Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting

Patricia L. St. John1, Ruixue Wang1, Yong Yin2, Jeffrey H. Miner3, Barry Robert1, and Dale R. Abrahamson1

1 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7400; and 2 Department of Anatomy and Neurobiology and 3 Renal Division, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6-7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

endothelial cells; glomerular basement membrane; podocytes


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DEVELOPMENT OF THE PERMANENT, metanephric kidney in the mouse begins at ~embryonic day 10 (E10), when the ureteric bud emerges from the Wolffian duct and projects dorsolaterally to engage mesenchymal cells of the metanephric blastema. Reciprocal signals from the ureteric bud and blastema, respectively, induce growth and repeated branching of the bud and formation of mesenchymal cell aggregates at each branch tip (13, 27). Subsequently, each mesenchymal aggregate converts to an epithelial vesicle, which elongates to form a short tubule that then develops a crevice to resemble a comma-shaped figure. The glomerulus originates as endothelial cells migrate into this crevice, or vascular cleft. Together with mesangial progenitors, these cells give rise to the glomerular capillaries, which mark the capillary loop stage of glomerular development (reviewed in Ref. 2). Epithelial cells located immediately beneath the original vascular cleft differentiate into podocytes, and those above the cleft develop into tubular segments of the nephron, which join ureteric bud branches that go on to form the collecting system. The induction of nephric figures in mice continues until ~1 wk after birth and then ceases once the full complement of nephrons has formed (27).

Electron microscopic examination of the vascular cleft has shown that the early glomerular endothelial cells are resting on a subendothelial basement membrane that lies parallel to but is distinct from an overlying basement membrane beneath developing epithelial podocytes (2). Immunoelectron microscopy has also shown that both endothelial cells and podocytes are engaged in synthesis of the key basement membrane proteins: laminin, collagen type IV, and entactin at this stage (1, 8). As the glomerular capillaries begin to form, the endothelium flattens and forms fenestrae, a process that is coincident with development of foot processes by podocytes. The double basement membranes between the two cell layers fuse to produce the glomerular basement membrane (GBM), which is shared by the endothelium on the vascular surface and podocytes on the urinary surface (reviewed in Ref. 2). Mechanisms accounting for the union of the subendothelial and subpodocyte basement membrane layers into the GBM are not known, but the incorporation of intravenously injected, soluble mouse laminin into newborn rat GBM indicates that specific binding sites for laminin exist in the immature GBM (31). Hence, binding interactions occurring between basement membrane proteins in the separate layers of the dual basement membrane may promote GBM fusion, but the details of this process remain unclear (31).

As glomeruli mature, a complex series of laminin and collagen type IV isoform transitions occur in GBM composition (reviewed in Ref. 15). Specifically, laminin-1, consisting of the laminin alpha 1-, beta 1-, and gamma 1-chains, and laminin-8 (alpha 4-, beta 1-, and gamma 1-chains) are found in the most immature GBM region of vascular clefts (9, 18, 28). However, these laminins are replaced by laminin-11, containing the laminin alpha 5-, beta 2-, and gamma 1-chains, and this is apparently the only laminin isoform found in the fully mature GBM (15). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are gradually replaced by alpha 3(IV)-, alpha 4(IV)-, and alpha 5(IV)-chains (15, 19). In general, the appearance of the laminin alpha 5- and beta 2-chains, and the alpha 3-chain of type IV collagen, all first occurs in capillary loop stages of glomerular development, although trace amounts of laminin alpha 5-chain have been reported in earlier, S-shaped stages (9, 15, 28).

Although very little is known about the regulation of GBM protein substitutions at either the gene or protein level, these changes in matrix composition are extremely important functionally. Mice genetically deficient in laminin alpha 5- or beta 2-chains display prominent glomerular phenotypes. Beginning at the capillary loop stage, mutants for laminin alpha 5 display disorganization of the podocyte cell layer, absence of an ultrastructurally definitive GBM, and a failure of glomerular endothelial cells and mesangial cells to form a capillary tuft (17). In mice lacking the laminin beta 2-chain, the laminin beta 1-chain persists in the GBM, which appears ultrastructurally normal (21). However, the podocyte foot processes are abnormally broadened, and animals develop massive proteinuria (21). These findings therefore emphasize pivotal roles for laminin alpha 5- and beta 2-chains in GBM structure and function and an inadequacy of their forerunners, laminin alpha 1, alpha 4, and beta 1, for establishing and maintaining the normal glomerular filtration barrier.

