Slit Diaphragms Contain Tight Junction Proteins

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Hirotaka Fukasawa, Scott Bornheimer, Krystyna Kudlicka, and Marilyn G. Farquhar

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California.

ABSTRACT Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.

J Am Soc Nephrol 20: 1491–1503, 2009. doi: 10.1681/ASN.2008101117

Slit diaphragms are specialized cell-cell junctions in other junctions, have been identified as slit dia- located between mature podocytes that have fasci- phragm components. Slit diaphragms are currently nated cell biologists and nephrologists for more looked upon as signaling platforms in which neph- than 40 yr.1 In contrast to podocytes, most other rin and Neph1 transduce major signals that serve to epithelial cells have junctional complexes com- maintain the filtration slits and to regulate podo- posed of tight junctions and adherens junctions. Slit cyte shape through interaction of slit diaphragm diaphragms originate from typical apical junctional proteins with the actin cytoskeleton.10 Mutations in complexes between primordial epithelia of the early nephrin,7 Neph1,9 and podocin8 have been linked S-shaped body. These junctional complexes mi- to diseases associated with foot process effacement grate in a zipper-like fashion to the base of the cell and proteinuria. In addition to these specialized slit where tight junctions persist as interdigitation of diaphragm proteins, a number of other proteins the foot processes begins.2,3 Slit diaphragms appear that are associated with junctions in other locations during the capillary loop stage and gradually re- are also concentrated at the slit diaphragms, includ- place tight junctions. In many diseases associated with proteinuria and foot process loss or efface- Received October 27, 2008. Accepted February 17, 2009. ment, there is a rerun in reverse of this developmen- Published online ahead of print. Publication date available at tal sequence, and tight junctions reappear between www.jasn.org. adjoining foot processes.4–6 Correspondence: Marilyn G. Farquhar, Department of Cellular Major progress has been made recently in estab- and Molecular Medicine, University of California San Diego, 9500 lishing the molecular make-up of the slit dia- Gilman Drive, La Jolla, California 92093-0651. Phone: 858-534- phragms. Several integral membrane proteins, in- 7711; Fax: 858-534-8549; E-mail: [email protected] cluding nephrin,7 podocin,8 and Neph1,9 not found Copyright ᮊ 2009 by the American Society of Nephrology

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Figure 1. JAM-A and occludin are present between foot processes of podocytes of normal and PAN nephrotic rats. By immunofluorescence, both JAM-A (A, a, and B, b) and occludin (F, f and G, g) are distributed in a punctate pattern along normal rat glomerular capillaries and in glomeruli from 7 d PAN-nephrotic rats (PAN). The boxed areas in A, B, F, and G are enlarged in a, b, f, and g. (C) By double labeling, JAM-A (red) and ZO-1 (green) colocalize (yellow) along glomerular capillaries at the base of the podocytes. Note that parietal cells (arrowhead) stain for ZO-1 but not for JAM-A. Nuclei were stained with DAPI (blue). (D) JAM-A and occludin (H) (10 nm gold) are seen to be concentrated at the slit diaphragms (arrows) in normal glomeruli. In PAN glomeruli, JAM-A (E) and occludin (I) are concentrated along the extensive tight or occluding junctions (arrowheads) (Bar ϭ 10 ␮m in A-C and F-G; Bar ϭ 50 nm in D, E, H, and I) ing the adherens junction proteins P-cadherin,11 FAT,12 sion to understanding the organization and functions of these ␤-catenin,11 and p120 catenin;13 scaffold proteins such as ZO- junctions. 1,14,15 CD2AP,16 MAGI-2,17 and CASK;13 and actin binding proteins, including IQGAP17 and ␣-actinin 4.17,18 Because slit diaphragms share some morphologic features with adherens junctions and contain P-cadherin and catenins, slit dia- RESULTS phragms are assumed to represent modified adherens junctions.11 However, several scaffold proteins that are often asso- JAM-A is Present in the Foot Processes of Podocytes ciated with tight junctions (i.e., ZO-1,14,15 MAGI-1,19 MAGI- Previously, we established that the scaffolding proteins ZO-114 2,17 and CASK13) are present at slit diaphragms and have been and CASK13 are present in foot processes of podocytes. In other shown to associate with nephrin. Based on their derivation epithelial cells, JAM-A, an integral membrane protein of tight from typical tight junctions2 and the fact that they are replaced junctions,20 directly interacts with both ZO-121 and CASK.22 by tight junctions in nephrosis,4,6 we reasoned that slit dia- This led us to investigate whether JAM-A is also present in the phragms might also contain membrane proteins normally as- slit diaphragms of podocytes. Immunofluorescence labeling of sociated with tight junctions. semithin cryosections with a JAM-A-specific antibody (Figure In this paper, we used morphological, biochemical, and S1) revealed that JAM-A is found in glomeruli from both nor- bioinformatics techniques to investigate the expression of rep- mal and PAN-treated rats (Figure 1, A and B). By double label- resentative tight junction proteins in glomeruli in situ and in ing, we observed that JAM-A was distributed in a punctate slit diaphragm-enriched fractions. Here, we document the pattern along the foot process layer of podocytes where it cor- presence of several tight junction proteins in slit diaphragms responds in distribution to that of ZO-1 (Figure 1C). Immu- and demonstrate their interactions with slit diaphragm pro- noelectron microscopy established that JAM-A is indeed con- teins in both normal and PAN nephrotic rats. The presence of centrated along the slit diaphragms in normal glomeruli tight junction proteins in slit diaphragms adds a new dimen- (Figure 1D) and is associated with the tight junctions that ap-

