High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha1(IV) collagen expression in response to endothelin-1: role of specific protein kinase C isozymes.

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal-regulated protein kinase (ERK1/2) signaling and alpha1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1-stimulated alpha1(IV) collagen mRNA expression from 1.2 +/- 0.1-fold to 1.9 +/- 0.2-fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)-PKC-delta, -epsilon, or -zeta inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1-driven GAL4 luciferase activity to 11 +/- 1-fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC-delta, -epsilon, or -zeta attenuated this response to NG levels. HG enhanced ET-1-stimulated intracellular alpha1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN-PKC-delta, -epsilon, -zeta, as well as DN-PKC-alpha and -beta, attenuated the response. Thus, HG-enhanced ET-1 stimulation of alpha1(IV) collagen expression requires PKC-delta, -epsilon, and -zeta to act through an ERK1/2-dependent pathway and via PKC-alpha and -beta, which are independent of ERK1/2.

Glomerular mesangial cell altered contractility in high glucose is Ca2+ independent.

In diabetes, loss of renal arteriolar smooth-muscle cell contractility leads to intraglomerular hypertension. In glomeruli isolated from streptozotocin (STZ)-induced diabetic rats, the mesangial cells (smooth muscle-like) display loss of contractile responsiveness to angiotensin II. This study examines the mechanistic relationship between altered mesangial cell contractility and vasopressor hormone-stimulated Ca2+ signaling in high glucose. Glomeruli were isolated from normal or STZ-induced diabetic rats to observe ex vivo mesangial cell contractile function. Also, rat mesangial cells were cultured (10-20 passages) in normal (5.6 mmol/l) or high (10-25.6 mmol/l) glucose for 1-5 days. Reduction of glomerular volume and decreased planar surface area of cultured mesangial cells in response to vasoconstrictor stimulation over 60 min were measured by videomicroscopy and personal computer-based morphometry. Contraction of glomeruli isolated from STZ-administered rat in response to endothelin (ET)-1 (0.1 mumol/l) or the Ca2+ ionophore A23187 (5 mumol/l) was impaired significantly compared with that in normal glucose. In the presence of arginine vasopressin (AVP) (1.0 mumol/l) or ET-1 (0.1 mumol/l), mesangial cells demonstrated a dose-dependent loss of contractile response to increasing glucose concentrations (5.6-25.6 mmol/l) within 24 h of high-glucose exposure, which was sustained for 5 days. Mesangial cells in high glucose were consistently smaller in size compared with those in normal glucose. Mesangial cells were preloaded with myo-[2-3H]inositol and intracellular [3H] inositol phosphate release in response to AVP (1.0 mumol/l) was analyzed by Dowex chromatography. Comparing cells in normal (5.6 mmol/l) verus high (25.6 mmol/l) glucose, we observed no significant difference in stimulated inositol phosphate levels from 10 to 60 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated rat glomerular cells demonstrate L-type Ca(2+)-channel activity.

The presence of L-type calcium (Ca2+)-channels and the effects of Ca(2+)-channel antagonists on cells of rat glomeruli were investigated. Glomeruli were isolated by graded sieving and after preincubation (10 min) in zero Ca2+, the uptake of 45Ca2+ by glomerular cells was measured. Depolarization with KCl (50 mM) or the dihydropyridine agonist Bay K 8644 (10 microM) stimulated 45Ca2+ uptake by 13% and 24%, respectively, above control (100%), which was inhibited by nifedipine (Nif, 10 microM), P < 0.05, and by both S and R isomers of verapamil (Ver, 10 microM), P < 0.001. In a separate experimental preparation, isolated glomeruli were preloaded (45 min) with 45Ca2+. Following a 45 min perifusion (37 degrees C, CaCl2 1.26 mM, in the absence of 45Ca2+), both KCl (50 mM) and Bay K 8644 (10 microM) induced cellular 45Ca2+ efflux with peak values above control of 11% and 15%, respectively, (P < 0.05). Exposure to Bay K 8644 preceded by depolarization with KCl resulted in enhanced 45Ca2+ efflux identifying the presence of voltage-dependent Ca(2+)-channel activity. Cultured rat mesangial cells grown to confluence on coverslips were preloaded with Fura-2 and cytosolic Ca2+ was measured by microfluorometry. KCl (50 mM), gramicidin (2 microM) and/or Bay K 8644 (6 microM) stimulated Ca2+ influx which was inhibited by Ver (10 microM). Ver did not alter endothelin-stimulated Ca2+ signalling. We conclude that L-type Ca2+ channels are present on both rat glomerular (endothelial and/or mesangial) cells in vivo and on cultured mesangial cells, and their activation may be hormone specific.

