Mira Krendel*,
Sangwon V. Kim,
Tim Willinger,
Tong Wang,
Michael Kashgarian,
Richard A. Flavell,|| and
Mark S. Mooseker*,,¶
* Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, and Departments of Immunobiology, Cellular and Molecular Physiology, Pathology, and ¶ Cell Biology, School of Medicine, Yale University, New Haven, Connecticut; and || Howard Hughes Medical Institute, Chevy Chase, Maryland
Correspondence: Dr. Mira Krendel, Department Cell and Developmental Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210. Phone: 315-464-8527; Fax: 315-464-8535; E-mail krendelm{at}upstate.edu
Received for publication November 6, 2007.
Accepted for publication August 28, 2008.
Myosin 1e (Myo1e) is one of two Src homology 3 domain–containing"long-tailed" type I myosins in vertebrates, whose functionsin health and disease are incompletely understood. Here, wedemonstrate that Myo1e localizes to podocytes in the kidney.We generated Myo1e-knockout mice and found that they exhibitproteinuria, signs of chronic renal injury, and kidney inflammation.At the ultrastructural level, renal tissue from Myo1e-null micedemonstrates changes characteristic of glomerular disease, includinga thickened and disorganized glomerular basement membrane andflattened podocyte foot processes. These observations suggestthat Myo1e plays an important role in podocyte function andnormal glomerular filtration.
Myosins are molecular motors that translocate cargo along actinfilaments in an ATP-dependent manner. Members of the myosinsuperfamily, which includes at least 24 myosin classes,1 contributeto a variety of intracellular functions, including organelletransport, actin reorganization, and cell signaling. Geneticevidence in mice and humans links myosin mutations to hearingand vision defects, neurologic problems, cardiomyopathies, immunesystem disorders, pigmentation defects, and cancer.2
This study focuses on physiologic roles of myosin 1e (myo1e),a class I myosin. Class I myosins consist of a single motordomain, a neck domain that binds one or more calmodulin (orcalmodulin-like) light chains, and a cargo-binding tail domain.2Some class I myosins have tails that consist of a single domain,termed tail homology 1 (TH1) domain. This domain is basic incharge and may effect interactions with negatively charged phospholipids.Other class I myosins, including myo1e, have longer tails thatin addition to TH1 include a proline-rich TH2 domain and C-terminalSrc homology 3 domains. Humans and mice express eight myosinI isoforms (1a through h); functions of three myosin I isoforms(1a, 1c, and 1f) have been previously analyzed using geneticmanipulation in mice.3 Mice lacking myo1a, a myosin that isexpressed exclusively in intestinal epithelial cells, exhibitdefects in the organization of the intestinal brush border.4Myo1c has been implicated in the adaptation by the inner earsensory hair cells; the importance of myo1c for adaptation hasbeen established, in part, using transgenic mice expressinga mutant version of myo1c that could be selectively inhibitedusing a modified ADP analog.5 Knockout (KO) of myo1f, a long-tailedMyo1 closely related to myo1e, results in defects in neutrophilmigration, which are linked to changes in integrin exocytosisand enhanced cell-substrate adhesion.6
Myo1e is expressed in a wide variety of tissues, including spleen,kidney, small intestine, pancreas, brain, and the immune system.6,7 We previously determined that myo1e tail binds endocytic proteinsdynamin and synaptojanin and found that inhibition of myo1efunctions in cultured fibroblasts led to defects in endocytosis.8To analyze myo1e functions in vivo, we developed a myo1e-KOmouse and characterized its phenotype. Myo1e was expressed inthe glomerular visceral epithelial cells, or podocytes, whichplay a central role in glomerular filtration. Myo1e-null miceexhibited defects in glomerular filtration and organization,which were similar to glomerular defects observed in inheritedrenal diseases that have been linked to mutations in podocyte-expressedproteins in humans.9, 10 Thus, myo1e seems to be necessary fornormal renal filtration and disruption of myo1e functions maycontribute to renal disease.
