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J. Biol. Chem., Vol. 277, Issue 34, 31154-31162, August 23, 2002

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Determinants of Vascular Permeability in the Kidney Glomerulus^*

Yuki Hamano, James A. Grunkemeyer, Akulapalli Sudhakar, Michael Zeisberg, Dominic Cosgrove^§, Roy Morello^¶, Brendan Lee^¶, Hikaru Sugimoto, and Raghu Kalluri

From the  Program in Matrix Biology, Divisions of Gastroenterology and Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, ^§ Gene Expression Laboratory, Boystown National Research Hospital, Omaha 68131, Nebraska, and ^¶ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston 77030, Texas

Received for publication, May 16, 2002, and in revised form, May 29, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The human kidneys filter 70 liters of blood plasma every day. The hallmark of almost all kidney diseases, whether acquired or genetic, is the leakage of plasma proteins into the urine because of alterations in the glomerular filtration unit of the kidney. In this regard, the human mutations in nephrin, podocin, -actinin-4, COL4A3, and COL4A5 genes expressed in the glomeruli have been implicated to cause alterations in glomerular filtration apparatus. Nevertheless, the expression of these proteins in relation to each other in mouse models for glomerular vascular leak is unknown. Additionally, within the glomerulus, the central question of whether the primary filtration barrier is the basement membrane or the epithelial slit diaphragm remains ambiguous. Therefore, in this study, we examined the localization and expression of glomerular epithelial slit diaphragm and glomerular basement membrane proteins implicated in glomerular vascular leak using mice deficient in either the 3 chain of type IV collagen, the major constituent of glomerular basement membrane, or LMX1B transcription factor, which regulates the expression of key glomerular type IV collagen genes COL4A3 and COL4A4 or nephrin, a glomerular epithelial slit diaphragm-associated protein. This study demonstrates that decreased expression of slit diaphragm protein, nephrin, correlates with a loss of glomerular filter integrity. Additionally, we demonstrate that defects induced by proteins of glomerular basement membrane lead to an insidious plasma protein leak, whereas the defects induced by proteins in the glomerular epithelial slit diaphragms lead to a precipitous plasma protein leak.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The kidney is the site for the ultrafiltration of plasma, and this function is affected in many different pathological settings such as diabetes, lupus, polycystic kidney disease, hypertension, and several other acquired and genetic kidney diseases (1). Despite the prevalent nature of a defect in plasma ultrafiltration, which is common to many kidney pathologies, a central question regarding molecular cues that initiate plasma protein leak (proteinuria or glomerular vascular leak) in these etiologically diverse diseases still remains unanswered.

Every day, 70 liters of plasma is filtered by the 2.5 million renal glomeruli of the human kidneys (1). The glomerular filtration barrier of the kidney is a tuft of capillaries composed of a defined 300-nm thick basement membrane, which is lined on one side by fenestrated endothelium (facing the blood side) and lined on the other side by a specialized epithelium (facing the urinary space side) with long and extensive foot processes that give these specialized glomerular epithelial cells the name "podocytes" (2). Tracer experiments with ferritin have suggested that the fenestrated endothelium with a potential pore size of 500-1000-Å diameter is freely permeable to many large plasma proteins (3-11). Such studies have also implicated that the crucial barrier for plasma proteins larger than 70 kDa is maintained by the glomerular basement membrane (GBM)¹ and the glomerular epithelial slit diaphragms (GESD) (4, 5, 12-16). The question as to which of these two substructures is important for normal glomerular permselectivity remains open.

GBM has been implicated as an important component of the filtration apparatus (12, 13). Studies with dextrans have collectively suggested that the structural integrity of the GBM is pivotal for the maintenance of a highly regulated barrier permeable only to water, small solutes/ions, and smaller proteins but not plasma proteins larger than 70 kDa (4, 12, 13, 16-18). GBM is composed of several large molecules such as type IV collagen, laminin, heparan sulfate proteoglycans, perlecan, and nidogen (2, 19). These molecules assemble in an ordered fashion to provide a highly cross-linked insoluble compact structure with a net negative charge contributed predominantly by the proteoglycan moieties within the GBM (20). The importance of GBM was further validated when the human mutations in type IV collagen were identified as responsible for chronic renal injury associated with microhematuria and glomerular vascular leak (GVL) or proteinuria (21-23).

