Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs.

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated (22)Na uptake by 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 microM. Kinetic analyses showed that the extracellular Na+ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+ with a half-maximal activation value of pK 6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains.

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells.

BACKGROUND: Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited. METHODS: We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested. RESULTS: Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1-expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent. CONCLUSIONS: Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.

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

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.

Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.

BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms.

Functional and immunohistochemical studies were performed to localize and identify Na(+)/H(+) exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pH(i)) was measured with fluorescence microscopy by using 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na(+)-dependent pH(i) recovery from an acid load imposed by prior lumen and bath Na(+) removal. Removal of Na(+) from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pH(i). This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC(50) = 9.0 and 247 microM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC(50) = 0.86 and 7.6 microM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1-4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na(+) transport, pH(i), and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.

Nephrin localizes to the slit pore of the glomerular epithelial cell.

BACKGROUND: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein. METHODS: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed. RESULTS: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. CONCLUSION: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.

Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase.

Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.

Origins and formation of microvasculature in the developing kidney.

Regulation of microvessel assembly in the developing kidney is not known and may occur through vasculogenic, angiogenic, or both processes. To examine this question, we grafted rat and mice embryonic (E) day 12 (E12) kidneys, which have only a rudimentary vasculature, into anterior eye chambers of mouse and rat hosts. Species-specific, monoclonal anti-basement membrane antibodies showed that glomerular basement membranes, mesangial matrices, and microvessel basement membranes were always derived from the graft. When wild-type E12 mouse kidneys were grafted into anterior chambers of ROSA26 mice, in which the beta-galactosidase transgene is expressed ubiquitously, glomerular and microvascular endothelial cells stemmed from the graft, even after maintenance of kidneys in organ culture for 6 days before grafting. Immunolabeling with antibodies against the vascular endothelial growth factor (VEGF) receptor, Flk1, the EphB1 receptor, and its ligand, ephrin-B1, labeled discrete mesenchymal cells in embryonic and newborn kidney cortex, as well as developing microvessel and glomerular endothelium. In adult kidneys, Flk1 labeled glomeruli weakly, other vascular structures were unlabeled. When wild-type E12 kidneys were grafted under renal capsules of adult ROSA26 hosts, endothelium developing within the graft again came from the implanted kidney. In contrast, when E12 kidneys were grafted into renal cortices of newborns, glomeruli within grafts now contained host-derived endothelium. Similarly, when ROSA26 E12 kidneys were implanted into newborn wild-type hosts, chimeric vessels containing graft- and host-derived endothelium were seen in nearby host tissue. Our results indicate that cells capable of forming the entire microvascular tree of grafted metanephroi are already present in the E12 kidney. We hypothesize that Flk1/VEGF and EphB1/ephrin-B1 mediate renal endothelial mitosis-motility and cell guidance-aggregation behavior, respectively.

Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice.

Flk1, a receptor tyrosine kinase for vascular endothelial growth factor (VEGF), is the earliest known marker for endothelial precursors (angioblasts). We examined heterozygous mice in which the Flk1 gene was partially replaced by a promoter-less LacZ insert and used beta-galactosidase histochemistry to view cells transcribing Flk1. In day 10 (E10) embryos, a Flk1-positive network surrounded the metanephric blastema, and, at E11, a vessel entered the metanephros from its ventral aspect alongside the ingrowing ureteric bud. However, aortic branches did not engage embryonic kidneys at these time points. In newborns, beta-galactosidase was localized exclusively and intensely to endothelial cells of all vessels and glomeruli. In contrast, when E12 kidneys grown in organ culture for 6 days were examined, only scattered Flk1-positive cells were seen, glomeruli were unlabeled, and vessels were absent. When organ-cultured kidneys were then grafted into wild-type anterior eye chambers, numerous Flk1-positive endothelial cells in vessels and glomeruli were found, all stemming from the graft. Image analysis showed that grafts with the most abundant glomerulo- and tubulogenesis were also those with the richest expression of Flk1. We conclude that 1) kidney microvessels precede renal artery development, 2) angioblast differentiation is arrested in organ culture but released on grafting when vasculogenesis resumes, and 3) nephrogenesis and microvessel assembly are tightly coupled in vivo.

Binding of injected laminin to developing kidney glomerular mesangial matrices and basement membranes in vivo.