1492 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1491–1503, 2009 www.jasn.org BASIC RESEARCH pear between the podocytes in PAN nephrotic glomeruli (Fig- plexes.17,29 When we subjected glomerular lysates to velocity ure 1E).4 gradient centrifugation on sucrose gradients, we found that the tight junction proteins ZO-1, JAM-A, occludin, cingu- Occludin is Also Present at Slit Diaphragms lin, and CASK cosedimented in fractions 12 to 16 with Next, we investigated whether occludin, another tight junc- nephrin, podocin, and CD2AP (Figure 3). In contrast, cad- tion protein, is also present on foot processes of podocytes herin and ␤-catenin cosedimented in heavier fractions and obtained similar findings to those obtained for JAM-A. (fractions 19 to 23), and claudin-5, a specific marker for By immunofluorescence, occludin was distributed in a tight junctions of endothelial cells, sedimented in lighter punctate pattern along glomerular capillaries in both nor- fractions (fractions 7 to 10). These results suggest that tight mal (Figure 1F) and PAN-nephrotic (Figure 1G) rats. By junction proteins are present in the same protein complexes immunoelectron microscopy, occludin was concentrated at as slit diaphragm proteins. slit diaphragms between foot processes in normal glomeruli (Figure 1H) and at tight or occluding junctions in PAN Tight Junctions Proteins are Present in Nephrin glomeruli (Figure 1I). Multiprotein Complexes To establish whether tight junction and slit diaphragm pro- Tight Junction Proteins Cofractionate with Slit teins are found in the same protein complexes, we carried out Diaphragm Markers in Slit Diaphragm-Enriched pulldown and coimmunoprecipitation assays. GST-nephrin Fractions Prepared from Normal Glomeruli pulled down JAM-A, occludin, cingulin, and CASK, but not Using a procedure worked out for isolation of apical junctional crumbs 3 from glomerular lysates (Figure 4A). Moreover, we complexes from MDCK cells,23 we prepared slit-diaphragm- detected nephrin in immunoprecipitates obtained with either enriched fractions from normal rat glomeruli on self-forming JAM-A (Figure 4B) or occludin (Figure 4C) antibodies. Simi- linear 10 to 20 to 30% iodixanol density gradients. We cross- larly, we detected JAM-A and occludin in immunoprecipitates linked proteins before homogenization to avoid partial loss of obtained with anti-nephrin IgG (Figure 4D). These results peripheral membrane proteins and disassembly of protein demonstrate that JAM-A, occludin, and cingulin, but not complexes during preparation of junctional fractions. crumbs 3, are components of slit diaphragm protein com- We examined the distribution of 20 proteins in these gradi- plexes. ents that are expressed in different domains of the foot processes of podocytes (Figure 2A). Proteins in the same mem- Expression of Tight Junction Proteins is Increased in brane domains cofractionate in the gradient, whereas proteins PAN Nephrosis in different domains sediment at different densities.23,24 In Slit diaphragms are known to decrease in number and be re- normal glomeruli, the tight junction proteins JAM-A, occlu- placed by tight junctions in nephrosis.6,4 To find out whether din, cingulin, ZO-1, and CASK cofractionated in fractions 15 the expression of tight junction proteins is changed in nephro- to 18 with slit diaphragm markers, nephrin, podocin, CD2AP, sis, we performed immunoblotting on glomerular lysates from and Neph1 (Figure 2A). In contrast, adherens junction pro- normal versus PAN-treated rats (Figure 5). ZO-1, JAM-A, oc- teins (cadherins and catenins) were broadly distributed (frac- cludin, and cingulin were dramatically increased (approxi- tions 7 to 18) in the gradients and partially overlapped with slit mately 330%, 290%, 350%, and 320%, respectively) in PAN- diaphragm-enriched fractions (Figure 2B). Claudin-5, a spe- treated rats. By contrast, both crumbs 3 and nephrin were cific marker for endothelial cell tight junctions25 sedimented in decreased (approximately 70%) (Figure 5, A and B). much lighter fractions (fractions 1 to 12), and Crumbs 3, also a tight junction protein, sedimented in heavier fractions than slit Redistribution in Iodixanol Gradients of Tight Junction diaphragms. Podocalyxin, an apical membrane protein on foot and Slit Diaphragm Proteins Prepared from Nephrotic processes,14 and ␤1 integrin, a basal protein,26 were also dif- Glomeruli fusely distributed in the gradients. Podocalyxin cofractionated Next, we compared the behavior of junctional membranes pre- with its binding partner, ezrin,27,28 and ␣-actinin partially co- pared from PAN nephrotic versus normal rats after separation fractionated with its binding partners, ␣-catenin and ␤1 inte- on iodixanol density gradients. In glomeruli from 7 d PAN- grin. The finding that tight junction proteins are concentrated treated rats, ZO-1, JAM-A, and occludin as well as nephrin, in slit diaphragm-enriched fractions suggests that they are as- podocin, and CD2AP were less dense as they floated up and sociated with slit diaphragms. were broadly distributed across fractions 7 to 15 (Figure 6, A and B), whereas in normal glomeruli, they were restricted to ZO-1, JAM-A, Occludin, and Cingulin Cosediment in fractions 15 to 18 (see Figure 2). The distribution of cadherins Sucrose Gradients with Slit Diaphragm Proteins, but and catenins was unchanged as they were broadly distributed not Adherens Junction Proteins (Figure 6B). These results indicate that membrane domains Cosedimentation analysis of detergent-solubilized proteins containing the slit diaphragms and tight junctions obtained is commonly used to gain information on whether proteins from nephrotic glomeruli are less dense than those from nor- associate with one another in the same multiprotein com- mal glomeruli.

