Proposal for a unified CCN nomenclature.

A proposal is put forth to unify the nomenclature of the CCN family of secreted, cysteine rich regulatory proteins. In the order of their description in the literature, CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3) constitute a family of matricellular proteins that regulate cell adhesion, migration, proliferation, survival, and differentiation, at least in part through integrin mediated mechanisms. This proposal is endorsed by the International CCN Society and will serve to eliminate confusion from the multiple names that have been given to these molecules.

Glomerular mRNAs in human type 1 diabetes: biochemical evidence for microalbuminuria as a manifestation of diabetic nephropathy.

BACKGROUND: In patients with type 1 diabetes, some consider microalbuminuria to be a predictor of diabetic nephropathy while others believe it is an early feature of diabetic nephropathy. METHODS: Levels of mRNAs that are of pathogenetic relevance in diabetic nephropathy were compared in glomeruli isolated from microalbuminuric and overtly proteinuric subjects and in control normoalbuminuric diabetic subjects and living renal transplant donors. RESULTS: In subjects with microalbuminuria and overt proteinuria, glomerular mRNAs were virtually identical and approximately twofold higher for connective tissue growth factor (CTGF; P < 0.01) and collagen alpha2(IV) (P < 0.03) compared to living renal donors and normoalbuminuric patients. Glomerular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were not significantly different among the groups (P = 0.4). Weak but statistically significant correlations were noted between CTGF mRNA and albuminuria (assessed by rank), fractional mesangial surface area, and a composite renal biopsy index. Glomerular CTGF mRNA correlated inversely with creatinine clearance. Glomerular collagen alpha2(IV) mRNA levels correlated with albuminuria (by rank) and less strongly with fractional mesangial area. CONCLUSION: To our knowledge, these data provide the first biochemical evidence demonstrating that the glomeruli of microalbuminuric patients and those with overt proteinuria do not differ significantly. The data support the concept that microalbuminuria is not "predictive" of diabetic nephropathy, but rather is an earlier point in the spectrum of diabetic nephropathy.

Connective tissue growth factor and its regulation: a new element in diabetic glomerulosclerosis.

Connective tissue growth factor (CTGF), a member of the closely related CCN family of cytokines appears to be fibrotic in skin. To determine whether CTGF is implicated in diabetic glomerulosclerosis we studied cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to rhCTGF significantly increased fibronectin and collagen type I secretion. Further, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36-38 kDa). However, exposure to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in glomerulosclerosis, markedly induced the expression of CTGF transcripts. With all but mechanical strain there was a concomitant stimulation of CTGF protein secretion. TGF-beta also induced abundant quantities of a small molecular weight form of CTGF (18 kDa). The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta neutralizing antibody blocked this stimulation. In vivo studies using quantitative RT-PCR demonstrated that while CTGF transcripts were low in the glomeruli of control mice, expression was increased 27-fold after approximately 3.5 months of diabetes. These changes occurred early in diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (2-fold) observed in whole kidney cortices indicted that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation in both diabetic and non-diabetic glomerulosclerosis, acting downstream of TGF-beta.

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

F-actin fiber distribution in glomerular cells: structural and functional implications.

BACKGROUND: Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture. METHODS: Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy. RESULTS: The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli. CONCLUSION: Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.

Modelling the effects of vascular stress in mesangial cells.

It has recently been shown that mesangial cells are subjected to multiple forms of mechanical strain (fluid shear, hydrostatic pressure, and triaxial stretch) as a result of forces exerted by the vasculature. Nevertheless, the exact nature and the relative response to these stimuli have not been clarified. Although it is now well established that cyclic stretching of mesangial cells in culture results in the overproduction of extracellular matrix, indicating how intraglomerular hypertension may lead to glomerular scar formation, the contribution of different intracellular signalling mechanisms and extracellular mediators of the response are only now being identified. Recent studies point to a role for high glucose concentrations, transforming growth factor beta and its receptors, vascular endothelial growth factor, and connective tissue growth factor as important mediators, or modifiers of the response to mechanical strain. Although evidence exists for a role for protein kinase C, recent studies also implicate the mitogen-activated protein kinases along with enhanced DNA-binding activity of AP-1 as part of the signalling cascade altering matrix synthesis and cell proliferation in response to stretch. Finally, recent studies examining the effects of oscillating hyperbaric pressure demonstrate similarities, as well as differences, in comparison to those of cyclic stretch.

