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Background: Members of the bcl-2 family of proteins regulate the occurrence of apoptotic cell death. Apoptosis is a feature of both acute and chronic renal diseases. Diabetes mellitus modulates renal growth; it induces acute tubuloepithelial cell hypertrophy, leads to chronic glomerulopathy associated with tubular epithelial cell atrophy and interstitial fibrosis, and predisposes to renal cell loss from acute nephrotoxic injury. However, the mechanisms by which diabetes affects renal cell turnover are unclear., Methods: Northern analysis was performed on RNA isolated from murine tubular epithelial MCT cells grown in a medium with physiological or high glucose concentration (5.5 or 25 mmol/L, respectively) for 24 to 96 hours, and from kidney cortex of spontaneously diabetic db/db mice and their nondiabetic db/m littermates. Counting of dead cells in tissue culture and in the renal cortex was also carried out., Results: High ambient glucose downregulated the expression of the bcl-2 gene whose protein product is protective against apoptosis. Expression of the related bclx mRNA was also downregulated. However, the level of bax mRNA that encodes an apoptosis-promoting protein was transiently increased. Hyperosmolarity induced by D-mannitol did not reproduce these effects of high glucose. High glucose significantly increased the number of dead cells in culture. Thus, the net effect of high glucose concentration is to increase the expression of apoptosis regulatory genes and to favor apoptosis in proximal tubular epithelial cells. Correlative studies in vivo revealed decreased bcl-2 and increased bax gene expression in the renal cortex of diabetic db/db mice, and this was associated with increased apoptotic index in the tubular epithelium as compared with nondiabetic littermates., Conclusions: These studies suggest that the metabolic changes of the diabetic state modulate the expression of apoptosis-regulatory genes so as to favor apoptosis and that this modulation may be a factor in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy is one of the most frequent causes of chronic renal failure worldwide. Altogether, 35% of patients with insulin-dependent diabetes mellitus and a somewhat smaller percentage of patients with non-insulin-dependent diabetes mellitus ultimately develop diabetic kidney disease. Early diagnosis is of utmost importance since the development of diabetic nephropathy affects the general health, the carbohydrate metabolism of the patient, moreover it aggravates hypertension and accelerates atherosclerosis. Microalbuminuria is a sensitive but relatively late marker of diabetic kidney disease. Still, screening of diabetic patients for microalbuminuria is of great importance since there is no other screening test capable of diagnosing diabetic nephropathy at an earlier stage. The description of the genetic substrate of susceptibility to diabetic kidney disease would revolutionize the diagnosis and prevention of diabetic nephropathy. Until then, compliance with therapeutic guidelines outlined in milestone clinical studies of the last years may significantly decrease morbidity, the progression of, and the mortality associated with diabetic kidney disease.

Several systemic or intrarenal networks of cytokines and growth factors can be modulated by the diabetic state. We summarize the status of the renin-angiotensin system in diabetes mellitus and review the evidence of its involvement in the pathogenesis of diabetic nephropathy. Particular emphasis is placed on the nonhemodynamic properties of this vasoactive agent as both a renal growth factor and a profibrogenic peptide. Antagonizing the effects of angiotensin II with converting enzyme inhibitors is an established protective strategy in the management of diabetic nephropathy even in the absence of systemic hypertension. This and other indirect evidence from experimental animal studies suggest that the intrarenal concentration of angiotensin II may be increased as a result of increased synthesis and despite enhanced breakdown, that this peptide participates in the progression of diabetic nephropathy. However, down-regulation of angiotensin type 1 (AT1)-receptors is one of the abnormalities of both tubules and glomeruli in diabetic renal disease. A heightened bioactivation of the intrarenal angiotensin II system is therefore likely but not certain. Studies in cultured proximal tubular and glomerular mesangial cells have disclosed striking similarities between the effects of high glucose-containing medium and of treatment with angiotensin II on the growth properties and the induction of cytokines in these cells. There may also exist additive effects of angiotensin II and high glucose on signal-transduction pathways, such as activation of protein kinase C, although the contractile response to angiotensin II may be blunted by high glucose in mesangial cells. An important downstream mediator of the effects of both angiotensin II and high glucose is the activation of transforming growth factor-beta that can mediate at least some of the hypertrophic and profibrotic effects of either angiotensin II or high glucose in the diabetic kidney.

Diabetic nephropathy has become, one of the most frequent causes of chronic renal failure in the industrialized countries. Basic and clinical research aimed at the clarification of the pathogenesis of diabetic kidney disease is of utmost importance. The role of non-enzymatic glycosylation, the polyol cycle, oxidative stress, various hormones and cytokines in the development of diabetic nephropathy is very likely. However, there is still no unifying concept about the relative importance and interaction of these factors. This paper reviews the most important directions and results of basic research centering on diabetic kidney disease. The coming years will likely bring the elaboration of a detailed theory of the pathogenesis and an even tighter coordination of basic and clinical research.

One pathological feature of chronic cyclosporin A (Cs-A) nephrotoxicity is striped interstitial fibrosis, the pathogenesis of which is not completely understood. We have previously reported that Cs-A directly stimulates the synthesis of various collagens in a cultured murine proximal tubular cell line (MCT cells). In addition, we have recently observed that Cs-A stimulates the synthesis of transforming growth factor beta (TGF-beta) in MCT cells. To this end, we investigated whether Cs-A directly stimulates transcription of TGF-beta in this cell line. A single dose of 2,000 ng/ml Cs-A stimulates the expression of TGF-beta1 transcripts in MCT cells. This stimulation is abrogated in the presence of 150 and 250 ng/ml actinomycin D without significantly influencing basal mRNA expression, suggesting that the increase in mRNA is due to stimulated transcriptional activity. Furthermore, nuclear runoff experiments demonstrate that Cs-A directly stimulates the transcriptional activity of the TGF-beta1 gene in MCT cells. The results of transient transfection of reporter gene constructs containing regulatory elements of the murine TGF-beta1 promoter into MCT cells and subsequent treatment with Cs-A further suggest direct effects of Cs-A on TGF-beta1 gene transcription. This is the first report demonstrating Cs-A-mediated TGF-beta1 gene transcription in renal cells. Considering the well-known profibrogenic effects of TGF-beta, our results suggest a novel mechanism of Cs-A nephrotoxicity.