A different school of thought, which has been proposed for many years, includes a critical role of glomerular epithelial cells (podocytes) in the regulation of glomerular permeability barrier (5, 6, 8, 10, 11, 14, 16, 24, 25). A podocyte in the glomerulus can be divided into two different zones, a primary cell body and secondary processes, which constitute initial branching and again subsequent branchings (podocyte processes) that directly interact with the GBM (2). A key component of the epithelial barrier has been suggested to include a modified adherens junction between the foot processes of the podocyte called GESD (4, 14).

In 1974, Rodewald and Karnovsky (14) published a model of GESD based on electron microscopy studies. According to this study, the slit diaphragm is composed of rodlike structures connected to a central pole providing a zipper-like structure (14). Recent studies (26-29) have suggested that nephrin, a transmembrane protein localized specifically to the podocytes in the kidney, is an important structural component of the slit diaphragm. The human mutations in this protein have been associated with the congenital nephrotic syndrome of the Finnish type, and these patients exhibit a significant vascular leak of plasma proteins in the urine (26, 30). In addition, several others (31-34) recently identified glomerular epithelial proteins such as CD2AP, -actinin-4, podocin, and FAT have also been suggested as crucial for normal function of the glomerular filtration apparatus. Mice deficient in nephrin and cd2ap exhibit kidney defects associated with GVL (31, 35). Nephrin, CD2AP, and podocin have all been shown to interact with each other, further implicating a potential complex of these proteins in the regulation of glomerular permselectivity features (36).

Whereas several recent discoveries have been made regarding the molecular constituents of GBM and GESD, the specific role of each of these molecules in the regulation of glomerular vascular permeability has not been addressed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of Mouse Nphs 1 Gene and Construction of Targeting Vector-- The mouse Nphs1 genomic construct was cloned from a BAC library (Research Genetics, Huntsville, AL) isolated by hybridization to a 1.3-kilobase human nephrin cDNA probe representing exons 1-10 of the human cDNA. The BAC contained the entire mouse Nphs1 gene including the promoter region, the entire transcribed region from the transcription initiation site, and the 5'-untranslated region through the coding region, the 3'-untranslated region, and polyadenylation signal sequence. Subclones were cloned and sequenced. KpnI-linearized pBluescript II with nephrin genomic sequence plasmid was used for the gene-targeting construct (Fig. 4). It had a "short" flank (0.95 kb) containing the mouse Nphs1 promoter and the beginning of exon 1 in a frame with GFP-pA/PGK-Neo-pA cassette (2.8 kb). This was followed by a "long" flank (5.0 kb), which consists of the rest of exon 1 through intron 11. In total, 34 bp surrounding the Nphs1 ATG codon (from 1 to +33) were deleted and replaced with the GFP-pA/PGK-Neo-pA cassette. Adjacent to the "short" flank, a TK-DTA-pA cassette (1.2 kb) was inserted into a unique SacII site for negative selection in ES cells (Fig. 4).

Generation of Nephrin Knock-out (KO) Mice-- The KpnI-linearized targeting vector was electroporated into 129/Sv-derived ES cells. The cells were selected in the presence of G418 (Invitrogen). 12 ES clones were positive by PCR using two pairs of primers, a sense primer of the Nphs1 gene (5'-CAC AAG GGA AGA TGG AGG AGT TG-3') and an antisense primer of the Nphs1 gene (5'-TCC ACT CAC CTG TGG TCA GCA TTC-3') or an antisense primer of the GFP sequence (5'-AGT CGT GCT GCT TCA TGT GGT C-3'). PCR was performed under the following conditions: 95 °C for 40 s, 64 °C for 40 s, and 72 °C for 2 min with 40 cycles. Three clones from positive pools were confirmed by Southern blot with a probe described in Fig. 4A. These clones were used for BALB/c or C57BL/6 blastocyst injections, and 11 chimeras were obtained. They were mated with normal 129/Sv mice (Charles River Laboratories, Wilmington, MA), and three chimeras with satisfactory germline transmission were further evaluated. Three different lines of nephrin-deficient mice on pure 129/Sv background were obtained by intercrossing the F₁ heterozygotes independently for an individual line. The mice are genotyped according to the PCR described above.