Myo1e Is Expressed in Renal Podocytes
Staining of mouse kidney sections with antibodies against myo1erevealed that in the kidney, myo1e was predominantly expressedin the glomeruli (Figure 1). Myo1e partially co-localized withpodocyte-specific marker synaptopodin in podocyte foot processes(Figure 1A). In addition to foot process labeling, myo1e exhibitedprominent cytoplasmic staining. Endothelial marker VE-cadherinwas located in close proximity to myo1e but did not overlapwith myo1e (Figure 1B), indicating that myo1e is expressed inpodocytes rather than in glomerular endothelial cells.
Figure 1. Myo1e localization in mouse kidney. (A through C) Frozen kidney sections prepared from WT mice (A and B) and Myo1e-KO mice (C) were double labeled with antibodies against myo1e and either podocyte marker synaptopodin or endothelial cell marker VE-cadherin. Myo1e co-localized with synaptopodin (A) but not with VE-cadherin (B). No labeling with Myo1e antibodies was observed in the KO mice (C). Merged images show Myo1e in green and synaptopodin or VE-cadherin in red. Insets in A and B show enlargements of selected regions of the merged images. Bar = 20 µm.
Immunoblotting and immunofluorescence labeling of cultured mousepodocytes with antibodies against myo1e confirmed that it wasexpressed in podocytes (Figure 2). Myo1e was localized to punctatestructures, some of which were also labeled with anti-synaptopodinantibodies (Figure 2B). In some cells, myo1e was enriched inthe areas of cell–cell contacts, where it co-localizedwith cell adhesion protein β-catenin (Figure 2C). The observedstaining pattern was specific for myo1e because labeling withnonimmune IgG showed no enrichment in cell–cell junctionsor cytoplasmic punctae (Figure 2D), and labeling with antibodiesagainst nonmuscle myosin IIA (myo2a) revealed a different patternof localization, with myo2a being localized along actin stressfibers (Figure 2E).
Figure 2. Myo1e is expressed in differentiated mouse podocytes. (A) Western blotting of podocyte lysate using anti-myo1e antibodies reveals the presence of a protein band corresponding to myo1e. (B through E) Immunofluorescence labeling of mouse podocytes using polyclonal antibodies against myo1e (B and C) and myo2a (E) as well as rabbit nonimmune (NI) antibodies (D). Myo1e localizes to punctate structures (arrowhead in B) and cell–cell junctions (arrows in B and C). Myo1e partially co-localizes with synaptopodin (B) and adherens junction marker β-catenin (C). NI antibody labeling (D) and labeling using Myo2a antibody (E) show lack of co-localization with the junctional marker. Bar = 20 µm. Merged images show polyclonal antibody labeling in green and mAb (anti-synaptopodin or anti–β-catenin) in red.
Generation of Myo1e-KO Mice
To generate Myo1e-KO mice, we introduced loxP-recombinationsites into Myo1e gene by homologous recombination in embryonicstem cells. Cre-mediated recombination in germline cells wasthen used to remove loxP-flanked exon 4 from the mouse Myo1egenomic sequence (Figure 3A and Supplemental Methods). In themutant Myo1e allele, only the first 80 codons are expected tobe translated, followed by a stop codon. The KO approach wasdesigned with the goal of achieving a constitutive disruptionof myo1e protein expression in all tissues. Mice were genotypedusing genomic PCR, and the absence of myo1e protein was confirmedby Western blotting and immunofluorescence staining (Figures 1Cand 3).
