importance in maintaining the integrity of thepodocyte architecture. Furthermore, massive microvilli-likestructures developed in ILK-deficient podocytes, illustratingthat the submembranous actin skeleton architecture is profoundlychanged.
ILK has been implicated in the regulation of cell adhesion andcell survival (43,44). However, podocyte detachment and apoptosisseem not to be the initial cause of proteinuria in the podo-ILK–/–mice, because the podocyte numbers were preserved at the timepoint when severe albuminuria became eminent. Nevertheless,we cannot exclude the possibility that podocyte depletion viacell detachment and apoptosis in the podo-ILK–/–mice may deteriorate the proteinuria and accelerate the kidneydysfunction. Along this line, it is conceivable that podocyteloss from 4 wk onward may play a critical role in the developmentof glomerulosclerosis and kidney failure that eventually leadto animal death in podo-ILK–/– mice.
We have shown herein that ILK plays an essential role in podocytebiology in vivo. Through interacting with nephrin and -actinin-4,ILK builds a multicomponent complex that is essential for themaintenance of the podocyte function and glomerular filter integrity.Our findings have established that the cell–matrix integrinsignaling and the cell–cell adhesion SD signaling areintrinsically coupled through an ILK-dependent mechanism inpodocyte.
Acknowledgments
This work was supported by National Institutes of Health grantsDK061408, DK064005, DK071040 (Y.L.), and DK054639 (C.W.).
We sincerely thank Dr. Lawrence Holzman of the University ofMichigan for kindly providing the podocin-Cre transgenic mice.We are grateful to Ana Lopez, Mara Sullivan, and Marc Rubinof the Center for Biologic Imaging for technical expertise.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-actinin-4 in podocytes, whereas the localization of podocin and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated that ILK physically interacted with nephrin to form a ternary complex, and 
1-integrin complex that is present in the sole of the foot processes. Therefore, the cell–matrix integrin signaling also is indispensable for maintaining the delicate architecture of the glomerular filtration barrier. Consistent with this, mice that lack 