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Tight Junction and Slit Diaphragm Proteins Dissociate from the Actin Cytoskeleton in Nephrotic Glomeruli Next, we used sequential detergent extraction to assess the interactions between tight junction and slit diaphragm proteins and the actin cytoskeleton in normal and nephrotic glomeruli.30 Glomeruli isolated from normal and PAN-treated rats were sequentially solubilized in 0.5% Triton X-100 and RIPA buffer, and the distribution of junctional proteins in Triton X-100-soluble, RIPA-soluble, and RIPA-insoluble fractions was determined. In normal glomerular lysates we found nephrin, JAM-A, occludin, and CASK in all three fractions and ZO-1 and ␣-actinin mainly distributed in the RIPA-insoluble fraction (Figure 7). With PAN treatment, we saw a significant increase in the detergent extractability of nephrin, ZO-1, JAM-A, occludin, and CASK, as greater amounts of these proteins were detected in the Triton-soluble or RIPA-soluble fractions, and only ZO-1 was detected in the RIPA-insoluble fraction. By contrast, the distribution of ␣-actinin and actin did not change noticeably after PAN treatment. These results indicate that nephrin, ZO-1, JAM-A, occludin, and CASK (but not ␣-actinin) partially dissociate from the actin cytoskeleton in glomeruli from PAN rats. Subsequent results obtained after extraction with potassium iodide (KI), an actin depolymeriz- ing agent,27 supported this conclusion and showed that the ratios of actin-associated proteins were decreased in PAN glomeruli (Figure S3).

Analysis of Protein-Protein Interaction Map To better understand the molecular links between slit diaphragm, tight junction, and adherens junction proteins, we took advantage of the interologous interaction database,31,32 which is an amalgam of several databases containing protein- protein interactions validated from the literature and predicted interactions based on high throughput studies across multiple species. Our resulting protein-protein interaction map (Figure 8) contains only experimentally validated interactions. It shows that the closest link between nephrin and JAM-A, occludin, and cingulin is through Neph1 and ZO-1. A second possible connection is through PAR-6 and PAR-3. The Figure 2. Distribution of junctional fractions from normal rat glomeruli in iodixanol density gradient centrifugation. (A) A Post- entire group of tight junction proteins studied is on average nuclear supernatant (PNS) prepared from glomeruli isolated from only two protein-protein interactions away from a slit dia- normal rats was mixed with an equal amount of iodixanol [final phragm protein. This is in keeping with our finding that tight concentration 30% (wt/vol)] and overlaid with equal volumes of 20% and 10% iodixanol. After centrifugation at 350,000 ϫ g for 3hat4ЊC, fractions were collected from the top, separated by endothelial tight junctions, is concentrated in lighter fractions SDS-PAGE, and immunoblotted for the indicated 20 proteins (1–12). Podocalyxin, an apical domain protein in foot processes, expressed in foot processes of podocytes. In normal rat glomer- and ␤1 integrin, a basal protein, are broadly distributed in the uli, the tight junction proteins ZO-1, JAM-A, occludin, cingulin, gradient but are concentrated mostly in lighter fractions (1–13). and CASK cofractionate with the slit diaphragm enriched fractions Podocalyxin cofractionates with its binding partner, ezrin (8–14). marked by nephrin, podocin, CD2AP, and Neph1 in fractions 15 (B) Densitometric analysis showing the % of the total nephrin to 18. By contrast, the adherens junction proteins, cadherin and (triangles with dashed line), pan-cadherin (squares with dotted ␣-, ␤-, and p120 catenins, are broadly distributed in the iodixanol line), and ZO-1 (circles with solid line) present in the fractions gradients (fractions 7 to 18) and only partially cofractionate with shown in “A.” The distribution of ZO-1 (used as a tight junction slit diaphragm proteins. Crumbs 3, another tight junction protein, marker) in each of the fractions is similar to that of nephrin (slit is concentrated in heavier fractions (18–22) than other tight junc- diaphragm marker) but not to that of pan-cadherin (adherens tion and slit diaphragm markers. Claudin-5, a specific marker for junction marker).