Figure 3. Generation and characterization of Myo1e-KO mice. (A) Gene targeting strategy and probes for genotyping. Homologous recombination between the gene targeting vector and the WT Myo1e allele was used to create a targeted allele of the Myo1e gene. The targeted allele contained insertion of the LoxP sites and Neomycin resistance gene, but the exon structure of the Myo1e gene remained intact. Cre-mediated recombination in mice carrying the targeted allele resulted in germline deletion of exon 4 of Myo1e gene and formation of a mutant (functionally null) allele. Primers A, B, and C were used for genotyping by genomic PCR, with primers A and C designed to amplify a 140-bp fragment from WT allele and with primers A and B designed to produce a 260-bp fragment from mutant allele. (B) Characterization of Myo1e-KO mice by genomic PCR. Genomic DNA samples isolated from WT mice (+/+), mice homozygous for Myo1e mutant allele (–/–), and heterozygous mice (+/–) were used as a template for PCR with primer pairs A/C and A/B. PCR with the primer pair A/C produced a 140-bp band only in mice that carried at least one copy of the WT allele, and PCR with the primer pair A/B resulted in a 260-bp band in mice that carried at least one copy of the mutant allele. (C) Western blotting of tissue samples from WT and Myo1e-KO mice. Spleen and kidney samples were probed using anti-myo1e antibody. Antibody against -tubulin was used as a control for equal protein loading. No myo1e signal was detected in samples from the KO mice.
Myo1e-KO Mice Have Kidney Disease
Myo1e-KO mice did not exhibit any visible abnormalities at birth,their growth rates and lifespan did not differ from those ofwild-type (WT) mice, and no obvious defects were detected inany of their internal organs other than kidneys. Analysis ofurine samples collected from myo1e-null mice showed high levelsof proteinuria (Figure 4), as well as the presence of leukocytesand hemoglobin in the urine of KO mice. Possible causes of proteinuriainclude disruption of the glomerular filtration barrier or abnormalitiesin protein reabsorption in the renal proximal tubules. Undernormal conditions, proteins >40 kD are prevented from enteringtubular lumen by the glomerular filtration apparatus; thus,presence of high molecular weight proteins such as serum albuminin the urine is indicative of glomerular disease, whereas excretionof low molecular weight proteins suggests tubular absorptionabnormalities.11 SDS-PAGE analysis revealed that the major proteinfound in the urine of mutant mice was serum albumin (Figure 4A),indicating that the proteinuria was likely due to defects inthe glomerular filtration barrier. This is consistent with thepresence of blood/hemoglobin in the urine, which is also observedin some of the well-characterized glomerular diseases such asAlport syndrome as a result of the increased passage of redblood cells through the glomerular barrier.12 The amount ofalbumin excreted by Myo1e-KO mice within a 24-h period was significantlyhigher than in their WT littermates (P < 0.05; Figure 4).
Figure 4. Myo1e-KO mice have impaired renal function. (A) Myo1e-null mice exhibited massive proteinuria. Urine samples (2 µl) from WT and KO mice were separated by SDS-PAGE, and proteins were stained using Coomassie Blue. The prominent protein band corresponds to serum albumin. (B) Absence of Myo1e leads to albuminuria. Albumin concentration in 24-h samples of urine collected from WT mice and KO mice at the ages of 4 to 32 wk was measured using ELISA with anti-mouse albumin antibodies. Each dot represents one mouse (y axis is shown in logarithmic scale). Average albumin concentration was significantly higher in KO mice (P < 0.05). (C) Increased kidney weight in Myo1e-KO mice. (Top) Representative photographs of WT and KO kidneys; the kidney of a KO mouse is larger and paler than the WT. (Bottom) Kidney weight to body weight ratio comparison. Kidney weight and body weight were measured in five WT and six KO mice aged 12 to 18 wk; average kidney weight to body weight ratio was higher in the KO mice (P < 0.005). Error bars represent SD. (D) Myo1e-KO mice have increased BUN levels. The level of BUN was measured in serum samples from WT and KO mice. Each dot represents one mouse.
Visually, kidneys of Myo1e–/– mice appeared largerand paler than in their WT littermates (Figure 4C). In 8- to12-wk-old mice, kidney/body weight ratio was significantly higher(approximately 1.5 -fold) in –/– mice than in their+/+ littermates (P < 0.005; Figure 4C).