In previous work, we used a metanephric organ culture system in which avascular glomerular epithelial tufts form in vitro. By using metanephroi dissected from heterozygous Flk1-lacZ transgenic mice, endothelial cells expressing the vascular endothelial growth factor receptor, Flk1, are identified by the expression of the lacZ transgene. Defined glomerular endothelial cells are not seen in these cultures, but when cultured metanephroi are subsequently grafted into anterior eye chambers, graft-derived angioblasts differentiate into lacZ-positive endothelial cells and vascularize glomeruli that develop in oculo (23, 24). In the present study, we evaluated the laminin alpha 1-, alpha 5-, beta 1-, and beta 2-chains synthesized by the avascular glomeruli that form in organ culture, as well as in the vascularized glomeruli that develop after organ-cultured kidneys are grafted. Our results show that, despite significant podocyte differentiation in organ culture, incomplete laminin chain transitions occur in vitro. These errors are corrected, however, when organ-cultured kidneys are grafted and vascularized glomeruli develop. Our findings therefore implicate glomerular endothelial cells, and/or factors present in the circulation, as important mediators for normal GBM laminin assembly.


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Animals. Mice for these experiments came from our colonies of C57BL/6J and Flk1-lacZ transgenic (Flk1tm1Jrt) animals that we obtained originally from the Jackson Laboratory (Bar Harbor, ME). Flk1-lacZ trangenics were genotyped as before (24).

Antibodies. Monoclonal rat anti-mouse laminin-1 antibodies (MAb; 5A2 and 8B3) were prepared and characterized as previously described (3). Furthermore, MAb 5A2 has been shown to react specifically with the laminin beta 1-chain and not beta 2 (14). Similarly, MAb 8B3 binds specifically to laminin alpha 1-chain and does not recognize alpha 5 (16, 17). To define the MAb epitopes more precisely, we prepared and then screened a newborn mouse kidney lambda gt11 cDNA expression library with antibodies by using methods described before for preparing and screening a newborn rat kidney library with polyconal anti-laminin IgG (30). Positive colonies reacting with MAb 5A2 were then purified, expanded, and sequenced.

Further characterization of the laminin chain specificity of MAb 8B3 was carried out by binding of the antibody to cells transfected with laminin alpha 1-cDNA. In brief, the quail fibroblast cell line QT-6 (20) was transfected with a full-length mouse laminin alpha 1-cDNA expression vector (a gift from Peter Yurchenco, Robert Wood Johnson Medical School, Piscataway, NJ) or mock transfected. After 36 h, cells were rinsed in PBS, fixed in 1% paraformaldehyde with 100 mM L-lysine and 10 mM metaperiodate, rinsed again, and then permeablized with 1% Triton X-100. Cells were then incubated with MAb 8B3, washed, and treated with secondary antibody.

Chain-specific rabbit anti-laminin alpha 5 (18)- and guinea pig anti-laminin beta 2-antibodies (25) were characterized previously. Antibodies against nephrin (12) and glomerular epithelial protein 1 (GLEPP-1) (32) were gifts from Drs. Lawrence Holzman and Roger Wiggins, respectively (both at the University of Michigan, Ann Arbor, MI).

Experimental procedures. Timed pregnant mice at gestational days 12 and 13 (date of vaginal plug being day 0) and newborn mice were anesthetized and killed by cervical dislocation. For metanephric organ cultures, embryos were removed and kidneys were aseptically microdissected and cultured for 4-7 days (Table 1) as described before (24). In brief, metanephroi were placed in 12-well tissue culture dishes (Falcon, Oxnard, CA) containing 1:1 DMEM/Ham's F-12 medium (GIBCO-BRL Life Technologies, Grand Island, NY) supplemented with 5% FCS, penicillin G sodium, and streptomycin sulfate (both at 1,000 U/ml). Cultured metanephroi were incubated at 37°C in humidified air with 5% CO2. In some cases, wild-type and Flk1-lacZ kidneys that had been maintained in organ culture for 6 or 7 days were implanted into anterior eye chambers of C57BL/6J hosts as described before (24). Grafts of these previously cultured kidneys were then harvested 6 or 7 days later (Table 1).

