Prevention of mesangial sclerosis by bone marrow transplantation.

Previously we have shown that bone marrow (BM) transplantation (BMT) can attenuate progression of and even ameliorate mesangial sclerosis (MS) in Wt1-heterozygous mice. However, it is unclear whether BMT performed before the onset of disease will prevent the development of MS. To investigate whether intravenous (i.v.) or intrarenal (i.r.) administration of BM have equal effects on the progression of MS in Wt1-heterozygous mice, young Wt1-heterozygous mice that had not yet developed renal disease were used as recipients for BMT. After preconditioning with 750 cGy radiation, mice were transplanted with one million wild-type BM via i.v. or i.r. administration. All recipients and untreated controls were assessed for urinary albumin loss, renal pathology, and BM donor-derived renal cells over time. Representative kidney samples were subjected to transmission electron microscopy (TEM) analysis. Interestingly, i.r. and i.v. administration of BM cells gave comparable hematopoietic engraftment levels, and both were able to prevent the onset of MS as assessed by improved lifespan, renal function, renal histology, and TEM analysis. Taken together, we show for the first time that MS can be prevented if BMT is performed before disease onset. Similar therapeutic effects were obtained whether the BM was administered i.v. or i.r.

Morphology of isolated colonic crypts.

The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca2+ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca2+- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts - the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.

Regulation of DRA and AE1 in rat colon by dietary Na depletion.

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.

Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia.

The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.

Evidence that tumor necrosis factor triggers apoptosis in human endothelial cells by interleukin-1-converting enzyme-like protease-dependent and -independent pathways.

Cultured human endothelial cells (EC) resist tumor necrosis factor (TNF)-mediated apoptosis. However, the combination of TNF and the protein synthesis inhibitor cycloheximide (CHX) induces apoptosis in up to 50% of EC within 24 hours. TNF + CHX killing is effectively blocked by transfected CrmA protein or treatment with Z-VAD.fmk peptide-both inhibitors of interleukin-1-converting enzyme-like proteases-but not by transfected antiapoptotic proteins Bcl-2, Bcl-XL, or A1. C6-ceramide (cer) can also sensitize EC to TNF-induced apoptosis. TNF + cer killing, which can affect more than 50% of EC, is not effectively inhibited by CrmA or Z-VAD frank, but can be readily blocked by Bcl-2, Bcl-XL, or A1. Both TNF + CHX and TNF+ cer killing are induced by a TNF mutein that only interacts with the type 1 TNF receptor, and both responses can be inhibited by the antiapoptotic protein A20. These data suggest that TNF activates two biochemically separable pathways of EC injury.

The alpha3 isoform protein of the Na+, K(+)-ATPase is associated with the sites of cardiac and neuromuscular impulse transmission.

The alpha (catalytic) subunit of the Na+ pump (Na+, K(+)-ATPase) has three isoforms; alpha1 is ubiquitous, skeletal muscle expresses predominantly alpha2, and alpha3 has been localized to specific types of neurons and, possibly, to axonal processes. The alpha3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na+ pump alpha subunit and labeled alpha-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that alpha3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed alpha3 immunoreactive protein, as assessed by two isoform-specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that alpha3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na+,K(+)-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle.

Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

Na-K-ATPase alpha-isoform expression in heart and vascular endothelia: cellular and developmental regulation.