1494 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1491–1503, 2009 www.jasn.org BASIC RESEARCH junction proteins are enriched in the same fractions as slit diaphragm proteins. In contrast, adherens junction proteins have no direct link to slit diaphragm proteins. They are linked only indirectly through actin or ZO-1. On average, 3.5 protein-

Figure 4. Tight junction proteins form a protein complex with Figure 3. Tight junction proteins cosediment in the same protein nephrin but not crumbs 3. (A) JAM-A, occludin, cingulin, and complexes as slit diaphragm proteins, but not adherens junction CASK are pulled down with GST-nephrin tail from normal glomer- proteins. Glomeruli isolated from normal rats were lysed in 0.5% ular lysates. In contrast, crumbs 3 was not pulled down. Isolated Nonidet P-40 and 0.25% Triton X-100, and the lysate was loaded glomeruli from normal rats were lysed in 1% Nonidet P-40 with ontopofa5to25%discontinuous sucrose gradient and centri- 100 ␮mol/L potassium iodide. Twenty micrograms nephrin cyto- fuged as described in Concise Methods. Fractions were collected plasmic domain fused to GST (GST-nephrin) or GST alone (GST) from the top of the gradient and separated by SDS-PAGE fol- were incubated with 400 ␮g glomerular lysate at 4ЊC for 16 h and lowed by immunoblotting with the indicated antibodies. (A) ZO-1, immunoblotted with JAM-A, occludin, cingulin, and CASK anti- JAM-A, occludin, and cingulin are concentrated in fractions 12 to bodies. (B to D) Nephrin can be coimmunoprecipitated with 16 and cosediment with nephrin, podocin, CD2AP, and CASK. In anti-JAM-A (B) and anti-occludin (C) IgG. Similarly, JAM-A and ␤ contrast, cadherin and -catenin distributed in heavier fractions occludin can be coimmunopreciptated with anti-nephrin IgG (D). (19–23) of the gradient. Claudin-5, a marker for endothelial tight Isolated glomeruli from normal rats were lysed in 1% Triton X-100 junctions, is concentrated in lighter fractions (7–10). (B) Densito- and 0.5% Nonidet P-40. Immunoprecipitation was performed on metric analysis of ZO-1 (circles with solid line), nephrin (triangles 500 ␮g glomerular lysate from normal rats with JAM-A (␣JAM-A), with dashed line), pan-cadherin (squares with dotted line), and occludin (␣Occludin), nephrin (␣Nephrin), or preimmune (Pre) podocin (diamonds with hatched line). ZO-1 partially cosediments IgG. Glomerular lysates (25 ␮g) were included as controls. with nephrin and podocin but not with pan-cadherin.

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Figure 5. Comparative immunoblot analysis of expression of tight junction proteins in glomeruli from normal versus PAN nephrotic rats. (A) The protein levels of ZO-1, JAM-A, occludin, and cingulin are dramatically increased in glomerular lysates from Figure 6. Iodixanol density gradient centrifugation of postnuclear PAN-treated rats (PAN) compared with normal, whereas expres- supernatant prepared from glomeruli of PAN nephrotic rats. (A) In ␤ sion of crumbs 3 and nephrin are decreased. -actin was used as glomeruli from PAN-treated rats, fractions containing ZO-1, JAM-A, a control for protein load. Glomeruli isolated from two rats were occludin, and CASK and the slit diaphragm markers, nephrin, podo- ␮ lysed in RIPA buffer; 20 g glomerular lysate was immunoblotted cin, and CD2AP, float up to regions of lower density and are broadly with the indicated antibodies. We performed experiments three distributed in fractions 7 to 16. In normal glomeruli, they are re- times with similar results. (B) Densitometric analyses of data in “A” stricted to fraction 15 to 18 (compare with Figure 2). In contrast, the ␤ showing expression of tight junction proteins relative to -actin in distribution of adherens junction proteins (cadherins, ␣- and normal (open bars) and PAN (closed bars) glomeruli. Data are ␤-catenins) is similar in normal and PAN rats. (B) Densitometric Ϯ mean SEM values. analysis demonstrating that the distribution of nephrin and ZO-1 is protein interactions are required to reach the closest slit dia- shifted to lighter fractions in PAN (dashed lines) compared with phragm protein from an adherens junction protein, which is in normal (solid lines) glomeruli (data from Figure 2A). There is little change in the distribution of cadherins. keeping with our finding of a relative lack of enrichment of adherens junction proteins in slit diaphragm fractions (Figure 2). In addition to the direct interactions, a number of indirect DISCUSSION interactions occur among all three groups of proteins, espe- cially via kinases (not shown). Such signaling interactions im- In this study, we report data obtained from cell fractionation, ply coordination among the slit diaphragm, tight junction, and immunofluorescence, and immunoelectron microscopy es- adherens junction groups of proteins. tablishing the presence of the tight junction proteins JAM-A,