                              
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Table 1.   Ages and numbers of kidneys examined

For immunofluorescence microscopy, kidney tissues were placed in optimal cutting temperature compound (OCT; Miles, Elkhart, IN) and frozen in isopentane chilled in a dry ice-acetone bath. Individual, serially cut cryostat sections, ~4 µm thick, were picked up on separate slides, air-dried, fixed in 100% methanol at 0°C for 10 min (in most cases), and washed in PBS. Sections were then incubated with 50 µg/ml rat anti-mouse laminin MAb 5A2 or 8B3, followed by goat anti-rat IgG-fluorescein. Other sections were labeled with rabbit anti-laminin alpha 5-chain, anti-GLEPP-1, or anti-nephrin, followed by goat anti-rabbit IgG-fluorescein or -rhodamine (ICN Biomedicals, Costa Mesa, CA). Some sections were double labeled with both MAb 8B3 (anti-laminin alpha 1) and anti-laminin alpha 5-chain antibodies and appropriate fluorescein- and rhodamine-conjugated secondary antibodies. For labeling with anti-laminin beta 2-chain, cryostat sections were fixed with 4% paraformaldehyde (instead of 100% methanol) for 10 min at room temperature, blocked with 0.1 M glycine in PBS, and then treated with 0.1% SDS in PBS for 60 min at 50°C to expose the epitope. Sections were then incubated with guinea pig anti-laminin beta 2-chain, followed by goat anti-guinea pig IgG-rhodamine.

Serial sections of anterior chamber grafts of organ-cultured Flk1-lacZ kidneys were processed for immunofluorescence as described in Experimental procedures and for beta -galactosidase histochemistry as detailed before (24). Organ-cultured and cultured-grafted kidney tissues were also prepared for transmission electron microscopy as described previously (4).


    RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION
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Localization of the MAb 5A2 epitope to the NH2-terminal domain VI of the laminin beta 1-chain. Earlier work using Western blotting of porcine pancreatic and human neutrophil elastase digests of laminin had provided evidence that the 5A2 epitope resided on the E4 fragment of laminin, which corresponds to the NH2-terminal region of the beta -chain short arms (3, 29). Electron microscopy of rotary-shadowed immune complexes consisting of laminin and MAb 5A2 and rotary-shadowed eluates of laminin digests from MAb 5A2 affinity resins confirmed these results (3, 29). To define the 5A2 epitope more precisely, we prepared a newborn mouse kidney lambda gt11 cDNA expression library, and library screening with MAb 5A2 identified four immunoreactive clones. Computer sequence analysis throughout their open reading frames showed that all four clones corresponded precisely with published sequences for the laminin beta 1-chain (26) and encoded overlapping regions contained specifically within the NH2-terminal domain VI of the beta 1-chain short arm (Fig. 1). Clone 1a is 304 bp in length and is entirely included in clones 1b and 09. Clones 1b and 5 are 650 and 632 bp in length, respectively, and each contains 60-80 bp of the laminin beta 1-5' untranslated region, the 5' signal peptide, as well as the NH2 terminus of domain VI. Clone 09 is 487 bp in length and stretches from nucleotide +89 to +576 of laminin beta 1-chain domain VI. The alignment of the four clones is shown in Fig. 1. Together, they encode 162 of the 248 amino acids that constitute domain VI, which contains a mixture of short alpha -helices, beta -sheets, and random coils that form the NH2-terminal globular structure of the short arm of the laminin beta 1-chain (26).


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Fig. 1.   Diagram of laminin molecule showing location of region encoded by cDNA clones identified by anti-laminin monoclonal antibodies (MAb) 5A2. Clones 1b, 05, 09, and 1a all map to the NH2-terminal domain VI of the laminin beta 1-chain. The amino acid sequence in the overlap region among all 4 clones (dashed line) for the laminin beta 1-chain (accession number M15525) is shown and compared with the corresponding region of the laminin beta 2-chain (accession number NM-008483), which is unreactive with MAb 5A2 (14). Areas marked by bars show sites of sequence divergence between these 2 chains. One of these possibly represents the specific beta 1-chain epitope recognized by MAb 5A2.

