Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney.

The distribution of beta 1 and beta 3 integrins was studied in fetal and adult human kidneys by indirect immunofluorescence microscopy. In the developing kidney, the cells of the undifferentiated metanephric blastema displayed strong cell surface-confined beta 1 integrin immunoreactivity, whereas the cells of primary vesicles and comma- and S-shaped bodies reacted more weakly. In mature fetal as well as adult glomeruli, beta 1 integrins were distinctly localized, apparently confining to the basal cell surfaces of endothelial cells and podocytes abutting the glomerular basement membrane. In adult proximal tubules, beta 1 integrin immunoreactivity was strictly confined to the basal aspect of the epithelial cells, being absent laterally, which is unusual for membrane proteins of polarized epithelial cells. A more diffuse overall immunoreactivity was seen in distal tubules and collecting ducts. The epithelial cells of developing proximal and distal tubules displayed an overall distribution of beta 1 integrins. In each case, talin immunoreactivity followed that of beta 1 integrins. Compared with beta 1 integrins, beta 3 integrins showed a more restricted distribution, and differences were seen in the reactions of mono- and polyclonal antibodies. In developing glomeruli, beta 3 integrin immunoreactivity was prominently seen in the cells of Bowman's capsule, possibly revealing the presence of vitronectin receptor. Solitary cells, that reacted also with antibodies to the platelet glycoprotein IIb, were consistently detected in fetal glomeruli, suggesting the presence of megakaryocytes. The results show that during nephrogenesis, beta 1 integrins become distinctly polarized both in glomerular endothelial cells and podocytes, as well as in the epithelial cells of proximal tubules.

Immunohistochemical and scanning electron microscopic study of cytokeratin distribution in the collecting tubule of the rat kidney.

Cytokeratin distribution in the collecting tubules (CTs) of the rat kidney was studied by immunohistochemistry and scanning electron microscopy (SEM). After preparation of sections from formalin-fixed, paraffin-embedded tissue for immunohistochemistry, the remaining tissue was prepared for SEM. The cut surfaces of the tissue were examined by SEM and compared with sections stained with anti-cytokeratin antibody. The immunostained sections revealed positive staining along the entire CTs. However, in addition to diffusely stained cells, unstained and partially stained cells were seen. The latter were not distributed in inner medullary CTs, whereas the diffusely stained cells were observed in cortical, outer medullary and inner medullary CTs. This immunohistochemical heterogeneity of cytokeratin reactivity was prominent in the outer medullary and cortical CTs. From this comparative study of immunostained sections and SEM specimens, it was concluded that the heterogeneously stained cells correspond to intercalated cells, whereas the diffusely stained cells represent most principal cells. These results suggest that the difference in cytokeratin density among CT cells may represent different functional states, at least in intercalated cells.

Alpha-1-antitrypsin in the renal tubular epithelium in patients with or without alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin (AAT) globules were incidentally demonstrated in the kidney tubules from the autopsy material in patients with PiMZ, PiZZ and PiMM phenotypes in our laboratory. This study was designed to study the pathological significance of this phenomenon. A total of nine cases were selected from the autopsy file, two patients with AAT PiZZ, four with PiMZ and three with PiMM were studied. Sections of liver and kidney were stained for AAT by indirect immunoperoxidase method. All of the PiMM patients showed positive reactions in most of the proximal and some of the distal tubular epithelium. Of the six cases with abnormal Pi, one failed to show any reaction, four showed weak positive staining in some of the proximal tubules and one with high serum AAT revealed relatively strong activity in most of the proximal tubules. These findings suggest that AAT is present in the renal tubular epithelium. Whether AAT globules are produced in the renal tubular cells or secondarily absorbed from the glomerular filtrate is unclear. The significance of stainable renal tubular AAT in normal subjects, in patients with abnormal protease inhibitor types and in patients with renal disorders, needs further clarification and study.

Early expression of desmosomal components during kidney tubule morphogenesis in human and murine embryos.

