In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney.

The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding.

Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications.

Reduction of globotriaosylceramide in Fabry disease mice by substrate deprivation.

We used a potent inhibitor of glucosylceramide synthase to test whether substrate deprivation could lower globotriaosylceramide levels in alpha-galactosidase A (alpha-gal A) knockout mice, a model of Fabry disease. C57BL/6 mice treated twice daily for 3 days with D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidi no-propanol (D-t-EtDO-P4) showed a concentration-dependent decrement in glucosylceramide levels in kidney, liver, and spleen. A single intraperitoneal injection of D-t-EtDO-P4 resulted in a 55% reduction in renal glucosylceramide, consistent with rapid renal glucosylceramide metabolism. A concentration-dependent decrement in renal and hepatic globotriaosylceramide levels was observed in alpha-Gal A(-) males treated for 4 weeks with D-t-EtDO-P4. When 8-week-old alpha-Gal A(-) males were treated for 8 weeks with 10 mg/kg twice daily, renal globotriaosylceramide fell to below starting levels, consistent with an alpha-galactosidase A-independent salvage pathway for globotriaosylceramide degradation. Complications observed with another glucosylceramide synthase inhibitor, N-butyldeoxynojirimycin, including weight loss and acellularity of lymphatic organs, were not observed with D-t-EtDO-P4. These data suggest that Fabry disease may be amenable to substrate deprivation therapy.

Neutrophil-mediated endothelial injury in vitro mechanisms of cell detachment.

Neutrophil-mediated endothelial injury was assessed in vitro using assays of cell lysis and cell detachment. Activation of human peripheral blood neutrophils adherent to human umbilical vein endothelial cell monolayers by serum-treated zymosan produced dose-dependent endothelial cell detachment without concomitant cell lysis. This injury was inhibited by neutral protease inhibitors, but not by catalase or superoxide dismutase. Neutrophils from a patient with chronic granulomatous disease also produced endothelial cell detachment when activated by serum-treated zymosan similar to normal neutrophils. Endothelial detachment was also produced by cell-free postsecretory media from activated neutrophils or by partially purified human neutrophil granule fraction and was inhibitable by tryptic, elastase, and serine protease inhibitors, but not by an acid protease inhibitor. Analysis of iodinated endothelial cell surface proteins that had been exposed to partially purified neutrophil granule fraction showed complete loss of proteins migrating in the region of fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This result was prevented in the presence of neutral protease inhibitors. We conclude that neutrophil-derived neutral proteases mediate endothelial cell detachment in vitro through digestion of endothelial cell surface proteins including fibronectin.

Time course of stimulation of renal renin messenger RNA by furosemide.

Renin secretion responds rapidly to a variety of stimuli; however, reported changes in renal renin messenger RNA (mRNA) levels in vivo have been observed only after prolonged stimulation. Studies were designed to test whether rapid changes in renin mRNA levels can be produced in vivo. In the first series, Sprague-Dawley rats received furosemide (10 mg/kg) intraperitoneally and a low sodium diet (0.05% sodium); renin secretion was significantly stimulated at 8 or 16 hours after treatment, but renin mRNA levels did not change. In a second series, rats were pretreated with deoxycorticosterone acetate (200 mg/kg) and saline drinking water for 3 days and then killed 0, 2, 4, 8, or 48 hours after furosemide administration. The renin mRNA level was unchanged at 2 hours but was stimulated twofold at 4 and 8 hours and threefold at 48 hours. In additional animals, the response of renin mRNA 4 hours after furosemide was found not to be potentiated by the converting enzyme inhibitor quinapril (5 mg/kg). The results demonstrate that with acute stimulation, renin mRNA levels lag 2-4 hours behind the change in plasma renin levels.

Evidence for a stem cell common to hematopoiesis and its in vitro microenvironment: studies of patients with clonal hematopoietic neoplasia.

The origin and nature of cells forming the in vitro microenvironment in long-term cultures of human marrow were studied in five patients with clonal myeloproliferative disorders who were heterozygous for glucose-6-phosphatase dehydrogenase (G6PD). The results showed that cells in the adherent stromal layer forming the in vitro microenvironment were derived from the same clonal progenitors involved by the neoplasm in the four patients whose diseases originated in multipotent stem cells. In contrast, stromal cells were derived from normal progenitors in a patient with acute non-lymphocytic leukemia whose clone showed differentiative expression confined to cells in the granulocytic lineage. Mixing experiments demonstrated that the G6PD type displayed by the adherent marrow stromal cells was not obscured by contaminating non-adherent hematopoietic cells or marrow fibroblasts. The data suggest the existence of a pluripotent cell in normal hematopoiesis that gives rise to hematopoietic cells and to their micro-environment.