To characterize the severity of renal impairment in Myo1e-KOmice, we measured levels of blood urea nitrogen (BUN) in theserum of WT and KO mice. BUN levels in WT mice were normal,and there was little variation in BUN levels among individualmice (Figure 4D). The average BUN level in KO mice was higherthan in the WT mice (P < 0.05), and a wide range of BUN concentrationswas observed among KO mice (Figure 4D, Table 1). These resultsindicate that renal function in the KO mice is compromised butthe level of renal impairment varies among individual animals,an observation that is also confirmed by the variability inurinary albumin excretion level. Individual variability in theseverity of renal impairment was also observed in the mousemodel of Alport syndrome13; thus, such variability may be acommon feature of glomerular disease in mouse genetic models.
Table 1. Analysis of the renal phenotype in Myo1e-KO micea
Absence of Myo1e Leads to Defects in Glomerular Organization
Kidneys of Myo1e-KO mice exhibited signs of glomerular damage,including FSGS (glomeruli with thickened and closed capillaryloops, partially obstructed by extracellular matrix [ECM]) andexcessive ECM deposition in and around glomeruli (Figure 5,A through C; Table 1). Percentage of glomeruli that exhibitedsclerosis in the KO mice was 7.5 ± 4.5 (n = 3). We alsoobserved the presence of periodic acid-Schiff–positivecasts in renal tubules as well as periodic acid-Schiff–positiveprotein resorption droplets in proximal tubules (Figure 6, SupplementalFigure 1). Both of these defects may result from proteinuria,because excretion of high levels of protein in the urinary filtratepromotes both formation of proteinaceous casts and enhancedprotein degradation in proximal tubule cells. We also observedan increase in ECM deposition and cell infiltration in the juxtaglomerularand interstitial space, indicating the development of interstitialfibrosis, a common hallmark of renal injury (Figure 5).
Figure 5. Myo1e-KO mice develop renal inflammation and fibrosis. (A through D) Paraffin-embedded sections (A) or frozen sections (B through D) or of mouse kidneys were stained using Trichrome stain to reveal extracellular matrix deposits (A) or using antibodies against collagen V (B) and fibronectin (C) or mouse C3 (D). (A) Trichrome staining of kidneys from Myo1e-KO mice revealed juxtaglomerular deposition of collagen (blue) and cell infiltration into the interstitial space. (B and C) Immunofluorescence staining indicated increased deposition of collagen V (B) and fibronectin (C) in the kidneys of KO mice. (D) Proximal tubules of Myo1e-KO mice contained granular deposits of C3 (arrows). Complement deposits were not observed in the kidneys of WT mice. Bars = 20 µm.
Figure 6. Proximal tubule changes in the kidneys of Myo1e-null mice. (A) Periodic acid-Schiff staining revealed numerous periodic acid-Schiff–positive granules in the proximal tubules of Myo1-null mice, indicating increased protein degradation. (B) Examination of kidney sections from WT and Myo1e-null mice using EM showed that some proximal tubules in the Myo1e-null mice exhibited loss of the apical brush border (arrows) and the presence of numerous inclusions that may represent lysosomes or protein resorption droplets (arrowheads).