Developing kidneys of human and murine fetuses have been stained with monoclonal antibodies to desmosomal proteins 1 and 2 (desmoplakins) (dp 1&2), desmosomal glycoprotein 1 (desmoglein) and a polyclonal antiserum to desmosomal glycoproteins 2 and 3 (desmocollins). All three antibodies stain the mesenchymal condensates that represent the first stage in kidney tubule development, indicating that desmosomal antigens are expressed very early in tubule morphogenesis. Desmosomal antigens are continuously expressed throughout the developing tubule being concentrated at the apical and basal regions of the lateral membranes of cells. Staining is also present in both visceral and parietal membranes of the developing Bowman's capsule. In the mature tubule, desmosomal staining becomes restricted to a discontinuous apico-lateral ring around the cells. Staining is completely lost from the visceral membrane of the mature Bowman's capsule (the podocytes) but persists in the parietal membrane. At the condensate stage, staining for dp1&2 is much more intense than staining for simple epithelial keratin. Electron microscopy showed the presence of small (ca 0.1 microns) punctate junctions in the developing tubule. These may be immature desmosomes. No fully mature desmosomes such as are present in mature kidney were found. The results suggest that desmosomal proteins and glycoproteins are involved in the early development of adhesive contacts between cells of the kidney tubule. The changing pattern of antigen expression, the loss of desmosomal staining from the podocytes and the immaturity of junctions suggest that desmosomal adhesion is labile during tubule morphogenesis, perhaps in order to facilitate changes of cell-cell contact.

Cellular distribution of alpha B-crystallin in non-lenticular tissues.

alpha B-Crystallin is a subunit of alpha-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the cellular distribution of alpha B-crystallin in rat organs was examined in detail using immunohistochemistry. Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle's loop and medullary collecting duct of the kidney, Schwann cells of peripheral nerves, glia of the central nervous system, and decidual cells of the placenta. A close correlation with markers of oxidative activity suggests that alpha B-crystallin is expressed in cells that have high levels of oxidative function.

Evidence from functional and autoradiographic studies for the presence of tubular dopamine-1 receptors and their involvement in the renal effects of fenoldopam.

Evidence from receptor-ligand binding and biochemical studies seems to suggest the possible existence of tubular dopamine DA-1 receptors in the rat kidney. However, it is not yet clear whether these putative tubular DA-1 receptors are involved in the functional renal responses elicited during the administration of DA and DA receptor agonists. In the present study we have examined the renal effects of several doses of selective DA-1 receptor agonist fenoldopam in pentobarbital-anesthetized rats in an attempt to unmask a direct tubular DA-1 receptor-mediated diuresis and natriuresis. Additionally, we have performed receptor-ligand binding and autoradiographic studies to examine the presence and localization of DA-1 receptors in various regions of the rat kidney. At the highest dose studied (2 micrograms/kg/min), fenoldopam produced diuresis and natriuresis, which was accompanied by a significant decrease in blood pressure and also a significant increase in glomerular filtration rate. At 1 micrograms/kg/min, the diuretic and natriuretic effects of fenoldopam were observed in the absence of any changes in blood pressure and glomerular filtration rate, but there was a significant increase in renal blood flow. However, at 0.5 micrograms/kg/min, fenoldopam-induced natriuresis and diuresis was not accompanied by any changes in blood pressure, renal blood flow or glomerular filtration rate, implying a direct tubular effect. These effects of fenoldopam appear to be mediated via activation of DA-1 receptors, because they were antagonized by the selective DA-1 antagonist SCH 23390.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistological localisation of staphylococcal toxic shock syndrome toxin (TSST-1) antigen in sudden infant death syndrome.

A polyclonal antiserum to toxic shock syndrome toxin (TSST-1) and a standard immunoperoxidase technique were used on formalin fixed tissues from 50 cases of sudden infant death syndrome (SIDS) to determine if the syndrome was associated with bacterial infection. There was strong specific staining in the renal tubular cells in nine (18%) cases. A similar pattern of staining was seen in three of a series of 50 kidneys selected for comparison from a wide range of necropsy cases. The staining was finely granular within the cytoplasm of proximal convoluted tubular cells and diffuse in tubular cell nuclei. In an attempt to validate the staining pattern the immunoperoxidase technique was also performed on formalin fixed kidneys from rats which had been given intravenous injections of crude bacterial products containing TSST-1. These showed coarse granular cytoplasmic staining in proximal convoluted tubules with some diffuse nuclear staining. This pattern was not seen in controls injected with saline. These results indicate that TSST-1 might have a pathogenic role in some cases of SIDS.

Alterations of cytosolic calcium in LLC-PK1 cells induced by vasopressin and exogenous purines.