The glomerular distribution of type IV collagen and laminin in human membranous glomerulonephritis.

The glomerular distribution of type IV collagen and laminin, the major collagenous and noncollagenous components of the glomerular basement membrane, was studied by immunofluorescence microscopy in idiopathic and lupus membranous glomerulonephritis. Affinity-purified antibodies against type IV collagen reacted preferentially with the inner aspect and irregularly with the adjacent outer area of the thickened basement membrane. In contrast, laminin was detected along the inner aspect of the glomerular basement membrane, in subepithelial basement membrane protrusions ("spikes"), and in the newly formed basement membrane layer above the immune deposits. We conclude that type IV collagen and laminin do not codistribute in the newly formed matrix. This aberrant antigenic distribution may reflect a loss of coordinate biosynthesis or degradation of these matrix components by visceral epithelial cells.

Application of polymerase chain reaction techniques to study of rabbit renin gene expression.

Studies were performed to develop techniques to assess renin mRNA in a single microdissected juxtaglomerular apparatus (JGA) of rabbit using the polymerase chain reaction (PCR) method of amplification of cDNA sequences. In preliminary studies synthetic oligonucleotide primers corresponding to regions in the rat renin cDNA sequence, which are highly conserved between mouse, rat, and man, were found to yield good amplification efficiency with a rat renin cDNA template, but little product was observed with rabbit cDNA template. We therefore employed a nested primer PCR cloning technique to clone an 839 base pair portion of the rabbit renin cDNA to obtain species-specific sequence information for primer design. Here we report the nucleotide sequence of a partial rabbit renin cDNA clone and the use of species-specific primers that permit semiquantitative assessment of rabbit mRNA levels in the single JGA.

Human glomerular cells in vitro: isolation and characterization.

Human glomerular cells were isolated, identified, and propagated in vitro, and a number of features were found to be unique to glomerular visceral epithelial and mesangial cells. Epithelial cells are identified by their "epithelial" morphology in vitro, growth pattern, presence of C3b surface receptors, response to mitogens, synthesis of a single collagen type (IV), and nearly exclusive synthesis of one type of sulfated glycosaminoglycan, heparan sulfate. Mesangial cells differ from epithelial cells and other potential contaminating cells, such as fibroblasts or endothelial cells, by their characteristic light and electron microscopic morphology and their synthesis of specific collagen and glycosaminoglycan types. In addition, while they closely resemble vascular smooth muscle cells in many ways, they differ with respect to their response to mitogens. It should now be possible to study these cells for other features, including the presence of alloantigens. This property and their response to inflammatory mediators or cells are prerequisites to the determination of their role in the pathogenesis of renal allograft rejection.

Human glomerular visceral epithelial cells synthesize a basal lamina collagen in vitro.

Isolated human glomeruli were digested with purified bacterial collagenase yielding epithelial cells. These cells grew to saturation density and did not become multi-layered. They were identified as visceral glomerular epithelial cells by their morphologic appearance by phase and electron microscopy and by the presence of surface receptors for C3b. Neither Factor VIII antigen nor Fc receptors were observed. The glomerular epithelial cells synthesized a collagenous protein that was antigenically similar to human glomerular basal lamina. Proteins precipitated from visceral epithelial cell medium with affinity purified antibody against noncollagenous glomerular basal lamina antigens yielded a single collagenase labile protein that by sodium dodecyl sulfate/polyacrylamide gel electrophoresis migrated with an apparent Mr of 168,000 in the presence of reducing agents. Analysis of hydroxyproline isomers yielded a ratio of 3-hydroxyproline to total hydroxyproline of 0.17. Pepsin digestion yielded a disulfide-bonded multimer which, with reduction, migrated with an apparent Mr of 148,000. These data demonstrate that human glomerular visceral epithelial cells can be isolated and propagated in vitro and that they synthesize a collagen similar to that found in vivo.