The db/db mutant mouse is a rodent model of genetic diabetes that develops renal glomerular lesions with striking mesangial matrix accumulation by the age of 16 weeks, after 8-10 weeks of sustained hyperglycemia. However, abnormalities in renal function that antedate or accompany the appearance of these pathologic changes, which resemble those found in human diabetes, have not been delineated. We therefore examined renal function in young db/ db mice and their nondiabetic db/m littermates from the age of 8 through 15 weeks. Serum creatinine and blood urea nitrogen concentrations at the onset of diabetes in db/db mice did not differ significantly from mean concentrations in db/m controls. An elevated creatinine clearance, due in large part to increased body weight, and increased urinary albumin excretion were observed in db/db compared with db/m mice soon after establishment of sustained hyperglycemia. A relative reduction in creatinine clearance was demonstrable in db/db mice at the age of 15 weeks, coincident with the appearance of overt compromise in renal function manifested by frank increases in the serum creatinine and blood urea nitrogen. The findings indicate that the well-documented glomerular pathology in db/db mice is accompanied by definable alterations in renal function, which are similar in chronology and nature to those found in human diabetes.

Angiotensin II (Ang II) has growth-stimulatory properties on different renal cell types. However, possible growth effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvasculature have not been formally investigated. Therefore, we isolated and characterized primary cultures of rat glomerular endothelial cells. We used a simple technique in which collagenase-treated glomeruli were sparsely plated in several 96-well culture plates and microscopically screened for cobblestone-like outgrowth. After two limiting dilutions, homogeneous cultures were obtained. Cells were characterized by positive staining for the endothelial markers factor VIII, CD 31, endothelial leukocyte adhesion molecule-1, and the lectin Bandeiraea simplificifolia. Ang II stimulated the synthesis and release of endothelin-1 in culture supernatants. Moreover, in contrast to syngeneic mesangial cells, glomerular endothelial cells expressed angiotensin-converting enzyme. Ang II stimulated a mild but significant proliferation of quiescent cells, as measured by [3H]thymidine incorporation and direct cell counting. This mitogenesis was transduced by losartan-blockade angiotensin type 1 receptors. Moreover, Ang II mediated phosphorylation of mitogen-activated protein kinase 2 and induction of transcripts for the immediate early gene Egr-1. Our results indicate that Ang II is a moderate mitogen for primary cultures of rat glomerular endothelial cells and activation of these metabolically active cells may play a role in the pathophysiology of several types of glomerulonephritis. Moreover, remodeling of glomerular endothelial cells by Ang II may be important in the progression of structural renal damage during the course of hypertensive injury.

Diabetic nephropathy is characterized by renal hypertrophy, thickening of basement membranes, and accumulation of extracellular matrix in the glomerular mesangium and the interstitium. Our previous investigations have shown that high glucose concentration increases transforming growth factor (TGF)-beta1 mRNA in mesangial and proximal tubule cells and that treatment with anti-TGF-beta antibody results in prevention of the effects of high glucose on cell growth (e.g., induction of cellular hypertrophy) and the stimulation of collagen biosynthesis. We evaluated in vivo the functional role of the renal TGF-beta system in diabetic kidney disease by treatment of streptozotocin-induced diabetic mice with either a neutralizing monoclonal antibody against TGF-beta1, -beta2, and -beta3 (alphaT) or nonimmune murine IgG for 9 days. Diabetic mice given IgG demonstrated total kidney and glomerular hypertrophy, significantly elevated urinary TGF-beta1 protein, and increased mRNAs encoding TGF-beta1, type II TGF-beta receptor, alpha1(IV) collagen, and fibronectin. Treatment of diabetic mice with alphaT prevented glomerular hypertrophy, reduced the increment in kidney weight by approximately 50%, and significantly attenuated the increase in mRNA levels without having any effect on blood glucose. The antibody was without significant effect on mRNA levels in nondiabetic mice. This is the first demonstration that the early characteristic features of diabetic renal involvement, which include hypertrophy and increased matrix mRNAs, are largely mediated by increased endogenous TGF-beta activity in the kidney and that they can be significantly attenuated by treatment with neutralizing anti-TGF-beta antibodies.

Angiotensin II (Ang II) stimulates cellular hypertrophy of cultured murine proximal tubular cells (MCT cells). This Ang II-mediated hypertrophy depends on the endogenous induction and autocrine action of transforming growth factor-beta (TGF-beta). We have previously demonstrated that permanent transfection of MCT cells with the c-mas oncogene, whose protein product encodes a serpentine receptor-like moiety coupled to G proteins without an hitherto identified ligand, changes the hypertrophic actions of Ang II into a proliferative response (Am J Physiol 263: F931-F938, 1992). The present study demonstrated that Ang II failed to stimulate induction of TGF-beta 1 protein in c-mas transfected MCT cells under the control of SV 40 promoter/enhancer (pSV2mas) as measured by mink cell bioassay and specific ELISA for TGF-beta 1. Moreover, in contrast to either wild-type MCT cells or to cells permanently transfected with the SV 40 based expression plasmid only (pSV2 cells), Ang II stimulated gene transcription and mRNA expression of TGF-beta 1 were decreased in c-mas transfected cells. Our findings demonstrate that the Ang II-induced proliferation of c-mas transfected MCT cells most likely depends on failure of TGF-beta 1 induction in these cells. c-mas transfected cells are a useful tool to further investigate the complex relationships between activation of second messengers subsequent to binding of Ang II to AT1-receptors and gene regulation like transcription of TGF-beta 1.