Histology-- The excised kidneys were fixed in 10% buffered formalin and embedded in paraffin. Kidney samples were sectioned at 3-µm intervals, and three sections per slide were mounted. The sections were stained with hematoxylin and eosin and periodic acid-Schiff reagent. Transmission electron microscopy was done as described previously (37). For immunofluorescence analysis, fresh kidneys were removed and embedded in OCT aqueous compound (Sakura, Tokyo, Japan). They were snap-frozen in liquid nitrogen, and 4-µm sections were obtained using HM 505N cryostat (Microm International GmbH, Walldorf, Germany). The slides were fixed in cold (20 °C) 100% acetone for 3 min and then air-dried for 1 h. For COL4 stainings, cold 95% ethanol was used as fixative, and slides were denatured using 6.0 M urea and 0.1 M glycine, pH 3.5, at 50 °C for 1 h. Subsequently, the slides were washed three times in 10 mM PBS. Primary antibodies were diluted using 3% bovine serum albumin/PBS, applied to the sections, and incubated for 2 h at room temperature in a humid chamber. The slides were then washed three times with 10 mM PBS. Secondary antibodies were diluted together at 1:100 in 3% bovine serum albumin/PBS and applied for 1 h at room temperature. After four washes with 10 mM PBS, Vectashield (Vector Laboratories, App Imaging, Santa Clara, CA) anti-fade mounting medium was applied, and sections were coverslipped and imaged. The staining was analyzed by Eclipse TE300 fluorescence microscopy (Nikon, Tokyo, Japan). For controls, normal rabbit and mouse sera were used in place of primary antibodies, or sections were directly incubated with secondary antibodies.

Antibodies-- The polyclonal anti-nephrin, podocin, P-cadherin, FAT, and -actinin-4 antibodies were produced in the New Zealand White rabbits. The peptide sequences used as the antigens are as follows: nephrin, GPWDLHWPEDTYQDPRGI; podocin, SSSRESRGRGGRTPHKENK; P-cadherin, SDQDQDYDYLNEWGSR; FAT, YESGDDGHFEEVTIPPLDSQ; and -actinin-4, YYDSHNVNTRCQKICDQWDNLGS. KLH-conjugated peptides in Freund's complete adjuvant were injected into New Zealand White rabbits (Taconic, Germantown, NY). The rabbits received an injection of 250 µg of purified antigen intraperitoneally and were boosted at 2-week intervals. Six weeks after the first immunization and three boosters, the animals were killed, blood was harvested, and serum was separated from the plasma. The specificity of the antisera was tested by enzyme-linked immunosorbent assay, immunofluorescence using human and mouse kidney sections, and Western blotting of mouse kidney extracts (data not shown). The polyclonal anti-CD2AP antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal anti-ZO-1 antibody was purchased from Chemicon International, Inc. (Temecula, CA). The monoclonal anti-laminin 2 antibody was purchased from Sigma. The polyclonal anti-GFP antibody was purchased from CLONTECH Laboratories (Palo Alto, CA). The polyclonal anti-nephrin, anti-integrin ₃, nephronectin, and synapotopodin antibodies were gifts from Drs. Lawrence Holzman, Jordon Kreidberg, Louis Reichardt, and Peter Mundel, respectively (38-41). The anti-COL4A3, COL4A4, and COL4A5 antibodies were previously described by our laboratory (42).