During glomerular development, subendothelial and -epithelial basement membrane layers fuse to produce the glomerular basement membrane (GBM) shared by endothelial cells and epithelial podocytes. As glomeruli mature, additional basement membrane derived from podocytes is spliced into the fused GBM and loose mesangial matrices condense. The mechanisms for GBM fusion, splicing, and mesangial matrix condensation are not known but might involve intermolecular bond formation between matrix molecules. To test for laminin binding sites, we intravenously injected mouse laminin containing alpha1-, beta1-, and gamma1-chains into 2-day-old rats. Kidneys were immunolabeled for fluorescence and electron microscopy with domain-specific rat anti-mouse laminin monoclonal antibodies (MAbs), which recognized only mouse and not endogenous rat laminin. Intense labeling for injected laminin was found in mesangial matrices and weaker labeling was seen in GBMs of maturing glomeruli. These patterns persisted for at least 2 weeks after injection. In control newborns receiving sheep IgG, no binding of injected protein was observed and laminin did not bind adult rat glomeruli. To assess which molecular domains might mediate binding to immature glomeruli, three proteolytic laminin fragments were affinity-isolated by MAbs and injected into newborns. These failed to bind glomeruli, presumably owing to enzymatic digestion of binding domains. Alternatively, stable incorporation may require multivalent laminin binding. We conclude that laminin binding sites are transiently present in developing glomeruli and may be functionally important for GBM assembly and mesangial matrix condensation.

Diabetes induces changes in glomerular development and laminin-beta 2 (s-laminin) expression.

Offspring of diabetic mothers have developmental renal abnormalities; thus, we investigated the effects of the diabetic milieu on kidney development. Four groups of host rats, including insulin-deficient and insulin-treated streptozotocin-induced diabetic rats, normal controls, and insulin-treated nondiabetic rats, were prepared. After 38 days, rats received ocular implants of E14 fetal rat kidneys. Nine days later the fetal kidney grafts were harvested for analysis of glomerular development and expression of fibronectin, laminin, laminin-beta 2, and alpha-smooth muscle actin and m170, two additional markers of mesangial maturation. The rate of glomerular maturation was delayed in grafts placed in hyperglycemic, insulin-deficient diabetic rats. These glomeruli contained few mesangial cells or matrix, and laminin-beta 2 expression was reduced as compared with controls. Mesangial expression of alpha-smooth muscle actin and m170 was not detected. In contrast, grafts placed in insulin-treated diabetic animals had increased numbers of mesangial cells and expanded mesangial matrix. The content of laminin-beta 2 and expression of m170 and alpha-smooth muscle actin were also increased in these grafts. These data show that hyperglycemia and insulin status influence laminin isoform expression and play important roles in mesangial development.

Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules.

We have characterized the Na+/H+ exchanger (NHE) isoforms expressed in rat renal cortical tubule fragments. Amiloride sensitivity of the Na(+)-dependent intracellular pH (pHi) recovery in suspended tubules that had been acid loaded by an NH4+ prepulse was determined in nominally CO2/HCO3(-)-free solution, using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In the presence of 140 mM extracellular Na+, 800 microM amiloride inhibited the rate of Na(+)-dependent pHi recovery by only 65%, demonstrating the presence of a Na(+)-dependent amiloride-insensitive H+ extrusion system. This system was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Lowering extracellular Na+ concentration permitted 300 microM amiloride to completely inhibit Na(+)-dependent pHi recovery. These results can be explained by the expression of a Na+/H+ exchange with the pharmacological properties of NHE4. Using reverse transcriptase-polymerase chain reaction, we found specific mRNA for NHE1, NHE2, NHE3, and NHE4 isoforms in the renal cortex. Immunohistochemical studies using polyclonal antibodies against rat NHE4 peptide demonstrated that NHE4 is heterogeneously expressed on basolateral membrane domains of cortical tubules. These results strongly suggest that amiloride-insensitive Na+/H+ exchange expressed in renal cortical tubule suspensions is mediated by NHE4.

Human neutrophils do not degrade major basement membrane components during chemotactic migration.