The regulation of cytosolic calcium in LLC-PK1 cells by various agonists was characterized. Arginine vasopressin (AVP, 100 nM) rapidly increased cytosolic calcium (Caf) measured with fura-2 from a basal level of 65 +/- 5 to 516 +/- 102 nM followed by a return to a plateau level of 128 +/- 18 nM. Similar responses to 100 nM lysine vasopressin were seen. AVP also increased adenosine 3',5'-cyclic monophosphate (cAMP) as previously documented for these cells. A V2-selective AVP analogue increased cAMP without affecting Caf, whereas two V1-receptor antagonists prevented the Caf response to AVP without altering the cAMP response. Increasing cellular cAMP with forskolin, cholera toxin, or stable cAMP analogues did not affect Caf or the response of Caf to AVP. Both adenosine and ATP produced large Caf transients at concentrations of 1-10 microM in both calcium-containing media and after acute chelation of medium Ca with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The A1-selective adenosine analogue, (R-phenyl-isopropyl)-adenosine, and the A2-selective analogue, 5'-(N-ethyl)-carboxamido-adenosine, both produced Caf responses similar to adenosine. The Caf responses to adenosine and its analogues but not to ATP were blocked by the adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Islet-activating protein, pertussis toxin, inhibited the Caf response to adenosine and enhanced the cAMP response to AVP. Responses to all agonists were demonstrable in greater than 80% of single cells studied by microfluorometry, and individual cells responded to multiple agonists. These studies indicate that the Caf and cAMP responses to AVP in the LLC-PK1 cell line involve separate receptors, and they document the presence in this cell line of at least two types of receptors for exogenous purines.

Measurement of element content in isolated papillary collecting duct cells by electron probe microanalysis.

A method was developed to measure the element content of freshly isolated papillary collecting duct (PCD) cells by electron probe microanalysis in a scanning electron microscope. After isolation, the cells were transferred onto a Thermanox support by centrifugation and the extracellular medium was removed by brief exposure to buffered ammonium acetate; cryofixation, freeze-drying, and coating with carbon followed. Under visual control in the scanning electron microscope the Na, Cl, K and P content of cell clusters (about 30 cells/cluster) was then measured by X-ray microanalysis. Cells incubated in control medium showed potassium:sodium ratios identical to those determined previously in cryosections of the same cells. In ouabain-treated cells sodium influx and potassium efflux was demonstrated. Potassium left the cells with a t1/2 of 21.7 min. The t1/2 of Na influx was 12.6 min for the first 15 min of incubation, whereafter further influx was markedly slower. Ouabain-induced sodium influx was inhibited 40% by amiloride. These results indicate that X-ray microanalysis can be applied to analyze the ion content of isolated cell clusters derived from the papillary collecting duct. Using ouabain and amiloride as inhibitors the suitability of the method to identify transport systems is demonstrated.

Cholinergic receptors in renal medullary collecting duct cells.

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.

Fine-needle aspiration biopsy sampling in renal transplantation: interstitial cellular infiltrates and major histocompatibility complex class-II antigen expression in renal tubular cells.

The dynamics of major histocompatibility complex (MHC) class-II (DR) antigen products in renal tubular cells and cellular infiltrations in the interstitium of the kidney following renal transplantation without immunosuppressive therapy are presented. DR antigen in 27 normal kidneys and in 18 of 19 kidneys transplanted as autograft showed no DR-antigen expression on their tubular cells. These DR-antigen-negative tubular cells could be induced to express the antigen during courses of untreated rejection both in primary (n = 6) and repeat (n = 4) allografts. Parallel to the increasing DR-antigen expression in the allograft, the autologous kidney expressed this antigen with the same time dependency and with the same intensity. Graft infiltrating cells were evaluated by daily fine-needle aspiration biopsy and core biopsy. Induction of DR antigen was associated with a significant infiltration of blastogenic cells and monocytes/macrophages in the allograft. During rejection of the allograft no cellular infiltrate was noted in the autograft, but during an initial time period after allograft removal cellular infiltrates were seen. Following both courses of rejection DR antigen returns to negative by 6-8 days as did blastogenic cells and monocytes/macrophages in the autograft interstitium. Inducible antigen expression in normally DR-antigen-negative allograft parenchymal cells during rejection is shown to depend on inflammatory cells in the graft. The antigen expression is not restricted to the tissue transplanted, but also affects autologous tissue of the host organism.

Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules.

A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis.

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid.

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.

Co-localization of erythrocyte Ca++-Mg++ ATPase and vitamin D-dependent 28-kDa-calcium binding protein.