SDS-PAGE and Western Blotting Analyses-- From newborn nephrin +/+, +/, and / mice, urine was collected and 4 µl of unconcentrated urine was loaded to 8% SDS-polyacrylamide gel under reducing conditions. After electrophoresis, the proteins were visualized by Coomassie Blue staining. For Western blotting, the proteins were extracted from newborn kidneys of nephrin +/+, /, Lmx1b +/+, and / mice and from 8-week-old kidneys of 3 +/+ and / mice. They were homogenized with protease inhibitors in 10 mM PBS, suspended in 2% deoxycholate, and then stirred for 4 h at room temperature. The suspension was centrifuged, and the pellet was washed three times with ice-cold distilled water. The lysate was solubilized in the lysis buffer (0.05 M Hepes, pH 7.5, 0.01 M CaCl₂, 4 mM N-ethylmaleimide, 5 mM benzamidine HCl, 1 mM phenylmethanesulfonyl fluoride, and 25 mM -aminohexanoic acid), mixed with 5× SDS sample buffer, and electrophoresed on a 10% SDS-polyacrylamide gel. Immunoblots were blocked with 5% skim milk-containing TBST buffer (0.1% Tween 20, 20 mM Tris, pH 7.6, and 140 mM NaCl). Western blotting was performed by incubating with the indicated antibodies in TBST buffer followed by secondary antibodies conjugated with horseradish peroxidase and then developed by an enhanced chemiluminescence system (ECL kit, Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Type IV collagen in the glomerular basement membrane is predominantly composed of 3, 4, 5 chain isoforms (43). Among them, the 3 chain is one of the most abundant protein constituent of the GBM proper and assembles with the other two -chains to form higher ordered structures called protomers (44, 45). Protomers constitute the basic building unit of the GBM scaffold (46). Human mutation in the 3 chain results in some cases of autosomal recessive Alport syndrome, which is associated with microhematuria and GVL (22, 23). Mice deficient in the protein, 3KO, develop chronic renal failure associated with proteinuria and die at approximately week 14 after birth (47). The 3KO mice are normal until week 4 with the exception of sporadic ultrastructural defects in the GBM, a lack of 3 (the deleted gene), 4, and 5 chains of type IV collagen in the GBM, and alterations in the GBM laminin content (Fig. 1, data not shown) (47).

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Expression of 3(IV), 4(IV) and 5(IV) chains of type IV collagen. A, the renal sections from 3(IV), Lmx1b, and nephrin / KO mice are stained with indicated antibodies. The 3KO kidneys are from 4-week-old mice. The Lmx1b and nephrin KO kidney sections are from newborn mice. A GBM-staining pattern is seen by 3, 4, and 5 antibodies. B, electron microscope analysis of 3KO kidneys at 4 weeks (4W) and 8 weeks (8W) of age. Notice the presence of GESD in the kidney section of 4-week-old 3KO mice with ultrastructural GBM defects (red arrow). The blue arrows show normal GBM and GESDs. The green arrows show defective GESDs and GBM in the 8-week-old 3KO mice in comparison to wild type (WT) control mice, which exhibit normal glomerular architecture. The magnifications are as shown in the figure.

In this study, we demonstrate that up until week 4, the molecular make-up of the glomerular slit diaphragm is normal despite significant structural defects within the GBM as recorded by electron microscopy (Fig. 1). The normal expression of podocyte/slit diaphragm-associated proteins such as integrin ₃₁, nephrin, podocin, -actinin-4, CD2AP, synaptopodin, ZO-1, and P-cadherin are observed in the 3KO mice up until week 4 (Fig. 2, data not shown) (26, 31-33, 39, 40, 48). Mesangial matrix associated proteins such as laminin 2 chain, which is known to translocate to the GBM during GVL, also maintains normal expression pattern at this stage (Fig. 2) (37).