Intracellular signaling of transcription and secretion of type IV collagen after angiotensin II-induced cellular hypertrophy in cultured proximal tubular cells.

Physiologic concentrations of angiotensin II (AII) can induce cellular hypertrophy in murine proximal tubular epithelium (MCT cells). This response is characterized by an increase in cell size, new protein synthesis, and by the secretion of new basement membrane type IV collagen in the absence of cellular proliferation. The present study was undertaken to evaluate the second messengers of these AII-induced cellular events with special reference to the increase in type IV collagen secretion. In initial experiments we observed that pretreatment of MCT cells with agents that increase concentrations of intracellular cAMP, like forskolin, dibutyryl cAMP, and isobutyl-methyl-xanthine abolish AII-induced amino acid incorporation, but have no effect on control cells or on their proliferation. In addition, 10(-8) M AII significantly decreased the concentration of intracellular cAMP. Phorbolesters were without significant effect on the hypertrophy or proliferation of AII-stimulated MCT cells or their rested controls. The transfection of MCT cells with reporter genes containing regulatory elements for type IV collagen revealed that the stimulatory effects of AII on collagen type IV depend, at least to some extent, on an increase in gene transcription. Agents increasing intracellular cAMP concentrations inhibited the AII-induced increase in transcription and secretion of collagen type IV, but had no effect on MCT cells grown in media without AII. Our findings provide evidence that AII-induced changes in tubular epithelium leading to the secretion of type IV collagen are mediated by a decrease in intracellular cAMP.

Vascular adventitial cell expression of collagen I messenger ribonucleic acid in anti-glomerular basement membrane antibody-induced crescentic nephritis in the rabbit. A cellular source for interstitial collagen synthesis in inflammatory renal disease.

BACKGROUND: Scarring in the interstitial compartment of the renal cortex heralds a poor prognosis in many forms of renal injury, however, the mechanism through which glomerular inflammation leads to interstitial scarring is not understood. In a model of anti-GBM disease in the rabbit, development of crescentic glomerulonephritis is associated with marked interstitial fibrosis and decreased renal function. We previously demonstrated that collagen accumulation in the model was preceded by increases in collagen I and IV mRNA and that these changes were primarily extraglomerular at early time points when inflammation was predominantly intraglomerular. In order to identify the cellular origins of extraglomerular collagen synthesis in this model, in situ hybridization using an alpha 2(I) procollagen probe was performed. EXPERIMENTAL DESIGN: A 602 bp rabbit alpha 2(I) procollagen cDNA was cloned using a PCR strategy and sequenced. The nucleotide sequence of the coding region was 94% identical with the human alpha 2(I) procollagen sequence. Northern blots were performed to define conditions of specific hybridization of the anti-sense riboprobe. Tissue sections from normal rabbit kidneys and from kidneys 4, 5, 7, 10 and 14 days after injection of anti-GBM antibody were hybridized with 35S-labeled sense and anti-sense riboprobes. Cells containing alpha 2(I) mRNA were identified by autoradiography and mRNA abundance was quantitated by grain density. RESULTS: No specific hybridization was detected with the sense probe at any time. alpha 2(I) mRNA was undetectable with the anti-sense probe in normal kidney sections. In contrast, the anti-sense probe hybridized specifically at all time points after induction of anti-GBM disease. In agreement with previous filter hybridization studies, on day 4, when inflammation was predominantly intraglomerular, cells in the periarterial adventitial compartment of renal cortex hybridized strongly. At later time points, labeling was also present in the interstitial spaces, the periglomerular region, in Bowman's space and in the glomerular tuft itself. CONCLUSIONS: We conclude that perivascular adventitial cells are among the first to respond to glomerular inflammation and represent a pool of cells that subsequently contribute to interstitial and glomerular scarring.

Cholesterol emboli presenting as acute allograft dysfunction after renal transplantation.

Cholesterol emboli are a common complication of atherosclerotic vascular disease. A 40-yr-old renal transplant recipient who developed acute allograft dysfunction 1 day after the initiation of cyclosporine therapy and 6 days after transplantation is described. A renal allograft biopsy revealed cholesterol emboli in interlobular arteries and in glomeruli. Four previously reported cases of cholesterol emboli in renal allografts are described, and the cause and pathogenesis of atheroembolic disease are reviewed. Atheroemboli causing injury to the renal allograft may arise from either donor or recipient vessels. Vigilance for the occurrence of these emboli needs to be maintained when donor or recipient vessels demonstrate evidence of significant atherosclerotic vascular disease.