The role of angiotensin-II in progressive diabetic glomerulopathy in the rat.

The potential pathogenic role of angiotensin-II (AII) in early progressive diabetic and renal ablation-induced glomerulosclerosis was explored and compared in the Sprague-Dawley (SD) rat and the mongrel dog. Male SD rats were divided into control and streptozotocin-treated (65 mg/kg, iv) groups. Unilateral surgical nephrectomy (Nx) was performed in half of each group. Enalapril (E; 50 mg/liter in the drinking water) was administered to half of each subgroup. Enalapril (high E; 250 mg/liter) was given to another 13 streptozotocin rats. All diabetic rats were treated with sc NPH insulin (4 U/day), and blood glucose was 520 +/- 124 mg/dl (mean +/- SD). Microalbuminuria was measured by RIA in 24-h urine collections; wet kidney weights were compared. [125I]AII binding assays were performed on isolated glomeruli. In control rats the high affinity binding dissociation constant (Kd) was 0.59 +/- 0.15 nM (n = 26; mean +/- SD) and receptor number (Ro) was 732 +/- 195 fmol/mg glomerular protein. At 3 weeks, the diabetic glomerular AII receptor Kd was 0.38 +/- 0.07 nM (n = 6; P less than 0.02 vs. control) and Ro was 784 +/- 97 fmol/mg protein (P = NS vs. control); diabetic high E Kd was 0.39 +/- 0.06 nM (n = 6; P less than 0.02 vs. control), and Ro was 873 +/- 105 fmol/mg protein (P = NS vs. diabetes without E). By 10 weeks, a Kd of 0.49 +/- 0.12 nM (n = 32; P less than 0.01 vs. control) and a Ro of 780 +/- 174 fmol/mg protein (P = NS vs. control) were observed when all of the diabetic group data were pooled. Neither Nx nor low or high dose E altered Ro. This is evidence that intraglomerular AII levels are normal or reduced after diabetes, Nx, or both. In the diabetic group, low dose E partially prevented, and high E abolished, Nx-enhanced microalbuminuria and renal hypertrophy. In nine pancreatectomized insulin-treated mongrel dogs over a 12- to 24-month period, despite moderately poor glucose control (300 +/- 75 mg/dl) and combined unilateral Nx in five dogs (12 months), elevated microalbuminuria was not observed. [125I]AII binding to isolated normal and diabetic dog glomeruli revealed the Kd to be of low affinity (1.86 +/- 0.28 to 13.80 +/- 1.88 nM), identifying the presence of type B receptors. Hence, the SD rat and mongrel dog differ in susceptibility to glomerular AII receptor type and progressive diabetic glomerulopathy.(ABSTRACT TRUNCATED AT 400 WORDS)

Glomerular dysfunction in the aging Fischer 344 rat is associated with excessive growth and normal mesangial cell function.