View larger version (71K): [in this window] [in a new window]   Fig. 2.   Expression of podocyte and GESD proteins in 3(IV)-deficient mice. Immunofluorescence staining of 4-week-old 3KO / mice (4W) (A) and 8-week-old 3KO / (8W) (B) mice is shown. The renal sections are stained with indicated antibodies. The double staining with laminin 2 is done, and merged images (Merge) are shown on the right side of each horizontal panel. Nephronectin was used as a control to assess expression of noncollagen and laminin proteins in the GBM. The magnifications are as shown in the figure. C, Western blotting analysis of wild type (WT) and 8-week-old 3KO mice. The kidney cortex extracts from WT and 3KO kidneys are used for the immunoblotting with antibodies to nephrin, podocin, CD2AP, -actinin-4, and actin antibodies.

The inception of GVL starting week 5 in these mice is associated with slit diaphragm alterations, podocyte effacement, and a significant alteration in the expression of glomerular slit diaphragm-associated protein, nephrin (Fig. 2). The expression pattern of all of the other proteins tested was exactly as at week 4 prior to the inception of GVL (Fig. 2). Additionally, the expression of laminin 2, a mesangial matrix-associated protein, is now also expressed in the GBM as was reported previously for this model of GBM defect (Fig. 2) (37, 49). These results strongly implicate slit diaphragms as a crucial regulator of GVL.

In the LMX1B transcription factor-deficient mice (Lmx1b KO), the expression of 3 and 4 chains of type IV collagen is vastly diminished in the GBM because of the requirement of this transcription factor for binding to the adenine-thymine-rich FLAT sequence in the promoter region (Fig. 1) (50). Interestingly, in contrast to the 3KO with GBM defects, the Lmx1b KO mice die immediately after birth associated with massive GVL. An analysis of GBM suggests ultrastructural defects and also a lack of slit diaphragms and podocyte effacement (51). A further examination of these KO mice in the present study demonstrated additional alterations in the expression of slit diaphragm associated proteins such as nephrin, CD2AP, and podocin (Fig. 3). An evaluation of the promoter region within the genes of these proteins shows the presence of adenine-thymine-rich LMX1B binding site (data not shown). This finding suggests that along with 3 and 4 chains of type IV collagen, nephrin, CD2AP and podocin may also be subject to the transcriptional dysregulation in the Lmx1b KO mice (data not shown). GVL in these mice is associated with alteration in the GBM architecture and alteration in the expression of nephrin, CD2AP, podocin, and synaptopodin (Fig. 3). The laminin 2 transition from the mesangial matrix to the GBM was also observed in this model of GVL, consistent again with such transition in mice with primary GBM defects (Fig. 3). These experiments further suggest that the precipitous GVL observed in the Lmx1b KO mice may be associated with podocyte defects rather than the GBM defects, because the 3KO mice with a similar GBM defect do not develop GVL until 4 weeks after birth (Fig. 1 and 2).

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Expression of podocyte and GESD proteins in Lmx1b-deficient mice. Immunofluorescence staining of wild type (WT) (A) and Lmx1b / (Lmx1b KO) (B) mice is shown. The kidney sections are stained with indicated antibodies. The double staining with laminin 2 is done, and merged images (Merge) are shown on the right side of each horizontal panel. Nephronectin was used as a control to assess expression of noncollagen and laminin proteins in the GBM. The magnifications are as shown in the figure. C, Western blotting analysis of WT and Lmx1b KO. The kidney cortex extracts from WT and Lmx1b KO kidneys are used for the immunoblotting with nephrin, podocin, CD2AP, -actinin-4, and actin antibodies.

To further delineate the contribution of the GESD and the GBM properly in the regulation of GVL, we generated a nephrin-deficient mouse (nephrin KO). As previously reported for a similar knock-out mouse, our mice die at day 2 after birth, and the death is associated with massive GVL and an absence of GESD (Fig. 4) (35). The GBM architecture is normal by electron microscopy, and the expression of several GBM proteins such as type IV collagen and laminins are normal (Fig. 1, data not shown). An analysis of several podocyte-specific proteins in the nephrin KO mice demonstrates that their expression is normal (Fig. 5). In addition, the expression of ZO-1, P-cadherin, and FAT in the podocyte is also normal (data not shown) (34, 48). The lack of nephrin in association with a lack of GESD is the only molecular defect that we can identify in the kidneys of nephrin KO mice (Fig. 5).