The Na pump (Na-K-ATPase) is important for regulation of membrane potential and transport in smooth muscle and heart. The alpha (catalytic)-subunit of this pump has three isoforms: alpha 1 is ubiquitous, but alpha 2 and alpha 3 are mainly localized to excitable tissue. Physiological differences between isoforms are not completely understood, but alpha 3 pumps appear to have a lower affinity for intracellular Na and a higher ouabain affinity than alpha 1 pumps. The alpha 2-and alpha 3-isoform mRNAs are expressed at high levels in the normal adult rat cardiac conduction system. Although alpha 1 and alpha 3 are both globally expressed in neonatal rat myocardia, there is a switch in the myocardial isoform pattern from alpha 3 to alpha 2 after birth. There are also important species differences in cardiac isoform patterns. Furthermore, changes in Na-K-ATPase isoforms in heart and vascular tissue have been reported in association with hypertension, but little is known about isoform expression in normal endothelia. We therefore studied the cellular distribution of Na pump protein isoforms in neonatal and adult myocardia and endothelia. Immunohistochemical analysis of rat tissues showed that the alpha 1-isoform was expressed throughout atrial and ventricular myocardium, with alpha 1 the only isoform detectable in the adult t tubule system. Although alpha 2 was also present in ventricular myocytes, the signal was markedly stronger in conduction tissue and papillary muscle. In hearts from neonatal rats, the alpha 3-isoform predominated in the cardiac conduction system, whereas alpha 2 was not detectable in any structure except vascular endothelium. In tissues and in cell lines representing a variety of species and vessel sizes, endothelia of large vessels expressed primarily alpha 1, whereas alpha 2 could be detected in endothelia of small vessels in rat heart. No evidence of alpha 3 expression in endothelium was found. Thus the complex spatial and developmental regulation of Na pump isoform expression in cardiovascular tissues may provide additional correlates to distinct physiological roles of these transporters.

A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: In the intestine, the cystic fibrosis transmembrane conductance regulator (CFTR) has been localized to the apical pole of crypt epithelial cells. Recent data indicate that some villus cells may also express CFTR, although the identity of these cells has not been established. The aim of the current study was to characterize the distribution, morphology, and surface marker expression of CFTR-expressing villus cells. METHODS: Immunofluorescence and immunoelectron microscopy was performed using anti-CFTR and enzyme marker antibodies. RESULTS: In the rat and human proximal small intestine, a subpopulation of scattered villus and superficial crypt epithelial cells label brightly with anti-CFTR antibodies. The fluorescent signal is detected throughout the cells with its greatest concentration apically. At the ultrastructural level, labeling involves the brush border and a prominent subapical vesicular compartment. The cells resemble adjacent villus enterocytes in their abundance of mitochondria and expression of basolateral Na(+)-K(+)-adenosine triphosphatase yet differ in their absence of brush-border sucrase and lactase expression. CONCLUSIONS: A previously uncharacterized subpopulation of villus cells with high levels of intracellular CFTR expression exists in the proximal small intestine. Morphological and cytochemical studies suggest that this subset of villus cells has a unique transport function.

Germinal matrix microvascular maturation correlates inversely with the risk period for neonatal intraventricular hemorrhage.

The risk period for intraventricular hemorrhage (IVH) of the preterm neonate is the first 3-4 postnatal days. For infants of < 34 weeks' gestation, this risk period is independent of gestational age. We hypothesized that this risk period is attributable to the perinatal induction of maturation of the germinal matrix microvasculature and tested this hypothesis by examining changes in the classical ultrastructural features of the blood-brain barrier over the first ten postnatal days in the newborn beagle model for neonatal IVH. Newborn beagle pups (n = 6) were anesthetized and systemically perfused and the brains were removed and prepared for electron microscopic examination. Examination of electron micrographs from the germinal matrix of animals on the first, fourth and tenth postnatal days demonstrated no difference in perimeter lengths and capillary and endothelial cell areas; in contrast, luminal areas significantly decreased across postnatal age (P = 0.04). Significant increases were found in basement membrane area between days 1 and 4 (P = 0.01) and tight junction length (day 1 vs. day 10, P = 0.02). In addition, on day 1, 19% of germinal matrix capillary perimeter was determined not to be covered by supporting cell processes, while by day 10, only 5% was bare. In contrast, the microvessels of the white matter exhibited no changes in these parameters during these three time points. These studies are consistent with the concept that basal lamina deposition and organization precede increases in endothelial cell tight junction formation and coverage by supporting cells.

Expression and molecular regulation of Na(+)-K(+)-ATPase after renal ischemia.

Renal ischemia causes redistribution of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) to the apical membrane of proximal tubules. We determined the time course of regeneration of Na(+)-K(+)-ATPase polarity and sought evidence of increased enzyme production during recovery as a means to restore polarity. Anesthetized rats underwent 45 min renal ischemia and reflow of 15 min, 2 h, 6 h, and 24 h. Immunofluorescent and electron microscopy showed loss of strict basolateral localization of Na(+)-K(+)-ATPase at 15 min reflow with repolarization by 24 h in sublethally injured cells. Both alpha 1- and beta-subunits were only in microsomal fractions at all reflow intervals. Immunodetectable levels of both subunits declined to 60-70% of control by 24 h reflow. Levels of mRNA for each subunit declined in parallel through 24 h to 55% of control. Overall transcription was profoundly depressed through 6 h but had recovered to near control by 24 h. Specific transcription of alpha 1- and beta-subunit mRNA was markedly decreased after ischemia and only partially recovered by 24 h. These results suggest that recycling of misplaced units rather than new Na(+)-K(+)-ATPase production is the means by which renal epithelia initially repolarize after ischemic injury.