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they cosediment with nephrin in sucrose gradients and interact with nephrin in immunoprecipitation and pulldown assays. The presence of tight junction proteins in glomerular junctions has wide functional implications. Until now, the glomerular slit diaphragms have been regarded to be highly specialized adherens junctions whose unique permeability properties are created by the presence of nephrin, podocin, and Neph1–3 that are specific components of slit diaphragms.11 We have shown that three proteins that usually make up tight junctions, occludin, JAM-A, and cingulin, are also expressed in slit diaphragms, and yet morphologically recognizable tight junctions are absent. We have further shown that in PAN nephrosis protein levels of JAM-A, occludin, and cingulin (Figure 5) as well as ZO-115 are increased at a time when nephrin and podocin expression goes down,33,34 expression of claudin-6 increas- es,35 and tight junctions form. From these findings, it is tempting to speculate that the expression of slit diaphragm proteins and their incorporation into tight junction protein complexes late in glomerular development may facilitate the formation of mature slit diaphragms. Conversely, decreased expression of nephrin33 together with increased expression of occludin, JAM-A, cingulin, and claudin-6 may facilitate the assembly of typical tight junctions in PAN nephrosis. Based on the above, the discovery of tight junction proteins at slit diaphragms Figure 7. Sequential detergent extraction of proteins from glomeruli of normal and has led to the following working hypothe- PAN-treated rats. (A) Immunoblot of normal and PAN-treated glomeruli after sequen- sis: In developing glomeruli, nephrin, tial detergent extraction. In normal glomeruli, nephrin, JAM-A, occludin, and CASK distributed in all three fractions (lanes 1 to 3), whereas ZO-1 and ␣-actinin mainly podocin, and Neph1 are synthesized late in distributed in the RIPA-insoluble (RIPA-I) (lane 3) fraction. PAN treatment resulted in a the capillary loop stage, form complexes significant increase in the detergent solubility of nephrin, ZO-1, JAM-A, occludin, and with tight junction proteins, facilitate mat- CASK in that increased amounts of these proteins were found in the Triton-soluble uration of tight junctions into slit dia- (compare lanes 1 and 4) and RIPA-soluble fraction (compare lanes 2 and 5), and only phragms, and prevent assembly of tight ZO-1 was detected in the RIPA-insoluble fraction (lane 6). The distribution of ␣-actinin junctions in normal, mature glomeruli. In and actin in each fraction is similar in normal and PAN rats. Glomeruli isolated from PAN nephrotic glomeruli, slit diaphragm normal and PAN-treated rats were sequentially solubilized in 0.5% Triton X-100 and proteins are reduced, and tight junction RIPA lysis buffer and separated into Triton X-100-soluble (TX-S), RIPA-soluble (RIPA-S), proteins are increased, allowing tight junc- and RIPA-insoluble fractions, followed by immunoblotting. (B) Densitometric analysis tions to assemble. showing the % of total protein in the Triton X-100-soluble, RIPA-soluble, and RIPA- Some time ago, Caulfield et al4 found insoluble fractions in normal (open bars) and PAN rats (closed bars) in “A.” Each bar indicates the percent of the total band intensity in each fraction. that junctions formed in PAN nephrosis have the morphologic features characteristic of tight junction proteins by routine EM occludin, and cingulin in slit diaphragms of normal glomeruli. and freeze fracture. Our findings that the protein levels of We have also shown that these tight junction proteins are JAM-A, occludin, and cingulin as well as ZO-115 are increased found in the same multiprotein complexes as nephrin because in glomeruli from PAN-nephrotic rats is in keeping with the

