Cultured skin fibroblasts as an in vitro model to assess phenotypic features in subjects with diabetic nephropathy.
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Calcium↗
Biomedical subjects
D Batlle
Publications and source records attributed to D Batlle.
Suppression of parathyroid hormone secretion in hemodialysis patients: comparison of paricalcitol with calcitriol.
Paricalcitol was introduced recently as an effective alternative to calcitriol for the suppression of parathyroid hormone (PTH) in patients with end-stage renal disease. An international, multicenter, double-blinded, randomized, comparative study of intravenous paricalcitol and calcitriol was performed. Results from 38 patients at dialysis units affiliated with the Northwestern University Medical School (Chicago and Evanston, IL) are reported here while a report of the full clinical trial is being completed. Results were evaluated in terms of obtaining the following end points: decrease of at least 50% in baseline PTH concentration and the occurrence of hypercalcemia and hyperphosphatemia. Paricalcitol therapy was started at a dose of 0.04 microgram/kg and increased by 0.04 microgram/kg increments every 4 weeks to a maximum allowable dose of 0.24 microgram/kg or until there was at least a 50% decrease in serum PTH concentration. Calcitriol therapy was started at a dose of 0.01 microgram/kg and increased by 0.01 microgram/kg increments every 4 weeks to a maximum allowable dose of 0.06 microgram/kg or until there was at least a 50% decrease in serum PTH concentration. Mean baseline serum PTH, calcium, and phosphorus concentrations were similar. Reductions in PTH occurred more rapidly in subjects administered paricalcitol compared with calcitriol, with no difference in serum calcium levels throughout the study between groups. The percentage of subjects experiencing severe hyperphosphatemia (serum phosphorus >8.0 mg/dL) was greater in those administered calcitriol compared with paricalcitol. In conclusion, our data suggest that paricalcitol reduces PTH levels more rapidly, with fewer episodes of hyperphosphatemia, than intravenous calcitriol.
Calcitriol↗
Hereditary distal renal tubular acidosis: new understandings.
The primary or hereditary form of distal renal tubular acidosis (dRTA), although rare, has received increased attention recently because of dramatic advances in the understanding of its genetic basis. The final regulation of renal acid excretion is effected by various acid/base transporters localized in specialized cells in the cortical collecting and outer medullary collecting tubules. Inherited defects in two of the key acid/base transporters involved in distal acidification, as well as mutations in the cytosolic carbonic anhydrase gene, can cause dRTA. The syndrome is inherited in both autosomal dominant and recessive patterns; patients with recessive dRTA present with either acute illness or growth failure at a young age, sometimes accompanied by deafness, whereas dominant dRTA is usually a milder disease and involves no hearing loss. The AE1 gene encodes two Cl-/HCO3- exchangers that are expressed in the erythrocyte and in the acid-secreting intercalated cells of the kidney. AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several mutations in the AE1 gene cosegregate with dominant dRTA. The modest degree of hypofunction exhibited in vitro by these mutations, however, does not explain the abnormal distal acidification phenotype. Other AE1 mutations have been linked to a recessive syndrome of dRTA and hemolytic anemia in which hypofunction can be discerned by in vitro studies. Several mutations in the carbonic anyhdrase II gene are associated with the autosomal recessive syndrome of osteopetrosis, renal tubular acidosis, and cerebral calcification. Some of these individuals present with deafness of the conductive type. By contrast, more recent studies have shown that mutations in ATP6B1, encoding the B-subtype unit of the apical H(+) ATPase, are responsible for a group of patients with autosomal recessive dRTA associated with sensorineural deafness. Thus, the presence of deafness and the type provide an important clue to the genetic lesion underlying hereditary dRTA.
Acidosis, Renal Tubular↗
Mechanisms of chloride transport in thymic lymphocytes.
This study examined mechanisms of Cl- transport in rat lymphocytes under a variety of conditions. Basal intracellular Cl- concentration ([Cl-]i) was not different between cells assayed in the presence of HCO3- or its absence (HEPES). Removal of external Cl- resulted in a fall in [Cl-]i and a rapid rise in intracellular pH (pH(i)). Both Cl- efflux and the rise in pH(i) were blocked by DIDS or removal of external Na+ but were unaffected by furosemide. The mechanisms governing Cl- influx were assessed in cells that had been Cl- depleted for 1 h. Reexposure to Cl- resulted in a rapid rise in [Cl-]i that was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked Cl- influx. Cl- depletion caused a marked increase in pH(i) that rapidly declined toward normal when the cells were reexposed to Cl-. Preincubation with DIDS completely blocked this decrease in pH(i). In contrast, neither removal of Na+ nor preincubation with furosemide affected the decline in pH(i) when the cells were reexposed to Cl-. We conclude that, in thymic lymphocytes, Cl-/HCO3- (or Cl-/base exchange) regulates both Cl- influx and efflux. Cl- efflux is totally inhibited by DIDS and is mediated by a Na+-dependent Cl-/HCO3- exchanger. Cl- influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a Na+-independent Cl-/HCO3- exchanger and by a Na+-K+-2Cl- cotransporter.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Hypoxia and high glucose cause exaggerated mesangial cell growth and collagen synthesis: role of osteopontin.