Cyclosporin A stimulates transcription and procollagen secretion in tubulointerstitial fibroblasts and proximal tubular cells.

The brief study described in this report was undertaken to determine whether cyclosporin A had any direct effect on the expression of tubulointerstitial procollagens in cultured renal cells. Our findings indicate that murine tubulointerstitial fibroblasts secreted significantly more procollagen type I after the addition of cyclosporin A, whereas syngeneic proximal tubular cells expressed significantly more types I and IV procollagen after cyclosporin stimulation. These increases in procollagen gene product correlated concordantly with changes in the levels of cytoplasmic mRNA with procollagen-specific cDNA probes. Transfection of these fibroblasts and proximal tubular cells with chimeric gene constructs containing enhancer/promoter elements for alpha2(I) and alpha 1(IV) procollagen linked to a chloramphenicol acetyltransferase reporter gene indicates that the stimulatory effect of cyclosporin on procollagen expression depends, at least to some extent, on an increase in transcriptional activity.

Rat intestinal basement membrane synthesis. Epithelial versus nonepithelial contributions.

Mesenchymal-epithelial interactions play an important role during tissue differentiation and morphogenesis. The basement membrane, which separates these compartments, appears to be critical to these interactions by providing a substratum for cell adhesion, promoting cell polarity and the differentiated phenotype. Unlike other epithelia, gut enterocytes adhere to, and migrate along a thin basement membrane as they differentiate along the crypt-villus axis with a turnover rate of 48 to 72 hours (rat). The relative importance of the enterocytes or of the mesenchymal cells of the lamina propria to the maintenance of the basement membrane is unknown. As indirect indicators of basement membrane biosynthesis, we have measured, by filter hybridization with labeled cDNA probes, the relative abundance of mRNAs for laminin and collagen IV chains in enterocyte fractions representing the crypt-villus gradient of differentiation and in cells of the underlying lamina propria. In confirmation of a gradient, mRNA for histone H2B was present as a decreasing gradient from crypt to villus, the crypt fraction containing the mitotically active enterocytes being most enriched for this transcript and, in contrast, the mRNA for beta-actin was present as an increasing gradient from crypt to villus, paralleling the abundance of microvillus core structures. The mRNAs for alpha 1(IV) and alpha 2(IV) collagen and laminin B1 and B2 chains were most abundant in the lamina propria. Little, if any, collagen IV mRNA was detectable in the enterocyte fractions. In contrast, laminin B1 and B2 mRNAs were enriched in crypt enterocytes but the steady-state level of these transcripts decreased in the superficial villus enterocyte fractions. These data suggest that the components of the intestinal basement membrane are synthesized by both mesenchymal and entodermal-derived cells. Alterations in the intestinal basement membrane structure and in cell adhesion during enterocyte differentiation may be partly mediated by changes in laminin synthesis by the enterocyte.

Structure of the amino-terminal portion of the murine alpha 1(IV) collagen chain and the corresponding region of the gene.

Collagen IV, the major structural component of basement membranes, is composed of two genetically distinct polypeptide chains, alpha 1(IV) and alpha 2(IV). We have isolated a 522-base-pair (bp) cDNA to the 5' portion of the murine alpha 1(IV) chain mRNA from a library constructed by specific primer extension of poly(A)+ RNA from differentiated F9 cells. This cDNA includes 141 bp of 5' untranslated sequence and encodes a signal peptide plus a portion of the amino-terminal cross-linking (7 S) domain. This cDNA clone was used to obtain the 5' portion of the murine alpha 1(IV) gene from which the nucleotide sequence of exons 1-6 was determined. Exon 1 (234 bp) codes for the 5' untranslated sequence, and the first 28 residues of the protein. The 5' untranslated sequence is highly conserved between the mouse and human species and has the potential to form three mutually exclusive stem-loop structures which may play a role in post-transcriptional regulation. Exons 2-6, which code for the 7 S domain, were found to be 60, 90, 45, and 63 bp in size. The exon structure for the helical portion of the 7 S domain is different from that of the major helical domain, suggesting that they evolved differently.