View larger version (67K): [in this window] [in a new window]   Fig. 4.   Characterization of nephrin-deficient mouse. A, genomic structure of mouse nephrin gene and the targeting vector. Exons and introns are shown as filled boxes and lines, respectively. The targeting vector contains 1.2 kilobases of 5'-nephrin genomic sequence homologies and 5 kilobases of 3'-nephrin genomic sequence homologies. Exon sizes are not drawn to scale. B, identification of three independent ES clones (Clone 6, 7, and 8) with specific homologous recombination by Southern blotting (left panel) and PCR (right panel). For Southern blotting, wild type (3.4 or 2.2 kb) and mutant (6.2 or 2.9 kb) specific bands are obtained with DNA cut with BamHI or ApaI, respectively. For PCR, wild type (1107 bp) and mutant (1266 bp) specific bands are obtained with the primer sets for wild type (WT) and mutant (KO), respectively. C, identification of nephrin +/+, +/, and / mouse by PCR. The genomic DNA from mouse tail is used for PCR analysis with the same primer sets. D, propagation of three independent nephrin / mouse lines. The genomic DNA from three lines of chimera, F1 and F2 mice (A-C), is examined with PCR. E, GFP expression in the nephrin mutant glomeruli. GFP expression is examined with fresh frozen sections from nephrin +/+, +/, and / mice. F, development of GVL in nephrin / mice 1 day after birth. The urine from nephrin +/+, +/, /, and 8-week-old 3(IV) / mice were analyzed with SDS-PAGE. G, renal histology of representative nephrin mutant mouse. Bowman's space in nephrin / mice is enlarged, and tubules are dilated. Renal sections are subjected to periodic acid-Schiff staining (×400). H, electron microscopic analysis of glomeruli of nephrin +/+, +/, and / mice. Foot processes are positioned closer in nephrin / mice (arrowhead) compared with nephrin +/+ and +/ mice (arrow). The magnifications are as shown in the figure.

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Expression of podocyte and GESD proteins in nephrin-deficient mice. Immunofluorescence staining of wild type (WT) (A) and nephrin / (Nephrin KO) (B) mice. The renal sections are stained with indicated antibodies. The double staining with laminin 2 is done, and merged images (Merge) are shown in the right side of the horizontal panel. Nephronectin was used as a control to assess the expression of noncollagen and laminin proteins in the GBM. The magnifications are as shown in the figure. C, Western blotting analysis of WT and nephrin KO mice. The extracts from WT and nephrin KO kidneys are used for the immunoblotting with antibodies to nephrin, podocin, CD2AP, -actinin-4, and actin antibodies.

Collectively, our results using three different genetic mouse models for GVL unequivocally suggest that podocyte and the GESD-associated protein, nephrin, constitutes the most critical determinant for proteins greater than 70 kDa during the glomerular plasma filtration. Although GBM is also an important component of the filtration apparatus, it definitely plays more of a passive role in the maintenance of vascular permeability in the kidney glomerulus.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The debate over the importance of GBM and the GESD in the regulation of glomerular vascular permeability has spanned well over three decades now (4, 5, 8, 11, 12, 14, 17, 24, 25). Previous studies (4, 5, 12-16) demonstrate that both structures may be important for maintaining a stringent barrier for plasma proteins larger than 70 kDa. This study provides compelling evidence of the GESD and nephrin as the primary regulators of glomerular vascular permeability.

Studies with 3KO mice suggest that significant ultrastructural defects in the GBM along with molecular alteration within two of the most abundant proteins such as type IV collagen and laminin do not lead to a precipitous GVL. The primary defect in these KO mice leads to a gradual insidious GVL dependent on the eventual accumulation of defects in the GESD and podocyte architecture potentially mediated by nephrin (Fig. 2). The altered podocyte GBM interaction attributed to defective GBM might contribute to eventual nephrin dysregulation, but this needs further investigation. Nevertheless, these studies provide the first time proof for a secondary role for GBM in the regulation of GVL.