TGF-beta receptor expression and binding in rat mesangial cells: modulation by glucose and cyclic mechanical strain.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-beta, but may also involve alterations in receptor expression and binding. METHODS: Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-beta1 binding and expression of TGF-beta receptors at the gene and protein levels. RESULTS: Kinetic studies showed that MCs bound TGF-beta1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-beta receptors (betaR) corresponding to betaRI, betaRII, and betaRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of betaRI and betaRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-beta receptors, whereas ligands associated with betaRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) betaRI and betaRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with betaRs I, II, and III (100 to 250%). This same treatment also increased the levels of betaRI and betaRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced. CONCLUSION: These results suggest that the modulation of TGF-beta receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.

Characterization of the rat mesangial cell type 2 sulfonylurea receptor.

BACKGROUND: Sulfonylurea receptors are classified as either high-affinity type 1 (SUR1) or low-affinity type 2 receptors (SUR2), and the gene expression of SURs has recently been demonstrated in kidney. However, functional data regarding a renal SUR are lacking. We previously demonstrated that mesangial cell (MC) gene and protein expression of extracellular matrix components were up-regulated by the sulfonylurea, tolazamide. After noting this biological response, we next sought to investigate the presence of a sulfonylurea receptor in rat MCs. METHODS: Equilibrium binding studies employing [3H]glibenclamide as a ligand were performed on crude MC membrane preparations. Gene expression for SUR was explored by Northern analysis of cultured MCs and whole kidney tissue. The effect of sulfonylurea on intracellular Ca2+ in MCs was assayed by spectrofluorometry, and glibenclamide-induced changes in the contractility of MCs were assessed. RESULTS: MCs bound [3H]glibenclamide with a KD of 2.6 microM and a Bmax of 30.4 pmol/mg protein as determined by Scatchard analysis. Three SUR2 transcripts were detected in MCs. A major transcript was detected at 5.5 kb and minor transcripts at 7.5 and 8.6 kb. Following sulfonylurea treatment of MCs, real-time videomicroscopy revealed intense MC contraction, coinciding with oscillatory increments of intracellular Ca2+ concentration. Further evidence of sulfonylurea-induced MC contraction was demonstrated by glibenclamide-induced deformation of a silicone rubber substrate. CONCLUSIONS: These results demonstrate that SUR2 resides on MCs. Functional activation of this receptor by sulfonylurea induces Ca2+ transients that result in MC contraction.

Mechanical strain of glomerular mesangial cells in the pathogenesis of glomerulosclerosis: clinical implications.

Due to their elasticity, glomeruli will undergo excessive expansion and repetitive cycles of distension contraction under conditions of impaired glomerular pressure autoregulation and systemic arterial hypertension. These alterations in glomerular volume are associated with mesangial cell stretch which in turn stimulates the synthesis and deposition of ECM with eventual mesangial expansion and glomerulosclerosis. Hyperactivity of growth factors with prosclerotic activity is an important component in the translation of cellular mechanical strain into the abnormal metabolism of ECM components. Although mesangial cell mechanical strain is expected to occur in both remnant glomeruli and in glomeruli of diabetic kidneys, quantitatively different factors will determine the resultant metabolic consequences. In remnant glomeruli, the mechanical stretch is intense, being accounted for largely by the marked glomerular hypertrophy and increased glomerular compliance. In diabetic glomeruli, however, the mechanical stretch is less prominent but its effect on ECM synthesis is markedly aggravated by the presence of hyperglycaemia. There are presently no methods clinically available to diminish the prosclerotic action of growth factors at the glomerular level. In addition, there are no effective means to specifically improve glomerular pressure autoregulation. Therefore, current therapies must be aimed at decreasing systemic arterial pressure, blocking angiotensin II action and reducing glomerular hypertrophy. While there are effective drugs for the treatment of hypertension and for angiotensin II inhibition, protein restriction is the only measure available to diminish glomerular hypertrophy. Finally, in diabetes correction of systemic and glomerular hypertension should be coupled with strict glycaemic control to correct both glomerular autoregulation and increased ECM deposition.