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teins to the actin cytoskeleton.37,40 They are also closely associated with signaling molecules (e.g., aPKC, Rab3B, Rab13, G␣s, G␣12, GEFH1) and polarity complexes (e.g., PAR-3, PAR-6, Crumbs, PALS-1).37–39 Occludin and claudins are believed to be responsible for the clas- sical sealing function of tight junctions.41 Interestingly, occludin interacts with c-yes, a Src-family kinase, in MDCK cells,42 and c-yes can phosphorylate nephrin at slit diaphragms.43 JAM-A20 is believed to participate in both the es- tablishment of cell polarity and in tight junction formation.20,44,45 JAM-A can bind several PDZ domain proteins, including ZO-1 and CASK. Recently, several JAM family members, JAM-4 and the Coxack- ievirus/adenovirus receptor (CAR), were reported to be present in foot processes of podocytes,46,47 but Figure 8. Proposed protein-protein interaction map for the podoctye slit whether JAM-4 is associated with slit diaphragms is diaphragm. Interactions between proteins analyzed in this study are shown controversial.19,48 Cingulin interacts with several based on interactions listed in the I2D database32,69 and validated in the literature (references shown). The slit diaphragm (red) and tight junction junctional and cytoskeletal proteins including ZO-1, 49 50 (blue) groups of proteins are linked by a direct interaction between Neph1 ZO-2, ZO-3, AF-6, myosin, and actin. The func- and ZO-1. This is the shortest link between nephrin and occludin, cingulin, tion of cingulin has not fully been established, but it and JAM-A (shown in bold). An alternative interaction may occur between has been suggested to regulate cell proliferation.51 nephrin and JAM-A via PAR-3 and PAR-6 (shown with dashed line and Although claudins are expressed in developing and smaller font), which were not examined experimentally in the current study. PAN nephrotic glomeruli35 as well as the junctions In contrast to tight junction proteins, the adherens junction proteins studied between parietal epithelial cells,52 they have not been lack direct interactions with slit diaphragm proteins, but indirect interactions convincingly localized at slit diaphragms in normal ␣ are present (e.g., -catenin is linked indirectly to CD2AP through actin and glomeruli.21,22,53–55 indirectly to Neph1 through ZO-1). Recently, it was reported that in nephrosis the polarity of foot processes is lost,56 which correlates with presence of tight junctions. Furthermore, our results based on our finding of decreased levels of crumbs 3. Intriguingly, we sequential detergent extraction and KI treatment indicate that could not detect an association between crumbs 3 (a polarity nephrin, JAM-A, occludin, CASK, and ZO-1 partially dissoci- protein associated with the tight junction)57 and nephrin in ate from the actin cytoskeleton in PAN nephrosis. The associ- pulldown and cofractionation assays, and in contrast to the ation between slit diaphragm proteins and membrane proteins tight junction proteins studied, the expression of crumbs 3 is of the foot processes and the actin cytoskeleton is highly dy- decreased in PAN nephrosis. namic, and tight junction proteins can be added to the growing Our protein interaction map suggests that ZO-1 may be the list of membrane proteins whose attachment to actin is re- key protein that links tight junction proteins and slit dia- quired for maintenance of the normal organization of the foot phragm proteins and in turn links both to the cytoskeleton. We processes and filtration slits. Previously, nephrin,31 podoca- previously reported that ZO-114,15 and CASK13,17 are present at lyxin28 (apical domain protein), and ␣3␤1 integrin36 (basal slit diaphragms and foot processes of podocytes. Our results domain protein) were found to dissociate from the actin cy- indicate that CASK forms a complex with nephrin and behaves toskeleton under pathologic conditions associated with foot the same as tight junction proteins in cosedimentation assays process effacement, and it was suggested or implied that this is (see Figure 4). In other epithelia, these MAGUK scaffold pro- the cause of the structural changes (effacement) of the foot teins serve to cluster occludin,58 claudins,59 and JAMs21 at tight processes. junctions and to anchor them to the actin cytoskeleton.60–62 Until now, the regulation of the functions of slit diaphragms A key unanswered question that has puzzled us6 and others has been centered on nephrin signaling. However, the presence for many years is why increased permeability of glomeruli and of tight junctions has important functional implications for proteinuria are associated with conversion of slit diaphragms signaling from slit diaphragms, as tight junctions serve as sig- to tight junctions. Among the possible factors to be considered naling centers that play important roles in orchestrating cell are that 1) the tight junctions in nephrotic glomeruli are dis- polarity, cell proliferation, and differentiation as well as regu- continuous with gaps that allow protein leakage; 2) damage to lating paracellular permeability in other epithelia.37–39 Tight the GBM results in increased permeability of the GBM;6,63 3) junctions are composed of transmembrane proteins (occludin, damage to the GBM results in areas of focal detachment of JAMs, claudins) and scaffolding proteins (ZO-1, ZO-2, ZO-3, podocytes from the GBM; and 4) detachment of junctional cingulin, MAGI-1, 2, 3) that link the integral membrane pro- proteins from the actin cytoskeleton. Our current results as

1498 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 1491–1503, 2009 www.jasn.org BASIC RESEARCH well as those of others emphasize that detachment of tight Preparation of Glomerular Lysates junction and slit diaphragm proteins from the actin cytoskel- Glomerular fractions (containing Ͼ 95% glomeruli; see Supplemen- eton is a common denominator of the podocyte’s response to tary Figure S1) were isolated from kidney cortices of male Sprague- injury. Dawley rats (150 g; Charles River Laboratories, Boston, MA) by In conclusion, the present study documents the presence of graded sieving as described previously.27 Glomerular lysates were pre- tight junction proteins in the slit diaphragms and nephrin pared by incubation of isolated glomeruli in either 1% Nonidet P-40, multiprotein complexes, and thus, significantly extends cur- 20 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 100 mmol/L potas- rent information on the molecular composition of slit dia- sium iodide (KI)27 (for GST pulldown), in 1% TX-100, 0.5% Nonidet phragms by demonstrating that they are a highly specialized P-40, 150 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.6, 1 mmol/L variant of the tight junction. The tight junction proteins ethylenediamine-tetraacetic acid (EDTA), 1 mmol/L ethylene glycol- JAM-A, occludin, ZO-1, cingulin, and CASK are known to be bis(oxyethylenenitrilo)tetraacetic acid (EGTA)13 (for immunopre- components of unique and extensive signaling networks. cipitation), or in 0.1% SDS, 0.5% deoxycholate, 1% TX-100, 20 mM Thus, their presence in slit diaphragms as well as in the modi- HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA (for immunoblotting of fied occluding junctions seen in nephrosis provides mechanis- tight junction proteins) supplemented with 1x Complete, EDTA-free tic insights into the assembly of these junctions and greatly proteinase inhibitor cocktail (Roche, Mannheim, Germany), 50 extends their potential protein interactions and signaling rep- mmol/L sodium fluoride, and 1 mmol/L sodium vanadate at 4°C for ertoire. 60 min. Detergent-insoluble material was removed by centrifugation (10,000 ϫ g for 10 min).