At sites of inflammation, circulating neutrophils (PMNs) migrate through microvessel walls into the subendothelial interstitium. While endothelial passage is mediated by adhesion proteins, including those of the integrin, selectin and immunoglobulin superfamily classes, the mechanisms used to cross the subendothelial basement membrane (BM) are unclear. Studies examining tumour cell invasion and lymphocyte extravasation suggest several possible mechanisms, including proteolysis. Different cells, however, may use different mechanisms to effect passage. To examine neutrophil-basement membrane interactions in more detail, human PMNs were embedded within reconstituted BM (Matrigel) and used in migration assays. The integrity of the gel following migration was assessed by assaying for the release of incorporated radiolabelled products and by-immunoblotting for specific matrix molecule epitopes. PMNs migrated through Matrigel in response to the chemotactic peptide FMLP. Degradation products of laminin, heparan sulphate proteoglycan or of gelatin, however, were not detected. In contrast, phorbol ester, which triggers activation without migration, released approximately 40% of incorporated HSPG, 30% of gelatin and 20% of laminin as intact molecules or degraded fragments. Electron microscopy of migrating cells demonstrated pseudopodia associated with channels within the Matrigel. Although the serine proteinase inhibitor DFP, plasma and a specific anti-neutrophil elastase IgG blocked degradation, these agents failed to inhibit migration. Migration was inhibited, however, when the Matrigel concentration was increased to 10 mg/ml. Thus, although PMNs will degrade matrix components they do not do so during migration, and proteolytic remodelling of the BM is not a pre-requisite for neutrophil passage.

ELK and LERK-2 in developing kidney and microvascular endothelial assembly.

Eph family receptor tyrosine kinases direct neuronal cell targeting, bundling and intercellular aggregation activity, yet their role in mammalian kidney development has been unexplored to date. We recently identified expression of ELK (Eph-like kinase) receptors in cultured human renal microvascular endothelial cells (HRMEC), and showed that ELK mediates their in vitro assembly into capillary-like structures in response to the exogenous ligand, LERK-2. Here we identify expression of the ELK ligand, LERK-2, in HRMEC and in primitive vascular structures of developing murine kidney. ELK and LERK-2 are expressed on endothelial progenitor cells of primitive microvasculature in a pattern similar to that of the VEGF receptor, flk-1. ELK LERK-2 and flk-1 antigens are also displayed on the branching ureteric bud epithelium; ELK and LERK-2 expression persists in mature collecting ducts, glomeruli and arterioles. To explore whether renal-derived endothelial cells may distinguish LERK-2 from the angiogenic Eck ligand, LERK-1 (B61), and whether endothelial cells from different sources may distinguish among Eph receptor ligands, we compared HRMEC and human umbilical vein endothelial cell (HUVEC) responses in an in vitro capillary-like assembly assay. HRMEC endothelial cells assembled capillary-like structures in response to LERK-2, but not LERK-1, under conditions that promoted HUVEC to assemble in response to LERK-1, but not LERK-2. Therefore, responses mediated through specific Eph family receptors (ELK and Eck) are discriminated by endothelial cells from different vascular bed sources. ELK and its ligand, LERK-2, are spatially and temporally coordinated in expression and may function in morphogenesis of the renal microvasculature.

Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts.

Renal glomerular capillary tufts have been believed to arise from angiogenic ingrowth of extrinsic vessels. We found, however, that when embryonic day 12 (E12) mouse kidneys were maintained in culture for 6 days and then grafted into anterior eye chambers of adult transgenic ROSA26 host mice (which carry the beta-galactosidase transgene), glomerular endothelial cells within the grafts were predominantly of intrinsic, kidney origin. To identify potential endothelial precursors, we immunolabled kidneys with antibodies against the vascular endothelial growth factor receptor, flk-1. Numerous discrete cells expressing flk-1 were scattered throughout the nephrogenic mesenchyme of both E12 and newborn kidneys, and with development these cells became concentrated in microvessels, glomerular vascular clefts, and glomerular tufts. In adults, flk-1 was weakly expressed in glomeruli but absent elsewhere. To examine abilities of flk-1-positive cells to establish glomeruli, E12 kidneys were grafted into kidney cortices of adult and newborn ROSA26 hosts. Grafts into adults resulted in few glomeruli containing host-derived endothelium, whereas a majority of glomeruli grafted into newborns contained host cells. Cells of graft origin were found in vessels forming in renal cortices of newborn hosts, but not in adults. Our findings indicate that embryonic kidney cells expressing flk-1 are angioblasts that create microvessels and glomeruli by vasculogenesis.

Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys.

To address origins of glomerular endothelial and mesangial cells in embryonic mammalian kidneys, we established interspecies grafts between rats and mice, in which fetal kidneys were implanted into the anterior eye chamber of adult hosts. After 5-7 days, hosts bearing grafts received intravenous injections with species-specific monoclonal antibodies (MAbs) to matrix components. In all cases, glomerular basement membranes and mesangial matrices labeled solely for donor-derived matrix. Additionally, microvessel extracellular matrices within grafts were usually of donor origin. To examine directly the origin of glomerular endothelial and mesangial cells, we grafted embryonic gestational days 11-12 (E11-12) kidneys from normal mice into anterior eye chambers of host reverse-orientation splice acceptor 26 mice, which are transgenic animals that express beta-galactosidase in every cell. When grafts were developed for beta-galactosidase activity, host cells were seen in peripheral vessels, but the majority of glomerular endothelial cells were of donor, not host, origin. Where host-derived-endothelial cells were found in glomeruli, donor endothelial cells were present as well. Mesangial cells were always of donor origin. When E11 mouse kidneys were labeled with the endothelial cell-specific Bandeiraea simplicifolia isolectin B4, we determined that endothelial cells are present from the inception of metanephrogenesis. Together, the evidence shows that cells of endogenous kidney origin were almost entirely responsible for development of the glomerular microvasculature in oculo. External vessels from the host, although important for graft maintenance, were not major contributors to the glomerulus.