Immature renal tubules are resistant to prolonged anoxia.

Very few data are available regarding the decreased susceptibility of the developing kidney to anoxia. Therefore, the purpose of this study was to develop an experimental system that would allow comparison of an anoxic insult in immature and mature proximal tubule segments and to investigate the hypothesis that the developing kidney is resistant to anoxia as compared with the mature kidney. Suspensions of proximal tubules from immature (age 8-10 d) and mature (8-10 wk) rats were obtained. The purity of the tubule suspension from the immature rats was documented by villin staining. A common buffer solution was developed to compare results from the immature and mature tubules. To study the response of the tubules to anoxia, we subjected the tubule suspension from both the immature and mature rats to 15, 30, 45, and 60 min of anoxia. Lactate dehydrogenase release was measured to assess plasma membrane damage, and ATP levels were determined as an index of cellular energy. After a short anoxic insult (15 or 30 min), the percentage of lactate dehydrogenase release was not significantly different from mature tubules. After prolonged anoxia (45 and 60 min) lactate dehydrogenase release continued to increase, whereas membrane integrity stabilized in the immature tubules. ATP levels decreased in both immature and mature tubules after anoxia, but the decline of ATP was greater in the mature tubules, with a plateau at 20% of basal ATP levels as compared with 40% in the immature tubules. Therefore, the developing kidney is resistant to prolonged anoxia.

Induction and intracellular localization of HSP-72 after renal ischemia.

To determine whether heat shock proteins (HSPs) might be active in cellular recovery following transient ischemia, we examined rat kidneys for 70-kDa HSP (HSP-70) mRNA expression, protein elaboration, and intracellular localization after 45 min of renal ischemia and reflow of 15 min, 2, 6, and 24 h. Inducible HSP-70 mRNA is present at 15 min of reperfusion, peaks between 2 and 6 h, and falls by 24 h. Inducible 72-kDa HSP (HSP-72) protein accumulates progressively through 24 h and is found in both soluble and microsomal fractions following ischemia. Within proximal tubules, immunofluorescent localization of HSP-72 is restricted to the apical domain at 15 min, is dispersed through the cytoplasm in a vesicular pattern at 2 and 6 h, and has migrated away from the apical domain at 24 h. A portion of the vesicular HSP-72 is associated with lysosomes; no intranuclear HSP-72 is detected. The course of mRNA induction, protein elaboration, and HSP-72 localization coincides with previously described changes in proximal tubule morphology and polarity following sublethal ischemic injury. HSP-72 may be instrumental in cellular remodeling and restitution of epithelial polarity during recovery from ischemic renal injury.

Indomethacin promotes germinal matrix microvessel maturation in the newborn beagle pup.

BACKGROUND AND PURPOSE: Although indomethacin has been demonstrated to prevent germinal matrix and intraventricular hemorrhage in clinical and animal studies, the mechanism of action of this agent to prevent hemorrhage remains unclear. Previous studies have demonstrated both that the microvessels in the germinal matrix of newborn beagle pups undergo basement membrane maturation during the first 4 postnatal days and that indomethacin may promote laminin deposition in tumor cell culture systems. METHODS: We employed the newborn beagle pup model to test the hypothesis that indomethacin may stimulate laminin deposition in germinal matrix microvessels. Newborn pups were randomized to receive either 0.1 mg/kg/dose i.p. indomethacin or an equal volume of saline diluent. Pups received doses of study medication once a day for 1, 2, or 3 days and were studied on postnatal days 1, 2, 3, or 4. Pups were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. RESULTS: Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density among the postnatal ages studied; similarly, there were no differences in vessel density between saline- and indomethacin-treated animals at any postnatal age. Quantification of germinal matrix stained intensity by confocal microscopy demonstrated significant increases in indomethacin-treated pups for both laminin staining at postnatal days 2 (p = 0.05) and 3 (p = 0.0009) and type V collagen staining at postnatal day 2 (p = 0.011). Although staining for beta 1 integrins increased across postnatal ages, there were no differences between saline- and indomethacin-treated animals. CONCLUSIONS: These data suggest that indomethacin may stimulate basement membrane deposition in the germinal matrix microvessels of newborn beagle pups to prevent germinal matrix and/or intraventricular hemorrhage.