Examination of glomerular proteins using immunofluorescencemicroscopy showed normal expression and localization of majorglomerular markers, such as nephrin, podocin, and integrin 3 in the KO mice (data not shown). To determine whether renaldysfunction observed in Myo1e-KO mice was related to immunemechanisms, such as autoimmune disease or immune complex deposits,we stained kidneys of WT and mutant mice with antibodies againstIgA, IgG, and third component of complement (C3). No increasein the glomerular C3, IgG, or IgA staining (Figure 5D and datanot shown) and no electron-dense immune deposits (Figure 7)were observed in the glomeruli of mutant mice. Thus, immunedysfunction did not seem to contribute to the development ofglomerular disease in Myo1e-KO mice. Conversely, immunofluorescencelabeling revealed granular deposits of C3 and IgG in the proximaltubules of Myo1e-null mice (Figure 5D and data not shown). Thisis consistent with the published data indicating that in rodentmodels of proteinuria, exposure of proximal tubules to IgG andcomplement as a result of defective filtration leads to depositionof IgG and C3 in proximal tubules.14 Complement deposited inthe proximal tubules may then serve as a local proinflammatorysignal that promotes tubular injury and infiltration of inflammatorycells. Next, we characterized renal changes in mutant mice usingelectron microscopy (EM). Whereas podocytes in the glomeruliof adult (2 to 4 mo old) WT mice formed numerous well-definedfoot processes, in podocytes of myo1e-null mice, foot processeswere effaced (flattened; Figure 7, Table 1). The average numberof foot processes per unit length of the basement membrane wasdecreased 2.6-fold as a result of the flattening of foot processes.The average thickness of glomerular basement membrane (GBM)was increased nearly two-fold in Myo1e–/– mice,and the basement membrane organization was disrupted (Figure 7).Whereas GBM in WT mice showed typical three-layered organization,there were no easily identifiable layers in the GBM of mutantmice, and multiple electron-lucent areas were present withinthe GBM. Electron micrographs of proximal renal tubules in Myo1e–/–mice revealed signs of tubular injury, such as the loss of theapical brush border and presence of numerous electron-denseparticles (either protein resorption droplets or lysosomes)in some tubules (Figure 6B).
Figure 7. Characterization of the glomerular lesions in Myo1e-null mice by EM. (A and B) Low-magnification (A) and high-magnification (B) electron micrographs of glomeruli from 2-mo-old WT and KO mice. Myo1e-null mice exhibit thickening and disorganization of the basement membrane (BM) and effacement of podocyte foot processes (FP). (C) Additional magnification of the electron micrographs shown in B shows normal three-layered GBM organization in WT mice and disorganized GBM in the KO mice. (D and E) Quantitative analysis of the electron micrographs of renal sections collected from WT and KO mice. (D) Average thickness of GBM is increased in the KO mice. (E) Average number of foot processes per unit length of GBM is decreased in the KO mice. Error bars represent SD.
Myo1e-KO Mice Develop Glomerular Filtration Defects within the First Month of Life
To determine whether defects in glomerular filtration and organizationarose during early renal development or later in life, we examinedurine and tissue samples from mice at the ages of 1 and 3 wk.Renal development in mice is completed by approximately 2 wkafter birth; thus, the time points that we selected provideinformation about the state of the glomerular filtration apparatusboth during kidney development and immediately after glomerularmaturation is completed. At the age of 1 wk, urine of both WTand myo1e-KO mice contained a small amount of albumin (Figure 8A),possibly as a result of an incompletely developed and slightlyleaky filtration barrier; the degree of proteinuria was comparablebetween the WT and KO mice. By 3 wk of age, WT mice exhibitedno proteinuria, whereas myo1e-KO mice developed a high levelof proteinuria (Figure 8A). Whereas glomerular filtration defectsin myo1e-KO were not apparent at 1 wk of age, glomerular ultrastructurein the KO mice was abnormal even at this young age, with theaverage density of foot processes being significantly lower(P 0.001), and the average GBM thickness being significantlyhigher than in the WT mice (P 0.05; Figure 8B). By the ageof 3 wk, foot process effacement and GBM thickening were evenmore pronounced (P 0.001; Figure 8B). Overall, foot processarchitecture in younger KO mice (Figure 8) seemed somewhat morenormal than in the 2-mo-old KO mice (Figure 7), indicating thatglomerular architecture continues to deteriorate as the miceage. Slit diaphragms appeared normal in both 1- and 3-wk-oldmice (Figure 8 and data not shown), indicating that glomerularfiltration defects were not due to the absence of slit diaphragms.