The direct relationship between elevated glucose concentrations and accelerated protein glycation has implicated increased glycation as a potential mechanistic link between hyperglycemia and the pathogenesis of diabetic nephropathy. Albumin modified by Amadori glucose adducts has been shown to stimulate collagen secretion by mesangial cells in vitro, and to contribute to the overproduction of glomerular mesangial matrix in vivo. To delineate mechanisms responsible for these effects, we examined the influence of glycated albumin on transcriptional activation of the alpha 1 (IV) collagen gene in renal glomerular mesangial cells. These experiments used a stably transfected reporter mesangial cell line that exhibits responses to media manipulations that are directionally parallel with those of non-transformed mesangial cells, and that expresses luciferase driven by 5'-flanking and first intron regions of the alpha 1 (IV) collagen gene. In these cells, purified glycated albumin stimulated collagen IV gene transcription, whereas glucose-free albumin did not. Further, glycated albumin induced a significant increase in mesangial cell collagen IV mRNA, assessed by Northern blot analysis and quantified by calculation of the ratio of collagen IV mRNA to 18S ribosomal RNA after densitometric scanning. The stimulation of collagen gene transcription and mRNA expression were both prevented by monoclonal antibodies known to specifically recognize Amadori-modified albumin. The findings indicate that glycated albumin promotes mesangial cell transcriptional activation and mRNA expression of the alpha 1 (IV) collagen gene and further implicate increased glycated albumin in diabetes in the pathogenesis of diabetic nephropathy.

Renal cells are a rich source of transforming growth factor (TGF)-beta, and they serve as targets for its actions. Our hypothesis that activation of the TGF-beta system in the kidney is implicated in the development of diabetic renal disease stems from the close similarity of actions of TGF-beta and high ambient glucose on renal cell growth and extracellular matrix metabolism. Proximal tubule cells and glomerular mesangial cells cultured in high glucose concentration express increased TGF-beta 1 mRNA and protein levels, and treatment with anti-TGF-beta antibodies results in prevention of the effects of high glucose to induce cellular hypertrophy and stimulate collagen biosynthesis. Several in vivo studies by different groups of investigators have reported overexpression of TGF-beta in the glomeruli in human and experimental diabetes. We have also observed that the development of renal hypertrophy in the insulin-dependent diabetic BB rat and NOD mouse is associated with increased expression of TGF-beta 1 in the kidney and that short-term administration of antibodies capable of neutralizing the activity of TGF-beta in the streptozotocin mouse model of diabetes results in attenuation of whole kidney and glomerular hypertrophy and overexpression of mRNAs encoding matrix components. Together, these findings are consistent with the hypothesis that the diabetic state stimulates TGF-beta expression in the kidney and that in turn this growth factor may mediate, in an autocrine/paracrine manner, some of the principal early manifestations of diabetic renal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

The elevated mRNA levels encoding matrix components in glomeruli isolated from streptozotocin-induced diabetic rats provide evidence that stimulation of matrix synthesis is important in early phases of diabetic glomerulopathy. We and others have demonstrated that high glucose stimulates collagen mRNA levels in short-term mesangial cell culture. To test whether transcriptional activation is operative and to gain insights into the underlying mechanisms, we studied a murine mesangial cell line stably transfected with a minigene expressing luciferase driven by 5'-flanking and first-intron regions of the alpha 1(IV) gene. High glucose stimulated luciferase activity dose and time dependently, with optimal stimulation (two-fold) achieved after 48 h in 450 mg/dL glucose (G450) versus 100 mg/dL (G100). We next tested the involvement of protein kinase C (PKC) because high glucose has been shown to stimulate de novo synthesis of diacylglycerol (DAG). Increasing PKC activity by treatment with a DAG analogue or active phorbol ester stimulated luciferase activity preferentially in G100; addition of the PKC inhibitors staurosporine or calphostin C markedly inhibited luciferase activity preferentially in G450. Thus high glucose promotes transcriptional activity of alpha 1(IV) gene through PKC activation. We also tested the involvement of protein kinase A (PKA). Intracellular cyclic AMP levels were increased two fold after 48 h in G450 versus G100, and addition of 8-Br-cAMP (0.1 mM) preferentially stimulated luciferase activity by almost three fold in G100 versus only 1.2-fold in G450. Hence, the signal-transduction mechanisms underlying the transcriptional activation of alpha 1(IV) gene in mesangial cells by high glucose are mediated by pathways involving the PKC system and possibly the cAMP/PKA system.

Cloning studies have characterized a large gene family of facilitative glucose transporters that are expressed in a tissue-specific manner, and at least two sodium-glucose co-transporters in brush-border membranes of intestinal and renal epithelia. These different transporters play specific roles in glucose transport and homeostasis because of their different tissue distribution, kinetic properties, and regulation of function. This review discusses recent advances in the study of glucose transporters and the regulation of their expression in the normal kidney and in diabetes.

Atrial natriuretic peptide (ANP) is a growth suppressor for a variety of different cell types including cultured mesangial cells. This study examined the effects of ANP on the expression and synthesis of transforming growth factor-beta (TGF-beta) in a cultured murine mesangial cell line (MMC). Full-length rat ANP as well as 8-bromo-cGMP, but not ring-deleted ANP analogues, induced, in a dose-dependent manner, TGF-beta 1 mRNA expression in MMC. Moreover, full-length ANP and 8-bromo-cGMP stimulated the synthesis and release of TGF-beta 1 into cell culture supernatants as measured by a specific enzyme-linked immunosorbent assay and mink cell bioassay. The induction of TGF-beta 1 by 10(-6) M ANP and 10(-4) M 8-bromo-cGMP for 24 h was also documented by western blotting of MMC culture supernatants. However, transient transfection studies in MMC with three different murine TGF-beta 1 reporter gene constructs revealed only little stimulation of activity after ANP treatment, suggesting that posttranscriptional activation may mainly contribute to TGF-beta mRNA induction. Functional studies demonstrated that the ANP-mediated inhibition of mitosis, induced by 5% fetal calf serum, is partly abolished in the presence of a neutralizing anti-TGF-beta 1-3 antibody. In addition, the antiproliferative effects of exogenous 8-bromo-cGMP are also attenuated by anti-TGF-beta 1-3 antibody. These findings indicate that ANP stimulates TGF-beta expression in MMC and that the antiproliferative effects of ANP in this cell line may be mediated, at least to some extent, by the endogenous induction of TGF-beta. An increase in intracellular cGMP is most likely the mediator of this TGF-beta 1 induction. Considering the fibrogenic effects of TGF-beta, these findings may be important in the development of glomerulosclerosis in situations with an increase in local or systemic ANP.