BACKGROUND: Fischer 344 (F344) rats display focal and diffuse glomerulosclerosis with aging postulated to result from loss of normal mesangial cell intrinsic function, e.g., vasoactive hormone signaling, or preservation of normal responsiveness to extrinsic growth factors. METHODS: In 3-, 17-, and 24-month-old F344 male rats, glomerular structure, measured by PC-based morphometry, and function were compared. Immunoperoxidase staining of glomerular proliferating cell nuclear antigen (PCNA) detected cellular proliferation. Primary cultured mesangial cells from the 3 age groups were studied in parallel. Calcium (Ca2+) signaling, measured by Fura-2 fluorescence, contraction to vasopressin (AVP) 1 microM, measured by videomicroscopy, and proliferative response to platelet-derived growth factor-beta beta (PDGF) were compared. RESULTS: Proteinuria was 13 +/- 4, 38 +/- 17, and 110 +/- 35 mg/24 hours at 3, 17, and 24 months, respectively (n = 5, mean +/- SE, p < .01, 3 vs 24 months), with no change in 24-hour creatinine clearances. Glomerular volumes (n = 200/group) for 3, 17, and 24 months, respectively, were .30 +/- .01, .60 +/- .02, .74 +/- 0.2 x 10(6) micron3 (p < .001, 3 months vs 17 months, and 17 vs 24 months). Glomerular basement membrane (GBM) widths and fractional mesangial volumes increased significantly with aging. Glomerular cell PCNA staining remained positive at 24 months. Cultured mesangial cell Ca2+ signaling and contraction to AVP were unchanged with aging. Proliferation to PDGF, which was partially inhibited with verapamil, was similar at 3 and 24 months. CONCLUSIONS: In the Fischer 344 rat, mesangial cell Ca2+ signaling, contraction, and proliferation responsiveness are unchanged with aging. Continued growth is associated with the glomerulosclerosis of aging.

The role of percutaneous transluminal dilatation in the treatment of transplant renal artery stenosis.

The assessment of percutaneous transluminal dilatation (PTD) as a new therapeutic approach for transplant renal artery stenosis (TRAS) was prompted by allograft loss following surgical treatment. Seventeen (7.0%) of 243 allograft patients had TRAS greater than 80% diagnosed by angiography. The outcome of PTD in 4 TRAS patients was compared to antihypertensive drugs alone in 5 and attempted surgical repair in 8. Patients receiving antihypertensive drugs alone required 3.2 drugs at optimal doses to maintain diastolic BP less than or equal to 90 mm Hg. Malignant hypertension developed in 1 non-compliant patient. Three of the 8 surgical repairs resulted in loss of functioning grafts due to postoperative renal artery thrombosis; the remaining 5 had substantial reduction in diastolic BP (pre 112 +/- 8 mm Hg, post 93 +/- 8 mm Hg, P less than .05). Four patients with main renal artery stenosis had successful, uncomplicated PTD with no graft loss, a significant reduction in diastolic BP (pre 108 +/- 10 mm Hg, post 88 +/- 5 mm Hg, P less than .05) and less antihypertensive drug requirement (pre 3.0 +/- .8 drugs, post 1.0 +/- .8 drugs, P less than .05). Restenosis occurred in 1 patient during the 6 to 10 month follow-up period. PTD compared favourably with conventional management of TRAS as a new therapeutic approach.

Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression.

Excess collagen IV expression by mesangial cells contributes to diabetic glomerulosclerosis. We hypothesized that in high glucose reactive oxygen species (ROS) generation by NADPH oxidase is PKC dependent and required for collagen IV expression by mesangial cells. In rat mesangial cells cultured in 5 mM (NG) or 25 mM d-glucose (HG), RT-PCR and Western immunoblotting detected p22(phox) and p47(phox) mRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. With the use of confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1 h, sustained up to 48 h, and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Gö6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22(phox) and p47(phox) was increased within 3 to 6 h and inhibited by Gö6976. In HG, Gö6976 or transfection with antisense against p22(phox) reversed the 1.8-fold increase in collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22(phox) or p47(phox), prevented ROS generation and the 2.3-fold increase in collagen IV protein. Increased mitochondrial redox potential in HG was unaffected by transfection with antisense against p22(phox). We conclude that in HG, mesangial cell ROS generation by upregulated NADPH oxidase is dependent on conventional PKC isozymes and also required for collagen IV expression.