When the 102-amino acid consensus-overlapping sequence of laminin beta 1-chain recognized by MAb 5A2 was compared with the sequence in the corresponding region of laminin beta 2 (10), a striking level of homology was observed (Fig. 1). Areas of sequence divergence were clustered within just three sites, each consisting of five to seven amino acids. Because MAb 5A2 does not crossreact with the laminin beta 2-chain (14), we speculate that one of these sites on the laminin beta 1-chain represents the core epitope for this MAb. At present, we are carrying out additional studies to confirm this possibility. Nevertheless, these results are entirely consistent with our earlier immunomap of the MAb 5A2 epitope on laminin and show that this MAb recognizes the NH2-terminal domain VI on the laminin beta 1-chain, specifically.

Confirmation of the chain specificity of MAb 8B3. Screening of the newborn mouse kidney lambda gt11 expression library with MAb 8B3 was unsuccessful and failed to produce any immunoreactive clones. To provide additional evidence that this MAb binds specifically to the laminin alpha 1-chain, we therefore undertook a strategy similar to what was originally used to demonstrate that MAb 5A2 is specific for laminin beta 1-chain (14). In this case, quail fibroblasts were transfected with a full-length mouse laminin alpha 1-cDNA expression vector. Cells transfected with the laminin alpha 1-cDNA reacted intensely with MAb 8B3, whereas mock-transfected cells did not (Fig. 2). Together with evidence presented earlier (16, 17), this confirms that MAb 8B3 binds specifically to the laminin alpha 1-chain.


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Fig. 2.   Quail fibroblasts mock transfected (a) or transfected with mouse laminin alpha 1-chain cDNA (b). When cells transfected with anti-laminin alpha 1-chain cDNA are incubated with MAb 8B3, intense immunofluorescence is seen (b). No fluorescence occurs in mock-transfected control cells incubated with MAb 8B3 (a).

GBM laminin isoform transitions in vivo. There are striking transitions in laminin distribution within the GBM during normal glomerular development in vivo (5, 15) (Figs. 3 and 4). Specifically, labeling for the laminin alpha 1-chain is abundant in early GBMs within vascular clefts of comma- and S-shaped figures, but it completely disappears from GBMs of capillary loop-stage glomeruli (Fig. 3a). In contrast, double immunolabeling of the same sections showed that the alpha 5-chain, which is absent in vascular clefts, becomes abruptly prominent in early capillary loops (Fig. 3b). On the other hand, the laminin beta 1-chain is present in both vascular clefts and peripheral GBM of early capillary loop stage glomeruli (Fig. 4a). Laminin beta 2-chain is not expressed in vascular clefts, however, and becomes progressively detectable in GBMs of capillary loop-stage glomeruli (Fig. 4b). In later, maturing-stage glomeruli, laminin beta 1-chain disappears from the GBM but remains in mesangial matrices, whereas laminin beta 2-chain persists in GBMs of maturing glomeruli, but not mesangial matrices (5, 15).


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Fig. 3.   Double immunofluorescence image of cryostat section of newborn mouse kidney labeled with anti-laminin alpha 1-chain (a) and anti-laminin alpha 5-chain (b) primary antibodies, incubated with appropriate secondary antibodies, and examined with fluorescein and rhodamine filters, respectively. Anti-laminin alpha 1-chain labels vascular cleft (a, arrows) but is completely unreactive with glomerular basement membrane (GBM) of capillary loop stage glomerulus (a, *). In contrast, anti-laminin alpha 5-chain reacts intensely with capillary loop GBM (b, arrowheads) but does not detect vascular cleft GBM of early nephric figure (b, *).



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Fig. 4.   Separate cryostat sections of newborn mouse kidney labeled with anti-laminin beta 1-chain (a) and anti-laminin beta 2-chain (b). a: Anti-laminin beta 1-chain recognizes vascular cleft (arrows) and capillary loop-stage GBMs (arrowheads). b: Anti-laminin beta 2-chain reacts only with capillary loop-stage glomeruli (arrowheads), and GBMs of earlier nephric figures (arrows) are negative. C, capsule.

These results therefore show that the transition of laminin alpha -chains takes place earlier and occurs far faster than that for the laminin beta -chains. Whether the abruptly expressed laminin alpha 5-chain associates with the beta 1-chain synthesized at that time (together with the gamma 1-chain to form laminin-10), with the beta 2-chain (to form laminin-11), and/or with other laminin chains is not yet clear (11, 15, 18). Importantly, the fate of the original laminin alpha 1-chain, the process for its rapid and selective removal from the nascent GBM, and the molecular control of the upregulation of the laminin alpha 5-chain are also unknown.