CONCISE METHODS Induction of PAN Nephrosis Male rats (150 g) were injected once intraperitoneally with PAN (15 Materials mg/100 g body weight) as described previously. Animals were sacri- Chemical reagents were from Sigma (St. Louis, MO) or Fisher Biotech ficed on day 7 after injection. All animal experiments were done ac- (Tustin, CA), and detergents were from Sigma or Calbiochem (San cording to the NIH Guidelines for the Care and Use of Laboratory Diego, CA). Kodak Biomax MR film was obtained from Fisher Bio- Animals. tech. SDS-PAGE and Immunoblotting TM Antibodies Protein concentration was measured by Quick Start Bradford Dye Mouse anti-nephrin mAb (043; for immunoblotting)13 and rabbit Reagent (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins were anti-nephrin 029 (for immunoprecipitaton)64 were raised against the separated on 8 or 10% SDS-PAGE under reducing conditions and extracellular domain of rat nephrin (amino acids 749 to 1030) and the transferred to polyvinylidene fluoride (PVDF) membranes (Millipore cytoplasmic tail of nephrin, respectively. Rabbit anti-podocin Corp., Bedford, MA) using a Minigel-Transfer-Unit (Bio-Rad Labo- (P0372) and mouse anti-␤-actin mAb (AC-15) were from Sigma. ratories) as described previously.30 ␤-actin was used as an internal Rabbit anti-CD2AP polyclonal IgG (R209) was raised against amino control. Protein bands were detected by enhanced chemilumines- acids 331 to 637 of mouse CD2AP.65 Mouse anti-JAM-A mAb (for cence (Supersignal; Pierce Biotechnology Inc., Rockford, IL) and immunofluorescence) and rabbit anti-JAM-A (for immunoblotting) quantified using Image J analysis software (NIH, Bethesda, MD). were obtained from Dr. Charles Parkos (Emory University) and In- vitrogen (Carlsbad, CA), respectively. Rabbit anti-Neph1 and anti-␤- Immunofluorescence Microscopy catenin antibodies were obtained from Drs. Lawrence Holzman (Uni- Kidneys were immersion fixed with 2% paraformaldehyde (PFA) for versity of Michigan) and James Nelson (Stanford University), 30 min. For preparation of semithin sections, PFA-fixed kidneys were respectively. Affinity-purified rabbit anti-pan-cadherin (recognizes cryoprotected and frozen in liquid nitrogen.67 Semithin cryosections P-, N-, E-, and R-cadherins), anti-␣-catenin, anti-ZO-1, anti-occlu- (0.5 ␮m) were cut with a Leica Ultracut UCT microtome equipped din, anti-JAM-C, anti-CASK (calcium/calmodulin-dependent serine with an FCS cryoattachment at Ϫ100°C and incubated with primary protein kinase), and mouse anti-claudin-5 IgG were from Invitrogen. antibodies overnight at 4°C followed by detection with Alexa 594 goat Rabbit anti-cingulin and anti-crumbs 3 antibodies were obtained anti-rabbit and anti-mouse IgG in PBS containing 5% fetal calf serum from Dr. Sandra Citi (University of Geneva, Switzerland) and Dr. for2hatroom temperature. Samples were examined with a Zeiss Andre´Le Bivic (Faculte´des Sciences de Luminy, France), respectively. Axiophot microscope (Carl Zeiss Inc., Thornwood, NY). Images were Rabbit anti-JAM-B antibody was from Santa Cruz Biotechnology collected with the ORCA-ER camera (Hamamatsu, Bridgewater, NJ) (Santa Cruz, CA). Mouse anti-p120 catenin was from BD Transduc- using Scion Image Version 1.59 (Scion Corp., Frederick, MA) and tion Laboratories (BD Biosciences, San Jose, CA), and mouse anti- processed using Adobe Photoshop CS3 (Adobe Systems, San Jose, podocalyxin mAb 5A was described previously.66 Rabbit anti-ezrin CA). (3C12) was purchased from NeoMarkers (Fremont, CA), mouse ␣-actinin mAb (clone AT6.172) was purchased from Chemicon (Te- Immunoelectron Microscopy mecula, CA), and horseradish peroxidase-conjugated goat anti-rabbit Rat kidneys were fixed in 4% PFA for 45 min followed by 15 min in 8% and anti-mouse IgG were from Promega (Madison, WI). PFA, cryoprotected in sucrose and frozen in liquid nitrogen as above.