Effects of oral antihyperglycemic agents on extracellular matrix synthesis by mesangial cells.

BACKGROUND: Increased expression of the glucose transporter GLUT1 in mesangial cells (MCs) markedly stimulates glucose transport and the formation of extracellular matrix (ECM), even when ambient glucose concentrations are low. Certain antihyperglycemic agents cause GLUT1 overexpression and increase glucose transport in various tissues. However, their effects on the kidney are unknown. Because diabetic glomerulosclerosis is characterized by the accumulation of mesangial matrix, was studied the effects of antihyperglycemic agents on matrix metabolism in MCs cultured either in 8 or 20 mM glucose. METHODS: Membrane-associated GLUT1 was measured by immunoblotting. The initial rate of glucose transport was determined according to the 2-deoxy-D[14C(U)]glucose uptake. Collagen metabolism was studied by metabolic radiolabeling with [14C]-proline. Fibronectin in the medium was measured by ELISA. GLUT1 mRNA was estimated by Northern analysis. RESULTS: The sulfonylurea tolazamide increased GLUT1 protein expression by 107 and 69% in 8 and 20 mM glucose-grown cells, respectively. However, GLUT1 mRNA levels remained unchanged. Transporter-dependent deoxyglucose uptake was increased by tolazamide up to 184% in a dose-dependent fashion and was evident at both glucose concentrations after three or five days of exposure to the drug. Tolazamide significantly stimulated transforming growth factor-beta 1 (TGF-beta 1) secretion and the total synthesis of collagen and collagen and fibronectin accumulation in the medium of MCs maintained in high or low glucose concentrations. The biguanide metformin did not alter GLUT1 expression, glucose transport, fibronectin formation, or collagen metabolism, except at high concentrations. CONCLUSION: Tolazamide markedly enhances ECM synthesis and accumulation in MCs probably by stimulating GLUT1 expression, glucose transport and TGF-beta 1 secretion, irrespective of the ambient glucose concentration. This effect was dose-dependent and minimally inducible by metformin.

Mechanical strain- and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-beta.

Cultured mesangial cells (MC) exposed to cyclic mechanical strain or high glucose levels increase their secretion of transforming growth factor-beta1 (TGF-beta1) and collagen, suggesting possible mechanisms for the development of diabetic renal sclerosis resulting from intraglomerular hypertension and/or hyperglycemia. This study examines whether glucose interacts with mechanical strain to influence collagen metabolism and whether this change is mediated by TGF-beta. Accordingly, rat MC were grown on flexible-bottom plates in 8 or 35 mM glucose media, subjected to 2 to 5 d of cyclic stretching, and assayed for TGF-beta1 mRNA, TGF-beta1 secretion, and the incorporation of 14C-proline into free or protein-associated hydroxyproline to assess the dynamics of collagen metabolism. Stretching or high glucose exposure increased TGF-beta1 secretion twofold and TGF-beta1 mRNA levels by 30 and 45%, respectively. However, the combination of these stimuli increased secretion greater than fivefold without further elevating mRNA. In 8 mM glucose medium, stretching significantly increased MC collagen synthesis and breakdown, but did not alter accumulation, whereas those stretched in 35 mM glucose markedly increased collagen accumulation. TGF-beta neutralization significantly reduced baseline collagen synthesis, breakdown, and accumulation in low glucose, but had no significant effect on the changes induced by stretch. In contrast, the same treatment of MC in high glucose medium greatly reduced stretch-induced synthesis and breakdown of collagen and totally abolished the increase in collagen accumulation. These results indicate that TGF-beta plays a positive regulatory role in MC collagen synthesis, breakdown, and accumulation. However, in low glucose there is no stretch-induced collagen accumulation, and the effect of TGF-beta is limited to basal collagen turnover. In high glucose media, TGF-beta is a critical mediator of stretch-induced collagen synthesis and catabolism, and, most importantly, its net accumulation. These data have important implications for the pathogenesis and treatment of diabetic glomerulosclerosis.