The effect of cyclosporine administration on the cellular distribution and content of cyclophilin.

Cyclophilin (CYP), an intracellular protein sharing amino acid sequence identity with the enzyme peptidyl-prolyl cis-trans isomerase has become the leading candidate for the receptor responsible for cyclosporine biological effects. Avid binding of CYP to cyclosporine and immunosuppressive cyclosporine metabolites has been demonstrated, while nonimmunosuppressive cyclosporine metabolites have tended not to bind to cyclophilin. A previous immunohistochemical analysis documented that CYP localized principally to the cytoplasmic cellular compartment, but nuclear staining was observed among some cells. This study was undertaken to more precisely define the ultrastructural distribution of CYP, and to determine whether CYP cellular content was affected by CsA therapy. Untreated Wistar rats or those receiving 7 days of CsA (15 mg/kg/day, i.p.) were anesthetized, perfusion-fixed in situ, and sacrificed. Analyses of lymph node, spleen, thymus, kidney, liver, heart, brain, and ileum used an affinity purified, rabbit anticyclophilin IgG. Transmission electron microscopy was performed after staining with anti-CYP using a horseradish peroxidase/biotin/avidin technique. Quantitative immunofluorescence was measured by confocal microscopy using anti-CYP, with a biotin/avidin/phycoerythrin technique. Cyclophilin localized to the cytoplasmic compartment--however, association with mitochondria endoplasmic reticulum, Golgi, and with the nuclear membrane among lymphocytes, as well as cells from kidney, liver and ileum--was documented. Cyclophilin was not identified within the nucleus proper. Tissues obtained from animals receiving CsA exhibited a generalized increase in CYP content compared with tissues from untreated controls, suggesting the possibility that CsA may exert a regulatory influence upon CYP gene activation. Collectively, the data were consistent with the hypothesis that CYP exerts a central role in cellular metabolism, and that CsA-mediated biologic effects result from the CsA/CYP interaction.

Redistribution of cellular energy following renal ischemia.

In order to elucidate the pattern of redistribution of cellular energy and the restoration of basic cellular metabolism following an ischemic renal insult, suspensions enriched in proximal tubule segments were studied after 45 min of bilateral artery occlusion and 15 min and 2 h of reflow from rats given either normal saline (control), ATP-MgCl2 (which enhances postischemic recovery of ATP), or alpha, beta-methyl adenosine diphosphate (AMPCP), which inhibits nucleotide degradation during ischemia. In non-ischemic control animals, approximately half of the energy is distributed to functional pump activity and half directed for non-transport purposes. When cellular ATP is reduced to 56% of control values, functional pump activity is significantly reduced to 61% of control, while energy delegated for non-transport purposes is decreased by a significantly smaller increment to only 78% of control at 15 min of reflow. In animals given ATP-MgCl2, the cellular and metabolic profile at 15 min of reflow was no different from ischemic control animals with cellular ATP levels similar at 58%. However, by 2 h, cellular ATP levels had increased significantly to 74%, and this was associated with a redistribution of cellular energy to functional pump activity (119% of control) with little change in non-transport function (76%). In animals treated with AMPCP, the cellular ATP levels were 74% of controls, similar to ATP-MgCl2-treated rats after 2 h of reflow. Despite the differences in reflow interval, the distribution of cellular energy was similar (functional pump activity 120% and non-transport activity 79%). By 2 h, cellular ATP was at 95% and both functional pump activity and non-transport activity were 100%.(ABSTRACT TRUNCATED AT 250 WORDS)

Beagle pup germinal matrix maturation studies.