Laminin synthesis in metanephric organ cultures. When E12 mouse kidneys were placed in organ culture, organotypic tubulogenesis and glomerular epithelial tuft formation occurred under the conditions used here (Fig. 5). Standard organ culture conditions do not support endothelial differentiation, however, and the glomerular structures that developed in vitro appeared avascular (Fig. 5), which has been observed repeatedly (6, 7, 22, 23, 24). When sections of organ cultures were labeled with anti-GLEPP-1 and anti-nephrin antibodies, the glomerular epithelial tufts were positive for both of these podocyte-specific differentiation markers (Fig. 6) that are normally first expressed at early capillary loop stages (12, 32). Additionally, when cultures were examined by electron microscopy, a substantial amount of podocyte development was observed, replete with the formation of foot processes (Fig. 7) spanned by slit diaphragms (Fig. 8). Moreover, basement membrane material containing defined layers resembling typical laminae densae and laminae rarae were found beneath the developing foot processes (Fig. 8). These basement membrane structures were not organized into linear loops as they are in normal glomeruli, however, but instead occurred as segments or cords of material between podocytes (Figs. 7 and 8). These ultrastructural features therefore appear identical to those observed earlier when intact kidney rudiments or dissected metanephric blastema separated from ureteric buds are maintained in transfilter cultures with appropriate inducer tissues (6, 7, 22). Despite the extensive development and differentiation of podocytes in metanephric organ culture, sections of cultured Flk1-LacZ metanephroi showed that there was no corresponding development of Flk1-positive endothelial cells within the glomerular epithelial tufts. The absence of defined endothelial cells, mesangial cells, and mesangial matrices was confirmed by electron microscopy (Figs. 7 and 8).


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Fig. 5.   Sections of embryonic day 12 (E12) kidney before (a) and after (b) 6 days in culture. Note that several "glomerular" epithelial tufts develop in organ culture (b, arrows) but no defined vascular structures are seen.



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Fig. 6.   Cryostat sections of E12 kidneys grown in culture for 6 days and labeled with anti-glomerular epithelial protein 1 (GLEPP-1) (a) and anti-nephrin antibodies (b). Both podocyte-specific markers of differentiation are expressed by the "glomerular" epithelial tufts in vitro.



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Fig. 7.   Electron micrograph of organ-cultured kidney showing glomerular epithelial tuft. A number of visceral epithelial cells resembling podocytes (Po) are seen, as are segments or cords of basement membrane-like material (arrows). Note cells corresponding to parietal epithelium of Bowman's capsule (BC). Magnification: ×5,700.



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Fig. 8.   Higher power electron micrograph from organ-cultured kidney showing presence of foot process-like interdigitations. Basement membrane material resembling the GBM is found beneath the foot processes (*). Structures corresponding to slit diaphragms can be seen between many of the foot processes (arrows). Magnification: ×38,000.

Earlier reports have shown that podocytes in metanephric organ culture express collagen type IV and laminin (22). To examine whether the GBM laminin chain isoform substitutions that normally take place in vivo might also occur in vitro, cryostat sections of E12/E13 kidneys that had been maintained in organ culture for 4-7 days were labeled with chain-specific anti-laminin antibodies. In sections that were doubly labeled for laminin alpha 1- and alpha 5-chains, glomeruli from organ-cultured kidneys often contained both chains, sometimes in the same basement membrane segments (Fig. 9). There were segments within the same glomeruli that contained laminin alpha 5-chain alone, however (Fig. 9). The basement membranes that formed within the glomerular epithelial tufts in vitro were not arranged in defined, linear loops as they are in vivo but instead were assembled into broad segments or cords (Figs. 7-9). Also in contrast to what occurs by the capillary loop stage of glomerular development in vivo, most glomeruli that formed in organ culture retained abundant amounts of laminin alpha 1-chain-positive basement membrane material (Fig. 9). Although both laminin alpha 1- and alpha 5-chain expression were observed within the same glomeruli, laminin beta 1-chain was expressed almost exclusively; little or no immunolabeling for laminin beta 2-chain was seen in glomeruli that formed in organ cultures (Fig. 9). Results from examination of sections taken from eight separate metanephric organ cultures are shown in Table 2, in which there was usually coexpression of laminin alpha 1- and alpha 5-chains and almost a complete absence of the laminin beta 2-chain. Taken together, these results indicate that, although considerable podocyte morphogenesis took place in organ cultures, including the expression of GLEPP-1 and nephrin, normal laminin isoform switching did not occur in this environment.