Transforming growth factor-beta (TGF beta) is a multifunctional cytokine showing growth effects on many cell types. In the present study effects of TGF beta 1 on mitogen-induced Ca2+ responses were investigated in an immortalized murine mesangial cell line where TGF beta 1 effects on growth are inhibitory. TGF beta 1 was found to inhibit intracellular Ca2+ mobilization induced by platelet-derived growth factor (PDGF). This effect was completely reversed by previous addition of the non-specific serine/threonine kinase inhibitor H-7, but was unaffected by GF 109203X, a specific inhibitor of protein kinase C (PKC). These findings suggest that inhibition of mitogen-induced Ca2+ mobilization by TGF beta 1 appears not to involve prior activation of PKC, but may participate in the mechanisms whereby mesangial cell growth is inhibited by TGF beta 1.

Accelerated protein glycation in diabetes has been mechanistically linked to the pathogenesis of diabetic nephropathy. Because glycated albumin induces abnormalities in cultured mesangial cells that resemble those characterizing the glomerular mesangium in diabetes, and monoclonal antibodies (A717) specific for Amadori-modified glycated albumin prevent these abnormalities, we postulated that in vivo administration of A717 could retard the progression of diabetic nephropathy. To test this hypothesis, diabetic db/db mice and their nondiabetic db/m littermates were treated with eight consecutive weekly injections of 150 micrograms of A717 (Fab fragments) to reduce the elevated plasma glycated albumin concentration, or with irrelevant murine IgG (MIg). Relative to nondiabetics, diabetic mice (MIg treated) manifested proteinuria (3.35 +/- 0.15 vs 0.87 +/- 0.1 mg albumin/mg creatinine), 3.8-fold increase in mesangial matrix fraction, and renal cortical overexpression of mRNAs encoding alpha 1(IV) collagen (2.6-fold increase) and fibronectin (3.8-fold increase). Treatment of db/db mice with A717 significantly reduced the proteinuria (1.52 +/- 0.3 mg/mg creatinine), inhibited mesangial matrix expansion, and attenuated overexpression of matrix mRNAs. The nephropathic protective effects of A717 were independent of any change in blood glucose concentrations. Antibodies unreactive with glycated albumin did not duplicate the beneficial effects of A717. Thus, abrogating the biologic effects of increased glycated albumin with A717 has a salutary influence on the pathogenesis of diabetic nephropathy and has novel therapeutic potential in its management.

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited 3H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.

Increased blood flow and vascular permeability of early diabetes have been associated with increased nitric oxide formation in diabetic rats, but the specific nitric oxide synthase responsible is unknown. We examined the modulation of the induction and activity of the inducible NOS isoform by high glucose concentration in a murine macrophage cell line, RAW 264.7, and murine glomerular mesangial cells. Culturing both cell types in high glucose concentration led to significant increases in nitrite production and the mRNA encoding iNOS upon stimulation with LPS plus interferon-gamma, as compared with normal glucose concentration. High glucose also modestly enhanced LPS/IFN-gamma-induced stimulation of the iNOS promoter in transient transfection experiments in mesangial cells. Protein kinase C activation led to enhanced mRNA expression of iNOS, and inhibitors of protein kinase C blocked nitrite accumulation in mesangial cells. These findings suggest that high glucose in combination with stimulation by LPS plus IFN-gamma enhances iNOS expression, and protein kinase C activation may be playing a role in this enhancement.

Renal injury in diabetes mellitus is a major cause of morbidity and mortality in diabetic patients. There is a clear correlation between the degree of glomerular as well as tubulointerstitial lesions and the development of reduced glomerular filtration rate. The important role of hyperglycemia in the genesis of diabetic renal disease has been strengthened by the application of tissue culture techniques. Recent in vitro studies, first in tubular epithelial cells and subsequently in the three glomerular cell types, have provided supportive evidence that high ambient glucose per se stimulates the synthesis of extracellular matrix components. Increased matrix synthesis and decreased degradation are thought to contribute to matrix accumulation in diabetic nephropathy. These processes are not mutually exclusive and they may be operating simultaneously but at different rates, with increased synthesis predominating early and decreased breakdown later in the course of the disease. Likely mediators of the effects of high glucose involve activation of the polyol pathway, altered myo-inositol metabolism, increased protein kinase C activity, and/or nonenzymatic glycation of various matrix proteins. A role for various growth factors, especially transforming growth factor-beta, also seems likely. However, the details of the cell-signaling mechanisms and the putative molecular mediators of the effect of hyperglycemia remain to be firmly established.

Renal hypertrophy is an early feature of diabetes, and it may predispose the kidney to the eventual development of parenchymal dysfunction. Since we have previously demonstrated that short-term culture in high glucose concentration stimulates production of transforming growth factor-beta 1 (TGF-beta 1) in proximal tubular and glomerular mesangial cells, we postulated that this cytokine, which has potent regulatory effects on cellular growth and extracellular matrix production, is important in mediating diabetic renal disease. In this study we evaluated the gene and protein expression of TGF-beta 1 in the kidney of two rodent models of spontaneous insulin-dependent diabetes mellitus [the biobreeding (BB) rat and the nonobese diabetic (NOD) mouse]. In association with the appearance in both models of significant renal hypertrophy, TGF-beta 1 mRNA levels were increased threefold in the kidney of the diabetic BB rat after 3 days of diabetes and also threefold after 7-9 days in the NOD mouse. There was no increase in TGF-beta 1 transcripts in the livers of the diabetic animals, suggesting that this response is tissue specific. Immunohistochemical studies revealed that TGF-beta 1 protein is concordantly elevated in the cortical tubular cells of the diabetic kidney in both models. These results suggest that the stimulated expression of renal TGF-beta is an early manifestation of the involvement of the kidney by diabetes. Whether increased TGF-beta production in the diabetic kidney is a key promoter of diabetic renal manifestations (e.g., hypertrophy) deserves additional studies.