Figure 8. Characterization of glomerular defects during renal development in Myo1e-KO mice. (A) By the age of 3 wk, myo1e-null mice develop massive proteinuria. SDS-PAGE followed by Coomassie staining was used to analyze urine (1 µl of urine for each 3-wk-old KO mouse, 2 µl of urine for the other mice). (B) Morphometric analysis of glomerular ultrastructure shows an increase in GBM thickness and a decrease in foot process number in Myo1e-KO mice at the ages of 1 and 3 wk. Error bars represent SD. (C) Electron micrographs of glomeruli from 3-wk-old mice show normal foot process and GBM organization in WT mice and foot process effacement and GBM thickening in KO mice. Slit diaphragms (arrows) appear normal in both WT and KO. Bar = 0.5 µm.
Selective filtration in the glomerulus requires proper compositionand organization of the GBM and formation of filtration slitsbetween interdigitated podocyte foot processes. Podocytes arecentral to the establishment of glomerular filtration barrierbecause they are involved both in GBM secretion and filtrationslit assembly. Most glomerular diseases are characterized bydramatic changes in podocyte structure and functions,15 andmany inherited glomerular diseases in humans have been attributedto mutations in podocyte-expressed proteins.9, 10
In this study, we show that myo1e is expressed in podocytesand that KO of Myo1e gene in mice results in defects in renalfiltration and podocyte organization. In the absence of myo1e,podocytes exhibit effacement of foot processes, a structuralchange that is typically observed in glomerular diseases andmay represent either the cause of impaired filtration (as aresult of the disruption of filtration slit architecture) orthe consequence of defective filtration.16 Glomeruli of Myo1e-KOmice also exhibit GBM thickening and disorganization, whichis reminiscent of structural changes observed in some of theinherited renal diseases, such as Alport syndrome.17, 18 Analysisof renal function in Myo1e-KO mice revealed severe proteinuriaas well as elevated BUN levels, both of which indicate impairedrenal filtration. Although impaired glomerular filtration mayalso be caused by the deposition of antibodies or antibody–antigencomplexes in the glomerulus, we did not observe immune complexdeposits by EM. Thus, the observed renal defects do not seemto be immune mediated but rather result from impaired podocytefunctions.
Defects in renal filtration in Myo1e-KO mice are not congenitalbut rather develop during the first weeks of life, possiblyas the result of the increased hydrostatic pressure in the glomerulusafter birth. Although filtration slit assembly seems to be normal,both foot processes and basement membrane exhibit defects earlyin the course of renal development, suggesting that foot processeffacement, disruption of GBM assembly, or both events combinedmay be the cause of glomerular abnormalities in Myo1e-KO mice.
Kidneys of Myo1e-KO mice exhibited hallmarks of chronic renalinjury, including tubulointerstitial fibrosis and glomerulosclerosis.In addition, some of the proximal tubules in the kidneys ofMyo1e-null mice showed signs of excessive protein degradationand loss of the apical brush border. These changes in the proximaltubule organization are likely the consequence of the high proteinconcentration in the urinary filtrate, which results in enhancedprotein absorption and degradation in the proximal tubule cellsas well as the presence of complement and various serum-derivedgrowth factors in the tubulointerstitial space. Release of complementand growth factors may then promote inflammation and fibrosis.14
The only study to date to investigate myo1e functions in vivoused a gene trap approach to disrupt Myo1e gene in mice.19 Thisanalysis was conducted as a part of a large effort to identifygenes that act downstream of PDGF during pre- and postnataldevelopment and to analyze the effects of disruption of PDGFtarget genes on mouse development. In this study, we inactivatedMyo1e gene using homologous recombination in embryonic stemcells, an approach that resulted in the complete ablation ofmyo1e protein expression as confirmed by immunoblotting andimmunofluorescence staining. Myo1e-KO mice described in ourstudy exhibit significant renal impairment, whereas renal abnormalitieswere not detected in the gene-trap study.19 Gene-trap constructinsertion occurred in the first intron of Myo1e gene. The lackof protein expression in Myo1e gene-trap mutant mice has notbeen verified, and it is possible that gene-trap insertion produceda hypomorphic, rather than a null, allele. Because the firstexon of Myo1e gene encodes only one amino acid, it is possiblethat initiation of transcription from another methionine (22amino acids downstream of the first) or alternative splicingaround the gene-trap insertion site could have resulted in synthesisof partially functional protein. In fact, residual protein expressionin gene-trap mutants is a fairly common occurrence.20 In addition,our observations show that whereas an increase in urinary albuminexcretion is readily observed in all Myo1e-null mice, BUN levelsin Myo1e-null mice are highly variable, with some animals exhibitingnormal BUN concentration. This result suggests a possibilitythat the tests used to characterize Myo1e gene–trappedmouse mutant (hematoxylin-eosin staining to evaluate overallrenal morphology and BUN measurements to analyze renal functions)may have missed renal changes induced by Myo1e gene disruption.