Intraventricular hemorrhage, or hemorrhage into the germinal matrix tissues of the developing brain, remains a common problem of preterm infants. The "risk period" for this insult is the first 3-4 postnatal days. We hypothesized that this risk period for hemorrhage is related to rapid perinatal maturation of the germinal matrix vasculature and employed the newborn beagle pup model for the study of this maturation. Newborn beagle pups (n = 30) were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density between postnatal days 1 and 4. Germinal matrix sections were also stained for antibodies to alpha-smooth muscle actin, collagen IV, collagen V, desmin, factor VIII-related antigen, fibronectin, glial fibrillary acidic protein, laminin, transferrin, and vimentin. Vasculature staining by alpha-smooth muscle actin was not noted until postnatal day 10, and differential staining was detected for antibodies to laminin and collagen V. Quantification of staining intensity by confocal microscopy demonstrated a significant increase in both extracellular matrix components at postnatal day 4 compared with day 1 (p less than 0.05 for both). These basement membrane proteins may add sufficient structural integrity to germinal matrix vessels to prevent capillary rupture and thus intraventricular hemorrhage.

Metabolic alterations in proximal tubule suspensions obtained from ischemic kidneys.

A viable suspension of proximal tubules that had sustained an in vivo ischemic injury was harvested, and cellular integrity and viability were determined. The histopathological appearance of this preparation has characteristic features of an ischemic injury and ATP levels were comparable to those observed with nuclear magnetic resonance spectroscopy in vivo. Sprague-Dawley rats were subjected to 45 min of bilateral renal artery ischemia and the kidneys were allowed to reperfuse for either 15 min, 2 h, or 24 h before the harvest of the proximal tubule suspension. There was a decrease in base-line oxygen consumption from 34 +/- 0.8 nmol O2.min-1.mg protein-1 to 22 +/- 0.6 at 15 min of reflow. This decline in oxygen consumption persisted during the first 2 h of reflow and returned to control levels by 24 h. Residual respiration in the presence of ouabain was similar at all reflow intervals suggesting that the decrease in basal O2 consumption was related to decreased Na+-K+-ATPase in situ. In contrast, there was no significant difference in Na+-K+-ATPase activity when determined chemically under Vmax conditions in all experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells.

In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537). The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of approximately 1 ankyrin to 4 Na,K-ATPase's, with a kD of 2.6 microM. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) 125I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of approximately 240 kD that is associated with both erythrocyte and kidney membranes that binds 125I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.

Morphological heterogeneity of the rabbit collecting duct.

The localization of carbonic anhydrase by histochemistry, of Na-K-ATPase by immunocytochemistry and of rod-shaped intramembranous particles by freeze-fracture electron microscopy, was determined in the collecting duct of rabbits. In the cortical collecting duct (CCD), rod-shaped particles, which are abundant in intercalated cells were observed in both the apical and basolateral membrane of all intercalated cells examined. In the outer stripe of the outer medullary collecting duct (OMCDo) a high density of rod-shaped particles was found only in the apical membrane of intercalated cells. All cells of the inner stripe of the outer medullary collecting duct (OMCDi) had rod-shaped particles in the apical membrane but not in the basolateral membrane. As the collecting duct entered the inner medulla the density of rod-shaped particles decreased until they were virtually absent in the terminal segment. Na-K-ATPase, localized to the basolateral membrane, was more abundant in principal cells than in intercalated cells in the CCD. In the OMCDo, staining was equal in principal and intercalated cells. All cells of the OMCDi and the inner medullary collecting duct (IMCD) stained for Na-K-ATPase. Carbonic anhydrase in the CCD was localized to the cell membranes and cytoplasm of intercalated cells. Principal cells did not stain for carbonic anhydrase. A similar pattern was seen in the OMCDo. In the outer region of the OMCDi most cells did not stain for carbonic anhydrase, whereas in the inner region the apical and lateral membranes of all cells stained for carbonic anhydrase. Weak cytoplasmic staining was occasionally seen. A similar pattern was seen in the initial half of the IMCD, while the terminal half of the IMCD did not stain. In this study, the localization of enzymes and rod-shaped intramembranous particles associated with Na+, K+, and H+ transport shows both segmental and cellular heterogeneity, and correlates with the known transport properties of tubule segments. The distribution of these enzymes and rod-shaped intramembranous particles is different in rabbits and rats, and may explain some of the functional differences between homologous segments in these species.