The effect of hypoxia on the proliferation and collagen synthesis of cultured rat mesangial cells was examined under normal-glucose (NG, 5 mM) and high-glucose (HG, 25 mM)-media conditions. In addition, a role for osteopontin (OPN) in mediating these processes was assessed. Quiescent cultures were exposed to hypoxia (3% O(2)) and normoxia (18% O(2)) in a serum-free medium with NG or HG, and cell proliferation, collagen synthesis, and OPN expression were assessed. Cells exposed to hypoxia in NG medium resulted in significant increases in [(3)H]thymidine incorporation, cell number, and [(3)H]proline incorporation, respectively. HG incubations also produced significant stimulation of these parameters under normoxic conditions, which were markedly enhanced in cells exposed to hypoxia in HG medium. In addition, hypoxia and HG stimulated the mRNA levels of type IV collagen, and the combination of hypoxia and HG resulted in additive increases in type IV collagen expression. Hypoxia and HG also stimulated OPN mRNA and protein levels in an additive fashion. A neutralizing antibody to OPN or its beta(3)-integrin receptor significantly blocked the effect of hypoxia and HG on proliferation and collagen synthesis. In conclusion, these results demonstrate for the first time that hypoxia in HG medium produces exaggerated mesangial cell growth and type IV collagen synthesis. In addition, OPN appears to play a role in mediating the accelerated mesangial cell growth and collagen synthesis found in a hyperglycemic and hypoxic environment.
Animals↗
Hypoxia stimulates osteopontin expression and proliferation of cultured vascular smooth muscle cells: potentiation by high glucose.
We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O(2)) or normoxia (18% O(2)) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [(3)H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor beta3 integrin but not neutralizing antibodies to beta5 integrin prevented the hypoxia-induced increase in [(3)H]thymidine incorporation. Inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein (MAP) kinase also reduced the hypoxia-induced stimulation of proliferation and OPN synthesis. Exposure to high-glucose (HG) (25 mmol/l) medium under normoxic conditions also resulted in significant increases in OPN protein and mRNA levels as well as the proliferation of VSM cells. Under hypoxic conditions, HG further stimulated OPN synthesis and cell proliferation in an additive fashion. In conclusion, hypoxia-induced proliferation of cultured VSM cells is mediated by the stimulation of OPN synthesis involving PKC and p38 MAP kinase. In addition, hypoxia also enhances the effect of HG conditions on both OPN and proliferation of cultured VSM cells, which may have important implications in the development of diabetic atherosclerosis associated with arterial wall hypoxia.
Animals↗
Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK.
BACKGROUND: We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth. METHODS: Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed. RESULTS: Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth. CONCLUSIONS: These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.
Animals↗
The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal.
Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.
Adrenal Cortex↗
A subdepressor low dose of ramipril lowers urinary protein excretion without increasing plasma potassium.
Angiotensin-converting enzyme (ACE) inhibitors are increasingly administered to patients with chronic renal disease. One issue of concern with the use of ACE inhibitors in patients with impaired renal function is the possible development of hyperkalemia. We reasoned that the impact of ACE inhibitors on plasma potassium could be minimized by administering these agents at very low doses. To examine this issue, we investigated the effect of a low dose of ramipril (1.25 mg orally once daily) and an eight-fold higher dose (10 mg orally once daily) on plasma potassium in 13 patients with proteinuria and mild chronic renal insufficiency. The study was divided into four phases: placebo (4 weeks), low-dose ramipril (8 weeks), high-dose ramipril (8 weeks), and washout phase (4 weeks). With the low dose of ramipril, urinary protein excretion decreased significantly as early as after 1 week of administration (from 4.4 +/- 0.5 to 3.7 +/- 0.4 g/24 h; P < 0.025) and did not decrease any further thereafter even when the dose was increased eight-fold. Mean arterial blood pressure and plasma potassium did not change significantly with the low dose of ramipril, whereas with the higher dose, mean arterial blood pressure decreased significantly (from 107 +/- 2.0 to 100 +/- 2.0 mm Hg, P < 0.005), and plasma potassium increased significantly (from 4.53 to 4.78 mEq/L, P < 0.05). We conclude that a low dose of ramipril can reduce proteinuria to the same extent as an eight-fold higher dose without significantly lowering blood pressure or increasing plasma potassium. This latter feature may be advantageous for the treatment of patients at risk for hyperkalemia who require ACE inhibitors.