Upregulation of osteopontin gene expression in diabetic rat proximal tubular cells revealed by microarray profiling.

Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5-70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-beta1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-beta1) cDNA or antisense TGF-beta1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin-angiotensin system activation, TGF-beta1 expression, and PKC-beta1 signaling.

Upregulation of D-myo-inositol transport in diabetic rat glomerular cells.

In the streptozotocin (STZ)-treated diabetic rat, reduced glomerular arteriolar resistance leads to raised intraglomerular pressure. Because vasoconstrictor hormones, such as angiotensin II, stimulate arteriolar smooth muscle and mesangial cell contraction via the D-myo-inositol (MI)-dependent transmembrane signaling pathway, in diabetes extracellular D-glucose may inhibit MI transport causing MI depletion, reduced signaling, and hypocontractility. Therefore we studied the regulation of Na-dependent MI transport in the intact cells (mesangial and endothelial) of isolated glomeruli from STZ rats after 2 wk of diabetes, with and without insulin, compared with controls. Specific [3H]MI uptake per milligram glomerular protein (10-150 min, 37 degrees C) was observed in the presence of 0, 5.5, and 30 mM D-glucose using L-[14C]glucose as a marker of nonspecific uptake. D-Glucose competitively inhibited Na-dependent MI transport (maximum velocity) into diabetic and normal glomerular cells. At 5.5 mM D-glucose, MI uptake by diabetic non-insulin-treated glomeruli was increased to 191 +/- 14% (SE) above normal glomeruli. Insulin treatment resulted in less upregulation (116 +/- 11%) of normal MI transport. High glucose concentration did not alter the rate of [3H]MI efflux from preloaded glomerular cells. To determine whether diabetes alters available substrate for transmembrane signaling, after incubation for 120 min, the incorporation of [3H]MI into cellular membrane phosphoinositides and soluble D-myo-inositol phosphates of isolated diabetic and control glomerular cells was compared. Diabetic glomerular cells displayed a significant increase (P less than 0.005) in [3H]MI incorporation into these fractions compared with controls.

Transglomerular cationic macromolecular flux is mediated by a convection-binding mechanism.

Glomerular polyanion function was explored using charged and neutral [3H]dextrans in the multiple indicator-dilution experiment. Anesthetized dogs received an intrarenal bolus of 125I-labeled albumin (plasma reference), [14C]inulin (glomerular reference) and [3H]dextran (test solute), followed by rapid serial sampling of the renal venous and urine outflows. Reduced urinary recovery of cationic diethylaminoethyl dextrans (DEAE) [3H]dextrans [19.0- to 31.5-A Stokes-Einstein radius (SER)], compared with neutral [3H]dextran indicated intrarenal binding reversed by excess unlabeled cationic dextran. Tubular microperfusion with cationic [3H]dextran confirmed a pretubular binding site (presumed glomerular). The application of a computer-assisted mathematical model of convective flux plus reversible binding revealed that binding affinity increased with molecular size. In vitro high-affinity binding of the same cationic [3H]dextrans to isolated rat glomeruli was also found to increase with molecular size and was inhibited by protamine sulfate. Intrarenal polycation perfusion with protamine sulfate (1.0-3.8 mg/g kidney) or lysozyme (1.1-2.2 mg/g body wt) resulted in intraglomerular binding of anionic [3H]dextran without increased proteinuria or altered glomerular permselectivity to neutral [3H]dextrans less than or equal to 33.0-A SER. Hence, transglomerular cationic solute flux is mediated by a convection-binding mechanism that creates an effective polyvalent barrier.

Cellular mechanisms of glucose-induced myo-inositol transport upregulation in rat mesangial cells.