Role of glomerular mechanical strain in the pathogenesis of diabetic nephropathy.

Glomerular rigidity limits the glomerular expansion and mesangial cell (MC) stretch induced by variations in intracapillary pressure. In tissue culture, MC stretch stimulates synthesis of extracellular matrix components (ECM). Therefore, altered glomerular rigidity in diabetes may influence ECM accumulation by modulating the glomerular distention and MC stretch associated with glomerular hypertension. An ambient of high glucose concentration per se also enhances MC formation of ECM, possibly altering the cellular response to mechanical stretch. In this study, compliance was measured in isolated perfused glomeruli from streptozotocin-injected rats at four days (4d-D), five weeks (5w-D) and six months (6m-D) after induction of diabetes. In addition, collagen metabolism induced by stretch was investigated in MC cultured in 8 and 35 mM glucose concentrations. Glomerular compliance was normal in 5w-D rats and moderately increased in 4d-D (16%) and 6m-D animals (14%). As compared to static cultures. MC stretch increased total collagen synthesis (8 mM, 50%; 35 mM, 27%) and catabolism. However, while the fraction of newly formed collagen being catabolized increased in 8 mM-stretched cultures, in 35 mM-stretched it was unchanged. This resulted in marked increase in the net collagen accumulated in the incubation medium (4 vs. 24%) and cell layer 5 vs. 15%) only in the latter. In diabetes, the largely unaltered glomerular stiffness renders hypertension-induced MC stretch unopposed. More importantly, the accumulation of ECM caused by any degree of mechanical strain is greatly aggravated in a milieu of high glucose concentration.

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells.

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension.

Regulation of glomerular volume in normal and partially nephrectomized rats.

Glomerular extracellular matrix accumulation may derive from the stretching of mesangial cells caused by excessive glomerular dilatation. The relationship of glomerular volume (VG) to intraglomerular pressure, expressed as compliance or as mean VG in the isolated, perfused rat glomerulus, was used to analyze factors that regulate VG. Glomeruli were highly distensible over the normal and relevant abnormal range of pressure. Compliance increased directly with basal VG (P < 0.001), i.e., larger glomeruli dilated more than smaller ones at any given pressure. Perfusion with atrial natriuretic peptide did not alter compliance, and inhibitors of nitric oxide synthesis exerted only a trivial effect. VG expansion was consistently reduced by angiotensin II, but this effect was small (3.8%, P < 0.001). After subtotal nephrectomy, compliance increased by 59% in the remnant glomeruli (P < 0.001); 22% of this increase was attributable to structural changes, and the remainder was attributable to the large basal VG of the hypertrophied glomeruli. Thus the major determinants of VG expansion include capillary wall tension, basal VG, and intrinsic distensibility, which is markedly influenced by the character of the extracellular matrix and only slightly altered by an angiotensin II-modified mesangial cell tone.

Effect of Misoprostol on Mesangial Cell Growth and Collagen Metabolism.

Independent of etiology, progressive chronic renal failure is characterized by accumulation of mesangial matrix and collagenous materials leading to glomerular sclerosis and closure. Pharmacologic intervention aimed at ameliorating this process has significant potential for substantial clinical benefit. We, therefore, assessed the effect of misoprostol on glomerular mesangial cell growth and collagen metabolism. Studies were carried out using a rat glomerular mesangial cell line cloned in our laboratory. At the concentration tested (1 &mgr;M), neither misoprostol nor prostaglandin E(2) had any effect on glomerular mesangial cell proliferation. Misoprostol did not change the absolute synthesis rates for collagen or total protein when measured by (14)C-proline incorporation into protein-associated hydroxyproline and proline respectively. However, the amount of collagen extruded into the medium, as a percentage of protein synthesis, was decreased by 10% in misoprostol-treated cells (p = 0.042). In addition, collagen breakdown was 26% greater in misoprostol-treated cultures (p = 0.044). Misprosotol had no such effects on cell cultures subjected to mechanical stress applied as continuous stretch-relaxation cycles. These results indicate that misoprostol influences mesangial cell collagen metabolism by increasing the rate of endogenous breakdown and decreasing collagen export outside the cell. Misoprostol has no effect on mesangial cell proliferation.