Carboxy terminal sequence and synthesis of rat kidney laminin gamma 1 chain.

We used antibodies against mouse Englebreth-Holm-Swarm (EHS) tumor laminin to screen a newborn rat kidney lambda gt11 expression library and isolated three overlapping cDNA clones, termed 2b-11 (401 bp), 10-b7 (779 bp), and 2a (2,095 bp). DNA sequence analysis identified these cDNAs as encoding much of the carboxy terminal domain I/II of laminin gamma 1 chain (formerly referred to as B2e), and 1436 bp of the 3' untranslated region. In situ hybridization of fetal (E15) rat sections localized laminin gamma 1 chain mRNA primarily to meninges of the brain, auditory and peripheral nerve fibers, gastrointestinal system, and developing lung airway epithelium. Intense hybridization was also found in early nephric structures and glomeruli of fetal kidneys. In kidneys of three-day-old rats, hybridization persisted over early nephric figures, developing glomeruli, and collecting ducts, but considerably less hybridization was seen over tubules. On Northern blots of neonatal kidney RNA, the three cDNA clones hybridized to two species of 7.5 and 5.5 kb, suggesting that developing rat kidney laminin gamma 1 mRNAs are processed using two different polyadenylation signals.

Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6.

The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the developing glomerulus by immunolocalization and metabolic labeling. The localization of p51 was compared with that of ZO-1, a component of the cytoplasmic face of the epithelial slit diaphragm, and with that of podocalyxin, and apical marker of the podocyte. p51 first became faintly, but clearly, detectable on the basal and lateral sides of the developing podocytes at the S-shaped body stage. Staining intensity increased with further maturation and was restricted to the visceral epithelial cells. On immunoelectron microscopy, the antigen was seen along the basal and lateral surfaces below occluding junction at the early capillary loop stage and later, with the interdigitation of foot processes, became concentrated in the slit pores. At no stage was p51 seen on the apical surface. p51 and ZO-1 were closely localized in the mature glomerulus but arrived at their final positions from opposite directions. p51 was on basal and podocalyxin was on apical sides of the glomerular epithelium from the S-shaped body stage onwards. Metabolic labeling studies showed that p51 is actively synthesized during initial glomerular development and that the rate of synthesis declines substantially with maturation. We conclude that p51 is primarily synthesized during the initial glomerular development, becomes concentrated in the slit pores of mature podocytes, and serves as a basal differentiation marker for podocytes.

Cloning and biological function of laminin in Hydra vulgaris.

The Cnidarian, hydra, lends itself to studies related to the role of extracellular matrix (ECM) components in development because of its high regenerative capacity and its simple structure, which is organized as an epithelial bilayer with an intervening ECM termed the mesoglea. Previous immunocytochemical and biochemical studies have established that hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan) associated with the ECM of vertebrate and more complex invertebrate species. Additional studies have also established that ECM components have a critical role in hydra development as monitored during head regeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)+ RNA isolated from Hydra vulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studies have shown that mAb52 blocks hydra cell aggregate development and experiments in the current study have shown that mAb52 also blocks in vivo interstitial cell (I-cell) migration in hydra grafts. Sequence analysis of cDNA clones isolated using mAb52 indicated that the protein encoded by these clones had structural homology with mammalian and Drosophila laminin B1 chain and hybridized to a single 6.75-kb band on Northern blots of total hydra RNA. One interesting difference in hydra laminin B1 was the presence of a FTGTQ amino acid sequence in place of the vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same > 200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the subepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bound to a single band with a mass of approximately 200 kDa. In addition, FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesoglea and anti-FTGTQ antibodies inhibited hydra cell binding to substrates coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also inhibited in vivo I-cell migration in hydra grafts. Given the early divergence of Cnidarians during evolution, these studies indicate the highly conserved nature of laminin and provide additional information regarding the critical role of ECM components during hydra development.