Uptake of myo-inositol (MI) is necessary to maintain normal cellular phosphoinositide signaling and function. MI transport is up-regulated in the cells of diabetic rat glomeruli compared with normal rat glomeruli [C. I. Whiteside, J. C. Thompson, and J. Ohayon. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F138-F144, 1991]. To identify mechanisms associated with upregulation of MI transport, rat mesangial cells were cultured in high (25.6 mM) vs. normal (5.6 mM) glucose. Specific Na(+)-dependent [3H]MI uptake (> 97%), using L-[14C]glucose as the nonspecific marker, was linear for 120 min in high and normal glucose. In high glucose, compared with normal glucose, there was no change in Michaelis-Menten constant values [29.1 +/- 0.6 vs. 30.3 +/- 0.7 microM (SE)], whereas maximum velocity (Vmax) was increased (2.024 +/- 52 vs. 1.132 +/- 115 fmol.mg protein-1.min-1, P < 0.001). Mannitol (20.0 mM), used as an osmotic control, had no effect on the upregulation of MI transport. Maximum upregulation of MI transport measured by Vmax (control taken as 100%) was observed after 8 h of exposure to high glucose (222 +/- 6% above control, P < 0.0001) or galactose (20.0 mM) (194 +/- 6%, P < 0.0001) and was sustained for up to 48 h. The protein synthesis inhibitors cycloheximide (20 micrograms/ml) or actinomycin D (5 micrograms/ml), the F-actin depolymerizing agent cytochalasin D (2 micrograms/ml), and the aldose reductase inhibitor Tolrestat (0.3 mM) independently prevented glucose- or galactose-induced upregulation of MI transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane signalling of cholinergic-stimulated contraction in isolated rat glomeruli.

Exposure of isolated rat glomeruli to carbachol (Carb) 1 mM induces glomerular (mesangial cell) contraction which is inhibited by pirenzepine. To identify the cellular signalling mechanism, we analyzed Carb-activated phosphoinositide hydrolysis in myo-[2-3H] inositol preloaded cells of isolated rat glomeruli. Carb evoked a rapid (5 s, 30.4 +/- 8.3% of basal) and sustained (60 s, 157.2 +/- 1.2% of basal) inositol trisphosphate (IP3) response. Acetylcholine 100 microM (Ach) in the presence of eserine also evoked IP3 formation. Mobilization of Ca2+ was monitored by preloading glomerular cells with 45Ca2+ (200 microCi/ml) and analyzing the cellular efflux using a constant perifusion system. Both Carb and Ach induced transient 45Ca2+ efflux, with peak values above baseline of 69.0 +/- 19.9 and 71.5 +/- 7.9%, respectively. Incubation with pirenzepine or prior release of Ca2+ from an IP3-sensitive intracellular pool by Thapsigargin (2 microM) prevented Carb-evoked Ca2+ release (100 and 60%, respectively). Therefore, glomerular cells possess functional cholinergic receptors which stimulate contraction via IP3-mediated intracellular Ca2+ release.

In vivo characterization of insulin uptake by dog renal cortical epithelium.

In vivo 125I-labeled insulin uptake by dog renal tubular epithelium was studied using the single-pass multiple indicator dilution (MID) method and analyzed by a computer-assisted model of transcapillary exchange and substrate-cell interaction. Anesthetized dogs received an intrarenal arterial bolus of multiple tracers: [3H]dextran greater than 70 kDa (plasma reference), [14C]inulin (extracellular reference), and 125I-insulin. Rapid serial sampling of the renal venous and urine outflows was performed. The renal venous outflow curves of 125I-insulin fell below [14C]inulin implying postglomerular extraction and antiluminal membrane (ALM) uptake. The fractional urine recovery of 125I-insulin was less than 0.03, indicating luminal tubular uptake of filtered hormone. After intravenous infusion of unlabeled insulin, repeat MID runs with tracer revealed saturable ALM uptake as evidenced by the 125I-insulin renal venous outflow curves approaching [14C]inulin. Luminal tubular uptake was unchanged and therefore unsaturable. The 125I-insulin renal venous data were studied using three mathematical models, incorporating postglomerular reversible binding, irreversible binding or transport. The best fit was obtained using the transport model. The modeling analysis is consistent with either uptake into a virtual epithelial membrane space (i.e., insulin never enters the cell but binds to or is distributed along the ALM) or insulin actually enters the intracellular compartment. In vivo uptake of 125I-insulin ALM is characterized by a Km of 15.44 nM.