Immunogold localization of high-affinity glucose transporter isoforms in normal rat kidney.

BACKGROUND: Facilitative glucose transporters (GLUT) have unique kinetic characteristics and distributions suited to the functions of the tissues in which they reside. However, little is known about their individual roles in renal glucose metabolism, and previous investigations of renal GLUT expression have been extensive only with respect to their mRNA levels. We provide here a complete analysis of three GLUT isoforms along the nephron using a sensitive immunodetection method. EXPERIMENTAL DESIGN: Normal rat kidneys were harvested, fixed in paraformaldehyde, and embedded in either paraffin or resin as required for immunogold labeling of individual GLUT Isoforms 1, 3, and 4. Samples were evaluated by light microscopy and selected regions analyzed by high resolution optical scanning with computer-assisted detection of immunogold-labeled GLUT at the subcellular level. We describe, compare, and related to the local patterns of glucose metabolism the cellular and subcellular expression patterns of these GLUT along the nephron. RESULTS: GLUT1 was most intensely labeled in the medullary thick ascending limbs of Henle, cortical collecting ducts, and inner medullary collecting ducts. In contrast, GLUT3 was most prominent in the inner medullary collecting ducts and GLUT4 in medullary thick ascending limbs of Henle. All three GLUT were detected in glomerular tufts, and GLUT1 was also detected in parietal epithelial cells. The predominant subcellular distributions in tubule cells were: basolateral and basolateral/cytoplasmic for GLUT1; basolateral and cytoplasmic for GLUT3; and perinuclear/cytoplasmic for GLUT4. GLUT 1 and 3 expressions were confirmed in specific regions by immunoblotting. CONCLUSIONS: 1) GLUT 1, 3, and 4 are expressed in both glomeruli and renal tubules. 2) The unique GLUT expression patterns along the renal tubules suggests unique functional roles for these isoforms. 3) The renal cortex demonstrates lesser labeling intensity for the high-affinity GLUT compared with the medulla, where higher rates of glucose oxidation and glycolytic metabolism are paralleled by higher GLUT labeling intensities.

Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.

An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT1 gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial beta-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis.

Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat.

To define the interplay of glomerular hypertension and hypertrophy with mesangial extracellular matrix (ECM) deposition, we examined the effects of glomerular capillary distention and mesangial cell stretching on ECM synthesis. The volume of microdissected rat glomeruli (Vg), perfused ex vivo at increasing flows, was quantified and related to the proximal intraglomerular pressure (PIP). Glomerular compliance, expressed as the slope of the positive linear relationship between PIP and Vg was 7.68 x 10(3) microns 3/mmHg. Total Vg increment (PIP 0-150 mmHg) was 1.162 x 10(6) microns 3 or 61% (n = 13). A 16% increase in Vg was obtained over the PIP range equivalent to the pathophysiological limits of mean transcapillary pressure difference. A similar effect of renal perfusion on Vg was also noted histologically in tissue from kidneys perfused/fixed in vivo. Cultured mesangial cells undergoing cyclic stretching increased their synthesis of protein, total collagen, and key components of ECM (collagen IV, collagen I, laminin, fibronectin). Synthetic rates were stimulated by cell growth and the degree of stretching. These results suggest that capillary expansion and stretching of mesangial cells by glomerular hypertension provokes increased ECM production which is accentuated by cell growth and glomerular hypertrophy. Mesangial expansion and glomerulosclerosis might result from this interplay of mechanical and metabolic forces.