Podocyte structural organization is guided by the actin cytoskeleton,which provides the driving force for foot process formationand support for junctional complex assembly.21, 22 Disruptionof actin organization, as well as mutations or deletions ofactin-binding proteins, leads to structural and functional defectsin podocytes.23, 24 Our finding that myo1e plays an importantrole in podocyte functions further underscores the importanceof the actin cytoskeleton for podocyte biology. Identificationof an actin-dependent molecular motor as an important contributorto podocyte structure and functions adds a new player to thecast of molecular characters that already includes regulatorsof actin dynamics (Nck and synaptopodin), actin bundlers (-actinin-4),and actin-binding scaffold proteins (ZO-1) (reviewed in reference21). We previously determined that in addition to interactingwith actin via its head (motor) domain, myo1e binds dynaminvia its tail domain.8 Recent experimental evidence showed thatexpression of dominant negative dynamin constructs caused actinreorganization and cell shape changes in podocytes and thatproteolytic degradation of dynamin may be responsible for impairedpodocyte function in some types of renal diseases.25 It remainsto be determined whether the effects of Myo1e KO on podocytefunctions are mediated by changes in dynamin activity or resultfrom a direct effect of myo1e on actin organization in podocytes.This will be the focus of future studies. In conclusion, ourfindings show that myo1e is expressed in podocytes and playsan important role in glomerular organization and functions.
Animal Studies
Animal experiments were approved by the Yale Institutional AnimalCare and Use Committee. Mouse colonies were maintained in theYale Animal Resources Center. Mouse urine was collected usingmetabolic cages and tested for protein, blood, and leukocytesusing dipstick test strips. We conducted quantitative albuminmeasurements in 24-h urine samples using mouse albumin ELISAkit (Bethyl Labs, Montgomery, TX). We measured BUN levels usingQuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA).
Immunofluorescence Microscopy, Histochemistry, and EM
Mouse kidneys fixed by perfusion with periodate-lysine-paraformaldehydefixative26 were postfixed using periodate-lysine-paraformaldehyde(for cryosectioning) or 3.7% formaldehyde in PBS (for paraffinembedding). The list of antibodies used for immunofluorescencelabeling and information on sectioning, immunostaining, imagecollection, and analysis is provided in Supplemental Methods.
Mouse kidneys were fixed for EM by perfusion with Karnovskyfixative (1.6% paraformaldehyde, 3% glutaraldehyde, and 0.5mg/ml CaCl2 in 0.1 M Na-cacodylate [pH 7.4]) and processed forEM as described previously.27 We collected images using a JeolJEM-1230 electron microscope equipped with a digital camera.
Conditionally immortalized mouse podocytes were grown as describedpreviously.28 Differentiated podocytes were fixed using 3.7%formaldehyde in PBS, permeabilized using 0.2% Triton X-100 inPBS, and processed for indirect immunofluorescence.