We have previously shown that the human kidney distal convoluted tubule (DCT) contains epitopes of the human erythrocyte Ca++-Mg++ ATPase pump (J Clin Invest 80: 1225-1231, 1987). To determine whether vitamin D-dependent 28-kilodalton-calcium binding protein (28kDa-CaBP)and Ca++-Mg++ ATPase are present in the same cells of the human kidney, kidney tissue was examined for immunoreactivity with antibodies directed against these proteins. Double-label immunohistochemistry showed that a majority of the distal convoluted tubules contain epitopes to both of these proteins. portions of the distal nephron which were positive for 28kDa-CaBP did not show anti-Ca++-Mg++ ATPase antibody binding. All other portions of the nephron were negative for both proteins. Western blot analysis of kidney homogenates by 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), showed binding of an anti-Ca++-Mg ATPase monoclonal antibody to a major band of Mr = 140,000. Western blots of kidney homogenates by 10% SDS-PAGE also showed binding of an anti-28kDa-CaBP polyclonal antibody to a protein band at Mr = 28,000. Incubation of parallel blots from the same 10% gel with 45CaCl2 demonstrated that the Mr = 28,000 band binds calcium. This work demonstrates, for the first time, that epitopes of vitamin D-inducible 28kDa-CaBp and human erythrocyte Ca++-Mg++ ATPase pump are present in the same cells of the human kidney. Previous work in our laboratory has shown that 28kDa-CaBP binds calcium in a manner analogous to calmodulin, a known regulator of the erythrocyte Ca++-Mg ATPase pump (J Biol Chem, 1987).(ABSTRACT TRUNCATED AT 250 WORDS)

Anatomical relationship between kallikrein-containing tubules and the juxtaglomerular apparatus in the human kidney.

Current evidence suggests a functional and biochemical link between the renin and the kallikrein systems. The purpose of this work was to study the localization of kallikrein along the human nephron to elucidate whether there exists an anatomical base for such interrelation. Serial sections of human kidney tissue were stained by immunocytochemical methods with antisera against kallikrein. Kallikrein immunostaining was observed exclusively in segments of the distal nephron lying in the cortical labyrinths and forming arcades in its distal portion. Consistently the tubules containing kallikrein established a close anatomical relationship with the afferent arteriole of the juxtaglomerular apparatus providing an anatomical base for an interaction between the renin and kallikrein systems in the human kidney.

Immunocytochemical localization of band 3 protein in the rat collecting duct.

Band 3 protein is the major anion transport protein of the erythrocyte cell membrane where it catalyzes the exchange of HCO3- for Cl-. There is evidence that band 3 protein is present in the collecting duct of both the rat and rabbit kidney. We used colloidal-gold immunocytochemistry to determine the ultrastructural location of band 3 protein in the rat cortical (CCD) and outer medullary collecting ducts (OMCD). Kidneys of normal Sprague-Dawley rats were fixed by intravascular perfusion with 1% glutaraldehyde and embedded in Lowicryl K4M. Two polyclonal antibodies raised in rabbits were used as the primary antibody in separate experiments, one against the 43-kDa fragment of the cytoplasmic domain of human erythrocyte band 3 protein and the other against rat erythrocyte band 3 protein. This was followed by exposure to gold-conjugated goat anti-rabbit immunoglobulin G. Transmission electron microscopy revealed gold particles along the basal and lateral plasma membranes of all intercalated cells of the OMCD. In the CCD, the basal and lateral plasma membranes of the type A intercalated cells only were labeled with gold particles. The type B intercalated cells and principal cells were devoid of gold particles, as were all cells of the proximal tubule, the distal convoluted tubule, and the thick ascending limb of the loop of Henle. We conclude that a Cl(-)-HCO3- transporter is present in the basal and lateral plasma membranes of the intercalated cells in the OMCD and the type A intercalated cells in the CCD. These findings provide further evidence that these intercalated cells are involved in H+ secretion in the OMCD and CCD of the rat. We have no evidence for the presence of band 3 protein in the type B intercalated cells of the CCD, which supports the hypothesis that type B cells are functionally and structurally distinct from type A cells.

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.

Monoclonal antibodies against laminin A chain and B chain in the human and mouse kidneys.

Three monoclonal antibodies against laminin A and B chains derived from mouse EHS sarcoma were isolated and designated Lam 1, Lam 2, and Lam 3. Lam 1 recognized laminin A and B chains, whereas Lam 2 and Lam 3 reacted with only A chain. By using the immunofluorescence method, three monoclonal antibodies equally stained the human skin basement membrane and Descemet's membrane of cornea. In the kidney, however, these monoclonal antibodies showed the different distribution; Lam 1 stained the glomerular basement membrane, the mesangium, Bowman's capsule, and the tubular basement membrane in the mouse kidney. In the human kidney, Lam 1 stained the mesangium very slightly bud did not stain the glomerular basement membrane. Both Lam 2 and Lam 3 stained the mesangium, Bowman's capsule, and proximal tubular basement membrane in the mouse kidney. In the human kidney, however, Lam 2 stained the mesangium and slightly stained the glomerular basement membrane, although Lam 3 stained only the mesangium. Oxidation of the kidney sections by sodium periodate changed the staining pattern of Lam 2 and Lam 3 in the human kidney, whereas it did not change the staining pattern of Lam 2 and Lam 3 in the mouse kidney.