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Fig. 9.   Cryostat sections of E12 kidneys grown for 6 days in culture. Sections were doubly immunolabeled for laminin alpha 1- and alpha 5-chains, or serial sections were labeled for beta 1- and beta 2-chains, as indicated. Epithelial tufts that form in organ culture express both alpha 1- and alpha 5-chains, sometimes in the same basement membrane segments (arrows, top left and right). The laminin alpha 5-chain is seen in other segments exclusively (arrowheads, top right). In contrast, only laminin beta 1-chain is synthesized by glomerular epithlelial tufts in organ culture; laminin beta 2-chain is not detected in the same tufts.


                              
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Table 2.   Glomerular laminin chain expression in organ-cultured and cultured-grafted metanephroi

Laminin synthesis in organ-cultured and then grafted metanephroi. To assess the laminin isoform switching patterns that might occur in anterior chamber grafts, we dissected E12 kidneys and implanted these into anterior eye chambers for 6 and 7 days (Table 1). In all cases, the normal laminin substitution sequences occurred: laminin alpha 1-chain was replaced by laminin alpha 5-chain, and the joint expression of laminin beta 1- and beta 2-chains were seen in capillary loop GBMs. When E12 and E13 kidneys that had first been grown in organ culture for 6 or 7 days were grafted into anterior chambers and examined for glomerular laminin expression, results from double-labeled sections also showed that, in most glomeruli, laminin alpha 1-chain was almost entirely replaced by laminin alpha 5 (Fig. 10, a and b, and Table 2). Additionally, capillary loop-stage glomeruli within grafts also usually contained both laminin beta 1- and beta 2-chains (Fig. 10, c and d, Table 2), as is normally the case at this stage of development. In other words, laminin isoform switching, which was arrested in organ cultures, appeared to proceed normally after organ-cultured kidneys were grafted into anterior chambers.


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Fig. 10.   Immunofluorescence of E12 kidneys that were first grown in organ culture for 6 days and then grafted into anterior eye chambers. Grafts were harvested 6 days later. a and b: Double immunofluorescence image from the same section labeled with anti-laminin alpha 1- and alpha 5-chain antibodies, as indicated. After grafting, laminin alpha 1 disappears from capillary loop-stage glomeruli, whereas laminin alpha 5-chain is abundant, as occurs normally (cf. Fig. 3). c and d: Serial sections treated separately with anti-laminin beta 1- and beta 2-chain antibodies, as indicated. Note that the same 4 capillary loop-stage glomeruli (*) express both laminin beta 1- and beta 2-chains in normal patterns (cf. Fig. 4).

A role for endothelial cells in GBM laminin isoform substitution? Work we carried out previously with Flk1-lacZ kidneys has shown that metanephric mesenchymal cells expressing Flk1, which we operationally defined as angioblasts, no longer express Flk1 in organ culture (24). This may explain, in part, why kidney microvessels and glomerular endothelial cells fail to develop under typical culture conditions. On the other hand, when avascular, organ-cultured kidneys are then grafted into anterior eye chambers, Flk1 expression by endothelial progenitors is restored, and glomerular and microvascular endothelial cells develop organotypically within the grafts (24). To evaluate a possible role for glomerular endothelial cells in mediating laminin isoform switching, kidneys from E12 Flk1-lacZ mice were cultured for 7 days and then grafted into anterior eye chambers of wild-type host mice. Grafts were harvested after 6 or 7 days of growth in oculo (Table 1) and frozen. Because conditions for beta -galactosidase histochemistry were incompatible with the immunofluorescence procedures required for this particular set of antibodies, separate, serial sections were processed for beta -galactosidase and immunofluorescence.

The three panels shown in Fig. 11 reflect the distribution of lacZ-positive glomeruli (indicating the presence of endothelial cells expressing Flk1) in serial section 7, and laminin alpha 1- and alpha 5-chains, respectively, in the doubly immunolabeled serial section 10. Several glomerular structures containing endothelial cells (Fig. 11c) correspond precisely to those in which there are marked absences of laminin alpha 1-chain (Fig. 11a) but the presence of laminin alpha 5-chain (Fig. 11b). Similarly, when serial sections were processed for laminin beta 1- and beta 2-chain immunolabeling and lacZ histochemistry, glomeruli containing Flk1-positive endothelial cells coincided exactly with those in which laminin beta 1- and beta 2-chains were both expressed (Fig. 12).