Increased glomerular collagen IV mRNA in streptozotocin-diabetic rats and stimulation of matrix transcripts by high glucose levels in short-term mesangial cell culture provide evidence that stimulation of matrix synthesis is important in early diabetic glomerulopathy. To test whether transcriptional modulation of collagen IV genes is operative, we stably transfected a murine mesangial cell line with a "minigene" expressing luciferase driven by 5'-flanking and first-intron regions of the murine COL4A1 gene to assess the response to high glucose and the associated signaling pathway. Luciferase activity was stimulated in a dose- and time-dependent manner [near-maximal stimulation in 450 mg/dl glucose (G450) was more than twofold the level in 100 mg/dl (G100) at 48 h]; high concentrations of D-mannitol were without effect. Neither low (2 ng/ml) nor high doses (2 micrograms/ml) of insulin modified luciferase activity in either G100 or G450. We next studied whether activation of protein kinase C (PKC) mediates the effect of high glucose. Treatment with the active phorbol ester phorbol 12-myristate 13-acetate for 2-4 h or with a diacylglycerol analogue for 24 h significantly stimulated luciferase activity preferentially in G100; the PKC inhibitors staurosporine or calphostin C significantly reduced the activity preferentially in G450. Thus high glucose levels promote transcriptional activity of COL4A1 gene in this reporter mesangial cell line, perhaps through PKC activation.

The polyol pathway in diabetes is activated in tissues that are not dependent on insulin for glucose uptake. To examine the role of the polyol pathway in renal extracellular matrix accumulation, we incubated murine proximal tubule cells in either normal or high glucose concentration in the presence or absence of the aldose reductase inhibitor sorbinil. Rising medium glucose from 100 to 450 mg/dl for 72 hours increased cell sorbitol levels sevenfold. Addition of 0.4 mM sorbinil reduced sorbitol content to virtually undetectable levels as measured by gas chromatography. Sorbinil (0.1 to 0.2 mM) also reduced the secretion of collagens types IV and I in the high glucose concentration after 48 to 72 hours but had no appreciable effect in the normal glucose concentration. Concordantly, 0.1 mM sorbinil inhibited the high glucose-induced stimulation of alpha 1(IV) and alpha 2(I) mRNA levels without affecting levels in normal glucose concentration. To study the role of transcriptional activation of collagen genes, we transfected proximal tubule cells with a chloramphenicol acetyltransferase (CAT) reporter gene linked to the promoter and regulatory elements of alpha 1(IV) gene. CAT activity increased several-fold in the cells grown in the high versus normal glucose concentration; this transcriptional activation in culture media containing high glucose concentration was reduced by treatment of the cells with 0.1 mM sorbinil. Thus, high ambient glucose activates the polyol pathway in proximal tubule cells, and may mediate the high glucose-induced stimulation of gene expression for collagens types IV and I.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous investigations have demonstrated that growing mesangial cells in high glucose concentration stimulates extracellular matrix synthesis and also increases the expression of TGF-beta. We tested whether the stimulation of extracellular matrix production is mediated by autocrine activation of TGF-beta, a known prosclerotic cytokine. Addition of neutralizing anti-TGF-beta antibody, but not normal rabbit IgG, significantly reduced the high glucose-stimulated incorporation of 3[H]proline. Denaturing SDS-PAGE revealed that mainly collagen types I and IV were stimulated by high (450 mg/dl) D-glucose. This high glucose-mediated increase in collagen synthesis was reduced by the anti-TGF-beta antibody. Treatment of mesangial cells grown in normal (100 mg/dl) D-glucose with 2 ng/ml recombinant TGF-beta 1 mimicked the effects of high glucose. Furthermore, the anti-TGF-beta antibody significantly reduced the increase in mRNA levels encoding alpha 2(I) and alpha 1(IV) collagens induced by high glucose. Thus, the high glucose-stimulated increase of collagen production in mesangial cells is mediated, at least in part, by autocrine TGF-beta activation. We postulate that the interception of the glomerular activity of TGF-beta may be an effective intervention in the management of diabetic nephropathy.

Diabetic serum contains increased concentrations of glycated proteins, which are preferentially transported into the renal glomerulus. We investigated effects of Amadori glucose adducts in serum proteins, the predominant form in which circulating glycated proteins exist in vivo, on glomerular mesangial cells, where the lesion of diabetic nephropathy originates. [3H]-thymidine incorporation by murine mesangial cells was significantly inhibited when cells were grown in the presence of serum glycated by incubation for four days with 28 mM glucose or when cells were cultured in microtiter plates that had been precoated with glycated serum. This effect was prevented by a monoclonal antibody immunoreactive with Amadori adducts in glycated albumin, and unreactive with other glycated serum proteins or with advanced glycation end (AGE) products. Glycated serum stimulated Type IV collagen gene expression and increased Type IV collagen secretion, an effect also prevented by monoclonal antibodies reactive with Amadori adducts in glycated albumin. The glycation-induced changes in proliferation, collagen synthesis and collagen gene expression were observed in media containing normal glucose concentration and were exaggerated in media containing high glucose concentration. The data indicate that Amadori products of glycated serum proteins induce mesangial cell abnormalities that are highly relevant to the pathogenesis of diabetic nephropathy, and that these effects are accentuated when glycated serum proteins are presented in a hyperglycemic milieu. The data also suggest that mesangial cells specifically recognize Amadori adducts in glycated albumin.