Myo-inositol and D-glucose transport in rat glomerular and cultured mesangial cells.

Myo-inositol (MI) is the precursor of membrane-bound phosphoinositides important for transmembrane signaling. This study examines whether freshly isolated whole glomeruli can be used to characterize intact cell transport kinetics for MI and D-glucose. Transport properties of cultured mesangial cells are compared. Glomeruli greater than 95% tubule free were isolated from Sprague-Dawley rat kidney cortex by means of selective sieving. Scanning electron microscopy revealed substantial damage of glomerular epithelial cells, whereas endothelial and mesangial cells remained intact. Specific [3H]MI uptake (7-180 min, 37 degrees C) was observed in presence of 5.5 mM D-glucose when L-[14C]glucose was used as a marker of nonspecific uptake. With ouabain (3 mM), or when Na was replaced with N-methyl-D-glucamine, choline, or Li, specific MI uptake was reduced by 95%. A single high-affinity, Na-dependent MI transport site on glomerular cells with Km of 16.5 +/- 1.4 (SE) microM and Vmax of 947 +/- 56 (SE) fmol.mg protein-1.min-1 was observed for 0.75-100 microM MI. D-glucose competitively inhibited MI transport. Specific D-[3H]-glucose transport was Na independent. Phlorizin inhibition of D-glucose and MI uptake was in keeping with Na-independent D-glucose transport. Km and Vmax for MI uptake in cultured mesangial cells were 42.7 +/- 7.1 microM and 1,474 +/- 192 fmol.mg protein-1.min-1, respectively. We conclude that freshly isolated glomeruli can be used to study cellular transport function that may be modified in disease states.

Diabetic rat glomerular mesangial cells display normal inositol trisphosphate and calcium release.

Since diabetes may cause cellular myo-inositol depletion, we investigated whether the observed in vitro hypocontractile response of streptozotocin (STZ)-treated rat glomeruli to angiotensin II (ANG II) is associated with an alteration in inositol trisphosphate (IP3) mobilization of intracellular Ca2+. Contraction of diabetic isolated glomeruli induced by ANG II (5 microM), measured in vitro by changes in the planar area, was reduced by 60%, compared with normal up to 60 min (P < 0.05). In cells of isolated glomeruli, preloaded with myo-[3H]inositol, production of [3H]inositol phosphates ([3H]IPs) and [3H]inositol trisphosphate ([3H]IP3) was analyzed by Dowex chromatography. ANG II (1 microM) evoked an immediate peak (5-10 s) in total [3H]IPs of 60.5 +/- 18.8% (mean +/- SE) above basal (nonstimulated state) in normal glomeruli, and 88.4 +/- 19.4% in diabetic condition [not significant (NS), n = 8]. At 60 s, the normal and diabetic total [3H]IPs responses were not significantly different from each other. The immediate (10 s) [3H]IP3 response from normal glomeruli, 8.1 +/- 7.9% above basal, was not significantly different from that of diabetic glomeruli, 15.7 +/- 7.4%. ANG II receptor-mediated rise in cytosolic Ca2+ in the cells of normal and diabetic isolated glomeruli was compared by measuring the efflux of 45Ca2+. Isolated glomeruli were preloaded with 45Ca2+. Following ANG II stimulation, peak 45Ca2+ efflux values at 1 min were 141.7 +/- 15.9% (normal) vs. 143.7 +/- 7.8% (diabetic) of baseline (100%), respectively (NS, n = 4). Thapsigargin, 2 microM, specifically prevented ANG II-stimulated and IP3-mediated 45Ca2+ efflux (73% inhibition, P < 0.001) from cells of whole glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen type I enhances endothelin-mediated contraction and induces nonproliferating phenotype in mesangial cells.