Adult↗
Temporal profile of serum-induced S-phase entry and retinoblastoma protein phosphorylation in human skin fibroblasts.
Cultured skin fibroblasts from type 1 diabetic patients with nephropathy have a hyperplastic growth phenotype in comparison with diabetics without nephropathy and controls. We studied the G1 phase cyclins in skin fibroblasts from control subjects to define the temporal profile of serum-induced pRB phosphorylation, cyclin D1 protein expression, and cyclin D1/CDK4(6) kinase activity as well as S-phase entry by FACS analysis. Our preliminary studies indicate that cultured skin fibroblasts from type 1 diabetic patients with nephropathy have an enhanced pRB phosphorylation, cyclin D1 protein expression, and cyclin D1/CDK4(6) kinase activity. This finding may become useful to identify patients at risk for the development of nephropathy.
Blood Proteins↗
H+-ATPase in hyperkalemic distal renal tubular acidosis.
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Acidosis, Renal Tubular↗
On the mechanism of the effects of potassium restriction on blood pressure and renal sodium retention.
Dietary potassium restriction increases sodium and chloride retention, whereas potassium administration promotes both diuresis and natriuresis. In epidemiologic and clinical studies, potassium intake is inversely related to blood pressure and is lower in blacks than in whites. The present studies examined the mechanism by which potassium restriction fosters sodium conservation and the impact of race on this response. Twenty-one healthy black and white men and women ingested an isocaloric, potassium-restricted diet (20 mmol/d) containing 180 mmol/d of sodium with and without a potassium supplement (80 mmol/d) for 9 days on two occasions. Additionally, eight of these subjects ingested the same diets for 3 days followed by a water load to determine free water clearance before and during the early phase of dietary potassium restriction. During potassium restriction, mean arterial pressure (MAP) derived from 24-hour blood pressure measurements was higher (85.7 +/- 1.6 mm Hg v 82.0 +/- 1.3 mm Hg; P < 0.001), cumulative sodium excretion lower (984 +/- 59 mmol/d v 1,256 +/- 58 mmol/d; P < 0.001), and weight greater (71.1 +/- 2.1 kg v 69.3 +/- 2.2 kg; P < 0.001). Blacks displayed no greater increase in MAP, although they excreted less sodium overall and less potassium on the potassium-supplemented diet. After a water load, minimum urine osmolality (Uosm) was lower (53.0 +/- 3.0 mOsm/L v 65.6 +/- 3.5 mOsm/L; P = 0.01) and free water clearance greater (4.44 +/- 0.59 mL/min v3.72 +/- 0.58 mL/min; P = 0.009) during potassium restriction. In conclusion, in healthy, normotensive subjects, potassium restriction was associated with an increase in blood pressure and volume expansion effected by increased renal sodium and chloride retention. Potassium restriction was also associated with increased free water clearance and enhanced diluting capacity consistent with augmentation of Na+, K+:2Cl- cotransporter activity in the thick ascending limb of Henle. This mechanism may play an important role in the renal adaptation required for potassium conservation, but at the expense of sodium chloride retention and an elevation in blood pressure.
Adult↗
Renal pathology patterns in type II diabetes mellitus: relationship with retinopathy. The Collaborative Study Group.