In the subgroup of diabetic patients who are destined to develop the full spectrum of the clinical syndrome of diabetic nephropathy, the kidney is afflicted with a series of distinct structural lesions principally involving the extracellular matrices. Diabetic nephropathy is characterized by hypertrophy of both glomerular and tubular elements, progressive accumulation of extracellular matrix components in the glomerular mesangium, and thickening of the glomerular and tubular basement membranes. Albeit less well recognized, progressive tubulointerstitial fibrosis is also a feature of the syndrome. Irrespective of pathogenetic mechanisms (be they metabolic, hemodynamic, or genetic), the structural changes involving the renal extracellular matrix compartments are believed to be the basis for the appearance of overt dysfunction, namely, proteinuria, hypertension, and renal failure. Therefore, a full understanding of the mechanisms that culminate in irreversible kidney failure requires a closer inspection of the status of the extracellular matrix in diabetes. This review outlines the different structural changes that typically occur during the course of the disease. Both glomerular and tubulointerstitial changes are reviewed. Valuable structural-functional correlations have been derived from examining kidney specimens obtained from patients with a wide spectrum of disease stages. Experimental animal models, supplanted with recent investigations in tissue culture on the effect of high ambient glucose levels, have increased our understanding of the cellular mechanisms that underlie the disordered matrix composition. Alterations in the metabolism of the collagens, proteoglycans, and other matrix constituents are reviewed.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies by our group have demonstrated that angiotensin II (ANG II), as a single factor in serum-free medium, induces cellular hypertrophy of a cultured murine proximal tubular cell line (MCT). The present study was performed to test the hypothesis that this growth effect was mediated by activation of endogenous transforming growth factor-beta (TGF-beta). Exogenous TGF-beta 1 (1 ng/ml) mimicked the growth effects observed with 10(-8) M ANG II (inhibition of DNA synthesis and induction of cellular hypertrophy). A neutralizing anti-TGF-beta antibody attenuated the ANG II-induced increase in de novo protein and total RNA synthesis as well as total protein content. This antibody also abolished the ANG II-mediated inhibition of [3H]thymidine incorporation into quiescent MCT cells. Control IgG or an unrelated antibody had no effect. A bioassay for TGF-beta using mink lung epithelial cells revealed that MCT cells treated with ANG II released active TGF-beta into the cell culture supernatant. Northern blot analysis and semi-quantitative cDNA amplification demonstrated increases in steady-state levels for TGF-beta 1 mRNA after ANG II stimulation of MCT cells, but not in a syngeneic murine mesangial cell line. Our data indicate that the ANG II-induced hypertrophy in MCT cells is mediated by synthesis and activation of endogenous TGF-beta. It is intriguing to speculate that TGF-beta may play a role in the early tubular cell hypertrophy and the subsequent interstitial scarring observed in several models of chronic renal injury that are characterized by increased activity of intrarenal ANG II.

Nonenzymatically glycated proteins are preferentially transported across the glomerular filtration barrier, and the glomerular mesangium in diabetes is bathed with serum containing increased concentrations of glycated albumin. We investigated effects of glycated albumin on mesangial cells, which are involved in diabetic nephropathy. [3H]-thymidine incorporation was significantly inhibited when murine mesangial cells were grown in culture media containing human serum that had been nonenzymatically glycated by incubation for 4 days with 28 mM glucose. This inhibition was reversed when monoclonal antibodies that selectively react with Amadori products of glycated albumin were added to the culture media. Purified glycated albumin containing Amadori adducts of the glycation reaction induced significant inhibition of thymidine incorporation and stimulation of Type IV collagen secretion compared with cells cultured in the presence of purified nonglycated albumin. These changes were prevented when monoclonal antibodies specifically reactive with fructosyl-lysine epitopes in glycated albumin were added to the cultures. The antibodies had no effect on growth or collagen production in the presence of nonglycated albumin. The results provide the first evidence directly implicating Amadori adducts in glycated albumin in the pathogenesis of diabetic nephropathy, which is characterized by decreased cellularity in association with expansion of the mesangial matrix.

The pathogenesis of the multiple structural lesions in diabetic nephropathy remains debated, and likely is multifactorial. The uniform thickening of the renal basement membranes lining the glomerular and tubular elements appears to be a consequence of the metabolic perturbations which are directly related to hyperglycemia. While most investigations have focused on the increased accumulation of extracellular matrix in the glomerular basement membrane and the mesangium, and their relation to derangements in glomerular function, little is known regarding the pathogenesis and the significance of the tubulointerstitial changes and the thickened tubular basement membrane (TBM). It is possible that these latter changes are causally related to the cellular hypertrophy of the renal tubular epithelium that lines the TBM. It has been postulated that in the earlier stages of the disease, hyperglycemia induces renal tubular hypertrophy and stimulates the synthesis of the various matrix components which are normal constituents of the TBM. Later, the structural composition of the TBM is susceptible to further modifications by non-enzymatic glycation, and this aberrant process may impart a relative resistance to matrix degradation leading to a slow turnover. In vitro investigations on murine proximal tubule cells in culture have provided evidence that elevated ambient glucose is a sufficient stimulus for cellular hypertrophy and increased biosynthesis of collagen type IV, the predominant constituent of TBM. High glucose levels increase steady-state collagen IV mRNA, partly due to transcriptional activation of cis-acting elements of the gene which are controlled by putative glucose-responsive trans-acting proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of glucose concentration in serum-free media on the proliferative growth response of a cultured murine mesangial cell line. Raising the ambient D-glucose concentration from 100 mg/dl to 450 mg/dl stimulated cell proliferation after 24 to 48 hours but had a growth inhibitory effect after 72 to 96 hours of incubation. This biphasic proliferative response to high glucose concentration was not mediated by the elevated osmolarity of the medium and did not occur when L-glucose was used. The early phase of glucose-induced proliferation was associated with increased expression of the immediate early genes c-myc and egr-1 as well as with induction of the S-phase related proliferating nuclear cell antigen (PCNA). Several lines of evidence indicated that the late phase of glucose-induced growth inhibition was mediated by the bioactivation of endogenous transforming growth factor beta (TGF-beta). Neutralizing antibody against TGF-beta prevented the late inhibitory effects of glucose on proliferation. On the other hand, exogenous TGF-beta (1 ng/ml) significantly inhibited basal proliferation in mesangial cells. Furthermore, Northern blot analysis revealed that TGF-beta 1 mRNA was induced by 450 mg/dl glucose in the medium after 48 to 72 hours, but not after 24 hours. Cell cycle analysis demonstrated that mesangial cells incubated in high glucose for 24 hours have a higher percentage of cells in the S-G2 phase of the cell cycle compared with cells grown in normal glucose concentration. After 48 hours of culture in elevated glucose concentration, the percentage of cells in S-G2 phase was decreased, and became comparable to that of cells in normal glucose concentration. However, the addition of neutralizing anti-TGF-beta antibody stimulated the progression of cells towards S-G2 in high glucose medium after 48 hours. The findings of this study demonstrate a biphasic growth response of mesangial cells when they were cultured in high glucose concentration; initially there was a transient stimulation of replication for 24 to 48 hours followed by a sustained inhibition after longer incubation periods. This inhibition may be mediated by the glucose-induced synthesis and/or bioactivation of TGF-beta which can inhibit proliferation of mesangial cells in an autocrine fashion.