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Fig. 11.   Serial sections of cultured and grafted Flk1-lacZ kidney. a: Serial section 10 (s10) immunolabeled for laminin alpha 1-chain and viewed with the fluorescein channel. b: s10 immunolabeled for laminin alpha 5-chain and viewed with rhodamine. Note that between a and b, GBMs within 5 glomeruli (arrows) do not express laminin alpha 1-chain but do express laminin alpha 5-chain, reflecting the normal pattern of laminin transition in capillary loop-stage glomeruli. Tubular basement membranes that selectively express the laminin alpha 1-chain (double arrows in a) and laminin alpha 5-chain (double arrows in b) can also be discriminated. art, arteriole. c: Serial section 7 (s7), developed for lacZ histochemistry, which reveals sites of Flk1 transcription and thereby identifies endothelial cells. Four glomeruli are found in the same location of the section as those seen above (arrows), and each contains endothelial cells. A fifth glomerulus (arrowhead) is also seen, but there are no corresponding stuctures seen in the later sections in the series (a and b) that were immunolableled. Similarly, a glomerulus that is immunolabeled in a and b (right of center) cannot be resolved by beta -galactosidase staining in c. Also, note in c a cross section through an arteriole. This serves as an additional reference point for the arteriolar tissue observed in b, coursing through the bottom portion of the serial section.



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Fig. 12.   Serial sections of a cultured-grafted Flk1-lacZ kidney. a: Section 15 (s15) immunolabeled for laminin beta 1-chain. b: Section 14 (s14) immunolabeled for laminin beta 2-chain. c: Section 13 (s13), processed for lacZ histochemistry. A glomerulus containing endothelial cells also expresses both laminin beta 1- and beta 2-chains in capillary loop GBM.

Not all glomeruli viewed in the serial sections that were processed for immunofluorescence were also detected in sections developed for Flk1-lacZ, however, and a few lacZ-positive glomeruli were absent in the serial sections processed for immunofluorescence (cf Fig. 11). We attribute these disparities to the unavoidable differences that exist from section to section. Had we been successful in processing the same sections for Flk1-lacZ and for double-label immunofluorescence, we might have been able to distinguish between nonvascularized and vascularized glomeruli and could have determined definitively whether correct laminin isoform switching occurred only in glomeruli containing endothelial cells. On the other hand, almost all of the glomeruli that develop in anterior chamber grafts of cultured kidneys are vascularized, and avascular glomeruli are rare (23, 24).

To evaluate glomeruli that developed in organ-cultured-grafted kidneys in more detail, grafts were examined by electron microscopy. As shown in Fig. 13, perfused glomerular capillary loops containing erythrocytes were lined by a distinct endothelial cell layer. The overall glomerular architecture in these cultured-grafted kidneys was therefore strikingly similar to capillary loop-stage glomeruli that develop normally and very different from the avascular glomerular structures that form in organ cultures (Figs. 7 and 8 ). Of interest, a few tubular basement membranes within the grafts were intensely positive for the laminin alpha 1-chain, and others were selectively positive for the laminin alpha 5-chain (Fig. 11). These probably represent proximal and distal tubular segments, respectively (15, 18), and are further indicators of the level of nephron differentiation that is achieved in this ectopic, anterior chamber grafting site.


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Fig. 13.   Electron micrograph of glomerular capillary wall from an E12 kidney that was grown in organ culture for 6 days and then grafted into anterior eye chamber. The graft was fixed for microscopy 6 days after implantation. An endothelial cell layer (En) lines the capillary, and is closely apposed by overlying podocytes (Po). The capillary is perfused by erythrocytes (E). Magnification: ×9,700.

In summary, routine organ culture conditions support the differentiation of avascular glomerular tufts containing well-differentiated podocytes. In our analysis, these cells expressed the podocyte-specific differentiation markers GLEPP-1 and nephrin and assembled epithelial slit diaphragms and lengths of GBM-like material. Although podocytes that develop in organ culture displayed many features of their normal counterparts, the basement membrane material they assembled was compositionally distinct from normal GBM. Specifically, substitution of laminin alpha 1-chain by alpha 5-chain was incomplete, and the laminin beta 2-chain was not installed. Once avascular, organ-cultured kidneys were grafted into anterior eye chambers, however, normal laminin isoform-switching patterns were largely restored: the laminin alpha 1-chain disappeared from capillary loop-stage glomeruli, and GBMs contained both the beta 1- and beta 2-chains. These results correlated with the direct identification of graft-derived glomerular endothelial cells within the same glomeruli in which appropriate laminin isoform substitution occurred. Taking all the data together, we conclude that correct GBM laminin isoform switching occurred only when glomeruli became vascularized.