In the experiments with Lmx1b KO mice, we provide further evidence for the slit diaphragm defects as responsible for precipitous GVL. Although these mice have overlapping GBM defects as observed in the 3KO, the additional defects in the GESD lead to a precipitous GVL immediately after birth, potentially leading to the death of these mice. Again, these experiments provide additional support for the notion that GESD constitute the pivotal barrier for plasma proteins greater than 70 kDa.

The nephrin KO mice generated for this study provide the unequivocal evidence for the role of GESD and nephrin as the primary regulators of glomerular vascular permeability. In these KO mice, the expression of type IV collagen, laminin, nidogen, and perlecan is normal, and this is associated with the normal architecture of the GBM. The only ultrastructural defect observed in the kidney of these mice is the obvious absence of the glomerular slit diaphragm and podocyte effacement. An examination of the several different podocyte and GESD-associated proteins in the kidney of these KO mice reveals that nephrin deficiency does not lead to alterations in the expression of these various GBM and GESD proteins such as podocin, CD2AP, -actinin-4, synaptopodin, integrin ₃, and type IV collagen 3, 4, 5 chains (Figs. 1 and 5). These results lead to a strong suggestion that nephrin is a pivotal protein for the maintenance of normal GESD structure and in turn the proper function of the glomerular permeability barrier.

Interestingly, GESD-mediated GVL resulting from primary defects in GBM leads to a transition of laminin 2 from the mesangial matrix (normal distribution) to the GBM (abnormal localization). Direct GESD defect does not lead to such transition, suggesting that the primary contribution of aberrant cell matrix alterations is because of GBM defects in laminin 2 transition. An evaluation of the functional consequence of such transition attributed to GBM defects needs further explanation.

Thus, although GBM defects can lead to glomerular vascular leak in the kidney, the precipitous factor seems to involve a compromise of the GESD structure. How defects in the GBM lead to eventual GESD defects remains unanswered. Nevertheless, this study provides strong evidence for nephrin as a crucial determinant of glomerular vascular permeability.

	ACKNOWLEDGEMENTS

We thank Dr. Arlene Sharpe and the Brigham and Women's Hospital Transgenic and Knock-out facility for the generation of the nephrin KO mice. We also thank Lori Siniski for assistance in the preparation of this paper.

	FOOTNOTES

^* This work was supported in part by Grants DK 51711 and DK 55001 from the National Institutes of Health, the Program in Matrix Biology at the BIDMC, Japan Research Foundation for Clinical Pharmacology (to Y. H.), and National Research Service Award Grant DK 09946 from the National Institutes of Health (to J. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF191090 and AF190638.

To whom correspondence should be addressed: Dept. of Medicine, Harvard Medical School, Program in Matrix Biology, DANA 514, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-0445; Fax: 617-975-5663; E-mail: rkalluri@caregroup.harvard.edu.

Published, JBC Papers in Press, May 30, 2002, DOI 10.1074/jbc.M204806200

	ABBREVIATIONS

The abbreviations used are: GBM, glomerular basement membrane; GESD, glomerular epithelial slit diaphragms; GVL, glomerular vascular leak; GFP, green fluorescent protein; KO, knock-out; PBS, phosphate-buffered saline; 3KO, 3 chain of type IV collagen deficient mice; Lmx1b KO, LMX1B transcription factor deficient mice; nephrin KO, nephrin-deficient mice.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				M. P. Marin, G. Esteban-Pretel, R. Alonso, Y. Sado, T. Barber, J. Renau-Piqueras, and J. Timoneda Vitamin A Deficiency Alters the Structure and Collagen IV Composition of Rat Renal Basement Membranes J. Nutr., April 1, 2005; 135(4): 695 - 701. [Abstract] [Full Text] [PDF]

				A. Bjornson, J. Moses, A. Ingemansson, B. Haraldsson, and J. Sorensson Primary human glomerular endothelial cells produce proteoglycans, and puromycin affects their posttranslational modification Am J Physiol Renal Physiol, April 1, 2005; 288(4): F748 - F756. [Abstract] [Full Text] [PDF]