Polycystic kidney disease is an inherited disorder of parenchymal structure that leads to renal failure. Cysts begin as focal dilations in proximal tubules and collecting ducts, giving rise to cyst walls lined by a phenotypically disturbed epithelium that expresses dysfunctional transport and matrix proteins. We used an mRNA search protocol to probe efficiently for tissue-specific disturbances that might underlie the formation of cysts. This search assessed the relative abundance of transcripts encoding a variety of growth factors (transforming growth factor-beta 1, interleukin-6, tumor necrosis factor, and endothelin-1), structural proteins (collagen IV, nidogen, fibronectin, and laminins A and B1), and cell adhesion molecules (CAMs; E-cadherin, N-CAM, laminin receptor, and fibronectin receptor) in the cystic kidneys of cpk/cpk mice and uncovered a previously unrecognized early reduction in mRNA encoding N-CAM (54%) and E-cadherin (56%) (n = 5; P less than 0.001). Levels of transcripts for growth factors, structural proteins, and for fibronectin and laminin receptors in normal and cystic kidneys were generally similar. The reduction in transcripts for N-CAM and E-cadherin in kidneys from cystic mice was not observed in autologous liver. The immunofluorescent staining of cystic kidneys confirmed that the decrease in N-CAM and E-cadherin was generally confined to regions abundant in developing cystic epithelium. The presence of both N-CAM and E-cadherin appears to guide the sequential differentiation and polarization of normal renal epithelium, and their attenuated expression in the kidney of cpk/cpk mice may be a material factor contributing to the pathogenesis of cyst formation.

Hypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress. However, hypotonicity enhanced 10- to 20-fold the efflux of organic cell osmolytes taurine, betaine, and trimethyloxamine, and this accounted for the loss of osmotically obliged water during RVD. Enhancement of osmolyte efflux by hypotonic stress was abolished by readjusting the low-Na+ saline to isotonicity (approximately 900 mosM) with innocuous cations (choline+, Li+, or N-methylglucamine+). The results suggest that reduction of medium tonicity may be the determinant for the RVD response to hypotonic stress. The above properties of the observed RVD were also displayed when studying changes on cell F-actin at the basolateral cell face; hypotonic stress (medium with 135 mM NaCl) produced a rapid disappearance of fluorescence related to this cytoskeletal component, whereas no such changes were seen in low-Na+ salines made isotonic with choline or N-methylglucamine chloride nor in a saline made hyposmolar by omitting urea. Hence, hypotonicity is required to affect F-actin organization (depolymerization?). These changes of F-actin fluorescence are transient; they were completed within 5-10 min of hypotonic stress, and afterwards a gradual reconstitution of cell F-actin organization was seen. The above observations are consistent with the assumption that, in shark rectal gland cells, transient loss of cytoskeleton (F-actin) organization at the basolateral cell face, induced by hypotonicity, brings about a selective efflux of organic osmolytes, thus producing the observed RVD.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.

Our previous studies have demonstrated that raising ambient glucose from 100 to 450 mg/dl significantly inhibited the proliferation of mouse renal proximal tubule cells in culture. This effect was demonstrated after a latent period of 24 to 48 hours. Because transforming growth factor-beta (TGF-beta) inhibits cell proliferation in most epithelial cell lines, we hypothesized that the inhibitory effect of high glucose levels on cell proliferation may be mediated by TGF-beta. The present studies were performed to test the hypothesis that TGF-beta is an autocrine cytokine whose activity can be modulated by ambient glucose. Exogenous TGF-beta inhibited [3H]-thymidine incorporation in a dose-dependent fashion and with high affinity (picomolar range), but with slightly lower potency in high versus normal glucose media. Northern analysis of mRNA demonstrated that proximal tubule cells constitutively express TGF-beta 1 transcripts, and that the steady state level of TGF-beta 1 mRNA was, on average, 63% higher in the cells grown for 48 hours in high versus normal glucose media. Furthermore, the conditioned media of cells exposed to 450 mg/dl glucose exhibited endogenous TGF-beta bioactivity as measured by inhibition of cell proliferation. The addition of a rabbit antiporcine TGF-beta neutralizing antibody significantly increased basal thymidine incorporation in high glucose media to levels approaching those of cells grown in normal glucose media. In contrast, the anti-TGF-beta antibody did not have a significant effect on the growth of cells in the normal glucose media.(ABSTRACT TRUNCATED AT 250 WORDS)

Tubulointerstitial lesions often develop in the kidneys of patients and experimental animals with diabetes mellitus. In an in vitro model of diabetic renal disease, it has been previously demonstrated in this laboratory that elevated glucose levels stimulate procollagen transcription and secretion in proximal tubule cells in culture while inducing cellular hypertrophy and reducing cellular proliferation. Previous experiments in other tissues have suggested that myo-inositol supplementation, probably by reversing a disturbance in cell myo-inositol metabolism related to increased activity of the polyol pathway, reverses the effects of glucose on cell function. We tested the effect of myo-inositol supplementation on proximal tubule cells in culture in the presence of elevated medium glucose level. Incubation in 450 mg/dL of glucose media reduced cell proliferation; 450 mg/dL of glucose plus myo-inositol (800 microM) increased proliferation, returning the value to that seen in cells incubated in 100 mg/dL of glucose. Incubation in 450 mg/dL of glucose media increased type IV and type I procollagen mRNA levels and peptide secretion rates compared with those seen in cells incubated in medium containing 100 mg/dL of glucose. This glucose-induced stimulation of procollagen mRNA levels and procollagen secretion was not observed when the elevated glucose medium was supplemented with 800 microM myo-inositol. On the other hand, myo-inositol supplementation did not prevent the glucose-induced cellular hypertrophy: there was no reduction in the increased leucine incorporation and cellular protein content. Cell incubation in 450 mg/dL of glucose media did not lead to a measurable decrease in total cellular myo-inositol.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of cultured murine proximal tubular cells in serum-free media containing 450 mg/dl of glucose resulted in cellular hypertrophy as defined by an increase in cell size, total protein content, and synthesis after 72 h. 10 nM angiotensin II further increased this hypertrophy, but failed to have any effect on cells grown in 100 mg/dl glucose. This enhancement by angiotensin II was blocked by treatment with 1 microM of the angiotensin-receptor antagonist DuP 753. Although cells incubated in either glucose media exhibited similar high-affinity angiotensin II-receptors, the receptor density was elevated only in cells grown in the presence of high glucose. Stimulation of cells in high glucose for 60 min with 10 nM angiotensin II also reduced significantly intracellular cAMP concentrations. This was not the case for proximal tubular cells cultured in normal glucose. Our results indicate that high glucose and angiotensin II have additive effects on the induction of hypertrophy in renal tubular cells.