Between the organ culture and anterior chamber environments, there are many potentially important differences affecting kidney development, and we recognize that neither environment replicates normal metanephrogenesis in situ. Nevertheless, three of the most prominent differences are the absence in organ cultures of 1) vascular endothelial cells, 2) blood-borne growth factors/chemokines and other signaling molecules, and 3) biophysical effects contributed by hemodynamic pressures and glomerular ultrafiltration. Although all three of these parameters are present within grafts of organ-cultured kidneys, we do not know which one figures most importantly in mediating basement membrane assembly. Clearly, the grafts become well perfused, but the renovascular dynamics in the anterior eye chamber grafts are likely to be far different than in kidneys that develop in the normal, abdominal location. Specifically, given that vessels that deliver blood into the grafts originate chiefly from the iris, the glomerular filtration pressures in the grafts are a small fraction of what they are in normal kidneys. Although glomeruli in grafts are forming a modest ultrafiltrate, there is, of course, no elimination of the filtrate by normal mechanisms through the ureter. Hence there are very different physiological conditions that exist between glomeruli that form in oculo from those that develop normally; yet, in both cases, GBM laminin isoform transitions occur correctly. As a consequence, physiological, hemodynamic parameters are probably not major determinants mediating GBM laminin synthesis. This leaves circulating growth factors and/or endothelial cells themselves as the most likely candidates, and we believe that they both are probably crucial.

Identification of specific growth factors and other substances that selectively direct endothelial cell development would be very important to achieve. As seen earlier (23, 24), and observed here again as well, the glomerular endothelial cells that differentiate in the grafts are in fact derived from the engrafted kidney. Under conditions used here, the vascular endothelial growth factor receptor Flk1 is expressed by only a few metanephric cells in organ culture, and these do not establish a recognizable microvasculature in vitro (23, 24). Once avascular, organ-cultured kidneys are grafted, however, there is marked upregulation of Flk1 within the graft, and some of these cells go on to assemble the glomerular endothelium. The molecular signals for this Flk1 upregulation on grafting are not known, and multiple signaling pathways are undoubtedly involved. In addition, we suspect that at least some of the signals are derived from the circulation, but this may be a very difficult question to resolve further at present.

How might glomerular endothelial cells affect GBM laminin isoform substitution? First, endothelial cells may be a key biosynthetic source for certain laminin isoforms. Earlier work carried out before the advent of isoform- and chain-specific anti-basement membrane protein antibodies have clearly shown that endothelial cells of immature glomeruli, in conjunction with podocytes, secrete laminin-1, type IV collagen, and entactin (1, 8). Whether glomerular endothelial cells synthesize the separate laminin-1 and laminin-11 chains examined here, and whether they are the sole source for these chains, now needs to be determined. Second, endothelial cells (possibly along with circulation-derived factors) may provide signals that stimulate podocytes to alter their laminin synthetic programs, which then lead to the appearance of new GBM laminin isoforms. As mentioned earlier, what becomes of the laminin isoforms laid down during initial stages of glomerular development is not known, but endothelial cells might play a role in GBM turnover as well. Perhaps additional experiments with the organ culture-grafting system used here, which stops and then restarts kidney endothelial cell development, may provide new insights into mechanisms governing glomerular endothelial cell differentiation and function. We are presently using this and other technologies with the hope of understanding more about how the endothelial and podocyte cell layers each help establish and maintain the glomerular capillary wall.


    ACKNOWLEDGEMENTS

We thank Eileen Roach for expert technical help, Dr. Peter Yurchenco for the mouse laminin alpha 1-chain cDNA, and Drs. Lawrence Holzman and Roger Wiggins for antibodies against nephrin and GLEPP-1, respectively.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34972 and DK-52483.

Address for reprint requests and other correspondence: D. R. Abrahamson, Dept. of Anatomy and Cell Biology, Univ. of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7400 (Email: dabrahamson{at}kumc.edu).

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.

Received 21 September 2000; accepted in final form 5 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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