SDS-PAGE and Immunoblotting
Urine samples were boiled with SDS sample buffer, separatedusing SDS-PAGE, and stained with Coomassie Brilliant Blue. Toprepare tissue samples for immunoblotting, we homogenized cellor tissue samples in 5% TCA, washed them with cold water toremove TCA, and boiled then with SDS sample buffer. We performedSDS-PAGE and immunoblotting as described previously.4
This work was supported by grants from the American Heart Association(0335441T) and American Diabetes Association (1-07-JF-39) toM. Krendel and from the National Institutes of Health (GM073823and DK 25387) to M.S.M., DK 62289 to T.W., and HD32573 to M.Kashgarian. R.A.F. is an Investigator of the Howard Hughes MedicalInstitute.
We thank Peter Hegan, Sue Ann Mentone, Qingshang Yan, and ZhaopengDu (Yale University) and Sharon Chase, Karen Maass, and MasakoNakatsugawa (SUNY UMU) for technical assistance; all membersof Mooseker laboratory for valuable discussion and advice; andDrs. Joseph Madri (Yale University Medical School), Peter Mundel(University of Miami Miller School of Medicine), Lawrence Holzman(University of Michigan Medical School), and Michael DiPersio(Albany Medical College) for generously providing antibodiesand cell lines.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
M. Krendel and S.V.K. contributed equally to this work.
M.K.'s current affiliation is Department of Cell and DevelopmentalBiology, SUNY Upstate Medical University, Syracuse, New York.S.V.K.'s current affiliation is Helen L. and Martin S. KimmelCenter for Biology and Medicine at the Skirball Institute ofBiomolecular Medicine, NYU School of Medicine, New York, NewYork.
Supplemental information for this article is available onlineat http://www.jasn.org/.
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Abstract
Introduction
3 in the KO mice (data not shown). To determine whether renal dysfunction observed in Myo1e-KO mice was related to immune mechanisms, such as autoimmune disease or immune complex deposits, we stained kidneys of WT and mutant mice with antibodies against IgA, IgG, and third component of complement (C3). No increase in the glomerular C3, IgG, or IgA staining (Figure 5D and data not shown) and no electron-dense immune deposits (Figure 7) were observed in the glomeruli of mutant mice. Thus, immune dysfunction did not seem to contribute to the development of glomerular disease in Myo1e-KO mice. Conversely, immunofluorescence labeling revealed granular deposits of C3 and IgG in the proximal tubules of Myo1e-null mice (Figure 5D and data not shown). This is consistent with the published data indicating that in rodent models of proteinuria, exposure of proximal tubules to IgG and complement as a result of defective filtration leads to deposition of IgG and C3 in proximal tubules.14 Complement deposited in the proximal tubules may then serve as a local proinflammatory signal that promotes tubular injury and infiltration of inflammatory cells. Next, we characterized renal changes in mutant mice using electron microscopy (EM). Whereas podocytes in the glomeruli of adult (2 to 4 mo old) WT mice formed numerous well-defined foot processes, in podocytes of myo1e-null mice, foot processes were effaced (flattened; Figure 7, Table 1). The average number of foot processes per unit length of the basement membrane was decreased 2.6-fold as a result of the flattening of foot processes. The average thickness of glomerular basement membrane (GBM) was increased nearly two-fold in Myo1e–/– mice, and the basement membrane organization was disrupted (Figure 7). Whereas GBM in WT mice showed typical three-layered organization, there were no easily identifiable layers in the GBM of mutant mice, and multiple electron-lucent areas were present within the GBM. Electron micrographs of proximal renal tubules in Myo1e–/– mice revealed signs of tubular injury, such as the loss of the apical brush border and presence of numerous electron-dense particles (either protein resorption droplets or lysosomes) in some tubules (Figure 6B). 
0.001), and the average GBM thickness being significantly higher than in the WT mice (P 