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Ultrathin cryosections (80 nm) were cut and processed as described min at 4°C). Glomerular lysates were applied on top ofa5to10to15 previously.27 Sections were incubated with rabbit anti-occludin, to 20 to 25% discontinuous sucrose gradient. Sucrose solutions were JAM-A, or cingulin IgG for 16 h at 4°C followed by incubation with prepared in lysis buffer containing 0.05% Nonidet P-40 and 0.025% anti-rabbit gold conjugates (10 nm) for2hatroom temperature. Triton X-100. After centrifugation at 44,000 rpm (200,000 ϫ g) for Sections were then adsorption-stained and examined with a JOEL 15 h at 4°C in Beckman Coulter SW 60 Ti rotor, fractions were col- EX-II electron microscope.30 lected from the top of the gradient and their sedimentation coeffi- cients were assessed using protein molecular weight standards GST-Pulldown Assays (Sigma) with known S values. GST and GST-nephrin tail fusion proteins were expressed in Esche- richia coli BL21 (DE3) (Stratagene, LA Jolla, CA) and purified on glutathione-Sepharose beads (Amersham Biosciences) as described Differential Detergent Extraction of Glomeruli Sequential extraction of proteins was carried out as described previ- previously.13 Glomerular lysates were precleared for1hat4°C, fol- ously.30 In brief, glomeruli were lysed in 500 ␮l Triton X-100 lysis lowed by incubation at 4°C for 16 h with GST-nephrin tail or GST buffer (0.5% TX-100, 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM alone (20 ␮g each) immobilized on beads. Beads were washed five EDTA with 1x Complete, 50 mmol/L sodium fluoride, and 1 mmol/L times with lysis buffer and boiled in 2x sample buffer for immuno- sodium vanadate) at 4°C for 30 min and centrifuged at 15,000 ϫ g at blotting. 4°C for 30 min. The insoluble pellet was resuspended in the same volume of RIPA buffer (0.1% SDS, 0.5% deoxycholate, 1% TX-100, Coimmunoprecipitation 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA with 1x Com- Glomerular lysates were precleared with protein G plus/protein A- plete, 50 mmol/L sodium fluoride, and 1 mmol/L sodium vanadate) at agarose beads (Calbiochem, San Diego, CA) at 4°C for 1 h and incu- 4°C for 30 min and centrifuged as above. The resultant pellet was bated with anti-nephrin, anti-JAM-A, anti-occludin, or preimmune solubilized by sonication in the same volume of 1x sample buffer as IgG (2 ␮g) for 16 h at 4°C. Immune complexes were bound to protein the supernatant, and equal volumes of the fractions were analyzed by G plus/protein A-agarose beads at 4°C for 1 h, washed three times with immunoblotting. lysis buffer, and boiled in 2x sample buffer for immunoblotting.

Preparation of Junctional Fractions Actin Depolymerization Enriched junctional fractions were prepared on self-forming linear 10 Isolated glomeruli were extracted on ice for 30 min with lysis buffer to 20 to 30% iodixanol density gradients24 as reported previously for (1% Triton X-100, 20 mM Tris-HCl, pH 6.2, 10 mM NaCl, and 1.5 23 30 isolation of junctions from MDCK cells. Isolated glomeruli were mM MgCl2). Insoluble material was separated by centrifugation at chemically cross-linked with dithiobis (succinimidylpropionate) 15,000 ϫ g at 4°C for 30 min and then incubated with 0.6 M potassium (DSP; Pierce Chemical) before mechanical breakage of glomeruli.68 iodide (KI) in the same buffer to depolymerize F-actin.27,68 The su- Freshly prepared 200 ␮g/ml DSP in PBS was added to the isolated pernatants containing actin-associated proteins released by KI were glomeruli for 20 min at room temperature with gentle rocking fol- separated by centrifugation as above. The resultant pellet was solubi- lowed by quenching in quenching buffer (120 mM NaCl, 10 mM Tris, lized by sonication in the same volume of sample buffer, and equal ϩ pH 7.4, 50 mM NaH4Cl). After cross-linking, glomeruli were ho- volumes of each fraction were analyzed by immunoblotting. mogenized in 0.25 M sucrose in 20 mM HEPES-KOH, pH 7.2, 90 mM

KOAc, 2 mM Mg(OAc)2 buffer using a loose Dounce homogenizer (20 strokes). Glomerular homogenates were transferred to microcen- Creation of the Protein-Protein Interaction Map trifuge tubes and centrifuged at 1000 ϫ g for 10 min to obtain the The protein-protein interaction map was based on protein-protein 32,69 postnuclear supernatant (PNS). The PNS was mixed with equal interactions listed in the I2D database and validated in the litera- amounts of iodixanol [final concentration 30% (wt/vol); Nycomed, ture. For simplicity it was restricted mainly to direct protein-protein Oslo, Norway] and overlaid with equal volumes of 20% and 10% interactions between proteins studied in this manuscript. The layout iodixanol. After centrifugation at 58,000 rpm (350,000 ϫ g)for3hat of the map was designed using NAViGaTOR software (http:// 4°C in a Beckman Coulter SW 60 Ti rotor, fractions were collected ophid.utoronto.ca/navigator/). from the top of the gradient. Gradients were highly reproducible and fraction densities comparable between gradients. ACKNOWLEDGMENTS Cosedimentation Assays Velocity gradient centrifugation was performed as described previ- This work was supported by National Institutes of Health Grant ously.29 Isolated glomeruli were lysed in 0.5% Nonidet P-40, 0.25% DK17724 (to M.G.F). Triton X-100, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 1 mmol/L EDTA with 1x Complete, 50 mmol/L sodium fluoride, and 1 mmol/L sodium vanadate, homogenized with very loose Dounce homogenizer (3 strokes), and incubated at 4°C for 90 min. Detergent- DISCLOSURES insoluble material was removed by centrifugation (15,000 ϫ g for 15 None.

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