Early functional disturbances in nerve, retina, and lens in diabetes mellitus appear to result from a common mechanism involving increased polyol-pathway activity with an associated effect on tissue myo-inositol metabolism. We tested the role of increased polyol-pathway activity in the early glomerular hemodynamic abnormalities in experimental diabetes in rats with dietary myo-inositol supplementation or the administration of sorbinil, an aldose reductase inhibitor. Each maneuver prevented the glomerular hyperfiltration of early streptozocin-induced diabetes and reversed the hyperfiltration of established diabetes of 10 days' duration. We also found that the abnormal response to captopril in diabetic rats was improved by dietary myo-inositol supplementation or sorbinil administration. Although nonhypotensive doses of captopril lowered glomerular filtration rate (GFR) in diabetic rats on a 0.01% myo-inositol diet, GFR increased substantially after captopril infusion in diabetic rats treated with sorbinil or myo-inositol supplementation. These data suggest that normalization of tissue myo-inositol metabolism restores normal responsiveness to angiotensin II; this may contribute to the reduction in GFR with the two experimental maneuvers. We also tested the interaction between polyol-pathway activation and high dietary protein intake. Aldose reductase inhibition and dietary myo-inositol supplementation had no effect on the component of increased GFR due to 50% dietary protein intake but specifically inhibited the hyperfiltration attributable to diabetes. These results suggest that hyperglycemia acts through increased polyol-pathway activity and its effects on tissue myo-inositol metabolism to play a fundamental role in the pathogenesis of the glomerular hyperfiltration characteristic of early diabetes.

Tubulointerstitial changes in the diabetic kidney correlate closely with the decline in glomerular filtration. In this study, we used a cell culture system of mouse proximal tubule epithelial cells to test the effects of glucose on cell growth, size, and matrix biosynthesis. [3H]thymidine incorporation was significantly inhibited in cells grown in 450 mg/dl glucose, compared with cells grown in 100 mg/dl glucose. The cells grown in the higher glucose concentration were slightly larger, their protein content and the total protein synthetic rate were significantly increased, and they secreted approximately twice as much procollagens type IV and type I. Concordantly, steady-state procollagen mRNA levels were also increased: 2.6-fold for the alpha 1(IV) and 2.2-fold for the alpha 2(I) procollagens. Additionally, nuclear run-off studies demonstrated that procollagen gene transcription rate was stimulated approximately 50%; beta-actin transcription rate was not altered. We used chloramphenicol acetyltransferase (CAT) reporter gene constructs to determine whether the increased transcription rate of alpha 2(I) gene was associated with activation of its enhancer sequence. Cells transfected with the enhancer demonstrated more than fivefold increase in CAT activity when cultured in the high-glucose medium. These studies demonstrate a multitude of effects of high ambient glucose concentrations on proximal tubule cell growth and collagen biosynthesis; cell proliferation is decreased although cell hypertrophy occurs. Procollagen gene transcription rate is stimulated and this response contributes to the observed increase in procollagen mRNA content. Activation of an enhancer sequence may be one possible mode through which high glucose levels increase the transcription of procollagen type I, presumably involving trans-acting factor(s).

(1) 0.1-1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark (Squalus acanthias) rectal glands, with an uptake of cell Na+ and a loss of K+. In contrast, 1 mM N-ethylmaleimide (NEM) does not swell rectal gland cells (RGC), while affecting cell cations. (2) The slow entry of [203Hg]pCMBS is linearly related to its external concentration (10 microM-1 mM) and a small accumulation of pCMBS (apparent gradient about 3) in the cells occurs in 2 h. Cell 203Hg rapidly washes out of the cells (fast rate constant 0.153.min-1; slow rate constant 0.0067.min-1), and this efflux is accelerated by 1mM dithiothreitol. Thus, a major portion of pCMBS inter-acts rather loosely with cell components. (3) pCMBS and NEM share: (a) a negligible effect on the efflux of 86Rb+ and of [14C]urea; (b) a gradual inhibition of the cell Na+,K(+)-ATPase activity. (4) NEM as well as agents lowering cell glutathione accelerate and increase the pCMBS-induced cell swelling. Conditions inhibiting the Na+,K(+)-ATPase (ouabain, absence of Na+) have the same effect. (5) pCMBS, but not NEM produce a disappearance of the F-actin-phalloidin fluorescence independent of cell volume changes, particularly at the basolateral RGC membrane. (6) The data are consistent with the following set of events: (a) pCMBS (but not NEM) affects the cell membrane by increasing the efflux of the cell osmolyte taurine (Ziyadeh et al. (1988) Biochim. Biophys. Acta 943, 43-52 and unpublished data); (b) on entry into the cells, pCMBS and NEM interact with cell -SH, including those of the Na+,K(+)-ATPase; this action produces the observed changes in cell cations. Also, pCMBS, but not NEM, decrease F-actin at the membrane; (c) the inhibition of the Na+,K(+)-ATPase activity together with the decreased resistance of the cell membrane to stretch (absence of F-actin) produces the observed pCMBS-induced cell swelling by osmotic forces (intracellular non-diffusible anions).