Accumulation of glomerular extracellular matrix is a characteristic accompaniment of mesangial cell proliferation in progressive renal disease. We examined how growth on several matrices affected the proliferative phenotype of cultured rat mesangial cells. Compared with growth on plastic, Matrigel, or mesangial matrix, collagen type I caused a decreased cell number at 72 h, decreased total DNA per culture, and a decrease in the incorporation of [3H]thymidine during S phase in cells released from quiescence. These antiproliferative and antimitogenic effects of collagen type I required growth on a collagen gel; soluble collagen or collagen fragments were without effect. Because a number of agents elicit both proliferative and contractile responses in mesangial cells, we examined the effect of growth on collagen on contractility. Compared with plastic, cells grown on collagen type I were more contractile, showed a higher Ca2+ signal in response to endothelin, and responded to endothelin with a more rapid myosin light-chain kinase-dependent phosphorylation of myosin light chain. We conclude that growth on a collagen type I gel uncouples contractility from a proliferative response in mesangial cells, suppressing proliferation while enhancing contraction and Ca2+ signaling in response to endothelin.

Extracellular chloride regulates mesangial cell calcium response to vasopressor peptides.

The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, the components of vasopressor-stimulated calcium signaling were defined in rat mesangial cells cultured on coverslips and preloaded with fura 2. By spectrofluorometry, manganese uptake (reflecting divalent cation channel activation) was observed by quenching of fura 2, or intracellular cytosolic calcium concentration was calculated by dual-excitation ratiometric measurement. In cells depolarized with KCl (45 mM), enhanced manganese uptake or increased cytosolic calcium were inhibited with verapamil (10 microM). Pretreatment of mesangial cells with verapamil reduced the sustained calcium level in response to endothelin-1 (0.1 microM) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 microM) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement confirmed that endothelin-1 stimulated a sustained increase in cytosolic calcium or divalent cation entry only in the presence of simultaneous depolarization. In chloride-free buffer (chloride replaced with impermeant anions), sustained calcium response to endothelin-1 was reduced by 72 +/- 8 (n = 8) and by 65 +/- 4% (n = 8) in the presence of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 microM). In chloride-free buffer, cytosolic calcium (unstimulated) increased to > 200 nM by 30 min. These data indicate that reduced extracellular chloride increases mesangial cell basal cytosolic calcium and decreases the transient and sustained cytosolic calcium response to vasopressor peptides.

Epithelial cell detachment in the nephrotic glomerulus: a receptor co-operativity model.

Detachment of epithelial cells from the glomerular capillary wall correlates with the massive increase in protein leakage across the capillary wall that is characteristic of many renal diseases. We introduce the hypothesis that this detachment process involves three classes of physical events acting at the subcellular level: the receptor-mediated binding of epithelial cells to basement membrane, the transglomerular hydraulic pressure gradient acting to lift the cells off the basement membrane, and a receptor-receptor co-operativity induced by mechanical deformations of the epithelial cell surface. After presenting the available evidence, we explore the hypothesis by means of a simplified, quantitative model of the detachment process. The model is developed by mapping between the stochastic events of cell adhesion receptor binding and the equilibrium statistical mechanics of the Ising model. Monte Carlo simulations predict cell attachment under normal conditions, as expected from experimental data, and detachment at lower receptor binding affinity and/or increased pressure gradient. The normal attached state in the model is found to be particularly sensitive to changes in the receptor-binding affinity. The amount of resistance the cell surface offers to deformation forces is a key determinant of whether the detachment of small clusters of receptors spreads to involve large areas of the plasma membrane, precipitating bulk detachment.