BACKGROUND: The glomerular and retinal vessels are both affected in patients with type I and type II diabetes mellitus. However, the prevalence of the nodular form of diabetic glomerular sclerosis (Kimmelstiel Wilson lesion) and other forms of glomerular pathology including diffuse mesangial sclerosis and their clinical correlates in type II diabetes are less well known. In addition, although recent studies have suggested that non-diabetic glomerular disease was a common cause of proteinuria in type II diabetes, the prevalence of other diseases is unknown. The literature on this subject is clouded by clinical bias regarding patients with diabetes who undergo renal biopsy. METHODS: Glomerular and retinal pathology and clinical correlates were studied in 36 patients enrolled in a prospective clinical trial of patients with type II diabetes mellitus, proteinuria, renal insufficiency, and hypertension. RESULTS: Seventeen biopsies had diabetic glomerular sclerosis with Kimmelstiel Wilson nodules; 15 biopsies had glomerular changes characteristic of the diabetic state including enlarged glomeruli and an increase in mesangial matrix without Kimmelstiel Wilson nodules (mesangial sclerosis lesion); and two had other primary glomerular diseases (IgA and membranous nephropathy). Patients with Kimmelstiel-Wilson nodules had elevated serum creatinines compared to patients with mesangial sclerosis lesions, but there were no other significant differences. Patients with Kimmelstiel Wilson nodules had more severe overall retinopathy than those with mesangial sclerosis lesions (P = 0.0043): six of seven with proliferative retinopathy had Kimmelstiel Wilson nodules, and seven of the eight patients without retinopathy had mesangial sclerosis lesions. CONCLUSIONS: The two discrete patterns of glomerular pathology and the correlation between diabetic retinopathy and the Kimmelstiel-Wilson lesion but not the mesangial sclerosis lesion suggest that the Kimmelstiel Wilson and mesangial sclerosis lesions of diabetic glomerulosclerosis are caused by different pathogenetic mechanisms. In this study, diabetic glomerulosclerosis was responsible for the clinical renal abnormalities in 94%, of patients with type II diabetes mellitus.
Adult↗
NHE-1 protein in vascular smooth muscle and lymphocytes from the spontaneously hypertensive rat.
The present study examined the abundance of NHE-1 protein in cultured vascular smooth muscle cells (VSMCs), freshly isolated thymocytes, and fresh aortic tissue from spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Two sets of affinity-purified antibodies (Ab[765-778] and Ab[698-711]) against different epitopes of the NHE-1 isoform of the Na+-H+ antiporter were used. Each set of antibodies recognized a major protein band at 105 to 110 kD that was more abundant in protein lysates prepared from cultured VSMCs from the SHR than those from WKY rats (Ab[765-778] 0.047 +/- 0.011 vs 0.010 +/- 0.002 O.D. units/10 microg protein, P<.001 for SHR and WKY, respectively; and Ab(698-711) 0.173 +/- 0.026 vs 0.087 +/- 0.028 O.D. units/10 microg protein, P<.05, for SHR and WKY, respectively). The increase in NHE-1 protein abundance in cultured VSMCs from the SHR was associated with a greater Vmax of the Na+-H+ antiporter as compared to those from WKY rats (17.93 +/- 2.07 vs 8.16 +/- 1.05 mmol H+/min, P<.001, respectively). In contrast to cultured VSMCs, there was no difference in the relative abundance of NHE-1 protein in fresh aortic tissue (0.075 +/- 0.018 vs 0.083 +/- 0.017 O.D. units/10 microg protein, from SHR and WKY, respectively) or in freshly isolated thymocytes (0.158 +/- 0.046 vs 0.226 +/- 0.054 O.D. units/10 microg protein, from SHR and WKY, respectively). We conclude that the increase in the Vmax of the Na+-H+ antiporter in cultured VSMCs from the SHR, compared to those from WKY rats, is due, at least in part, to increased levels of NHE-1 protein.
Animals↗
Angiotensin II activation of cyclin D1-dependent kinase activity.
Angiotensin II (AII) binds to specific G protein-coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. Since the cyclin D1 gene encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), an essential and rate-limiting step in G1 phase progression of the cell cycle, we examined the effect of AII on cyclin D1 expression and CD1K activity in the human adrenal cell line H295R. AII (10(-6) M) stimulated G1 phase progression within 12 h, with a maximal effect after 72 h. This action was antedated by the induction of cyclin D1 mRNA (3-fold), cyclin D1 nuclear protein abundance (4-fold), and CD1K activity (4-fold). AII induced cyclin D1 promoter activity 4-fold, via the AT1 receptor through an enhancer sequence at -954 base pairs. c-Fos and c-Jun bound the cyclin D1 -954 enhancer sequence, and the abundance of c-Fos within this complex was increased by AII treatment. AII induced extracellular signal-regulated kinase (ERK) activity 7-fold, and dominant-negative mutants of either p21(ras) or ERK reduced AII-stimulated cyclin D1 promoter activity. These findings suggest that AII may stimulate mitogenesis by increasing CD1K activity through a p21(ras)/ERK/activator protein 1 pathway.
Angiotensin II↗
Underlying defects in distal renal tubular acidosis: new understandings.
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Acidosis, Renal Tubular↗
Growth phenotype of cultured skin fibroblasts from IDDM patients with and without nephropathy and overactivity of the Na+/H+ antiporter.
An increased activity of the Na+/H+ antiporter in cells from patients with insulin-dependent diabetes mellitus (IDDM) has been proposed as a potential marker of nephropathy. We evaluated Na+/H+ antiporter activity and its relationship to DNA and protein synthesis in cultured skin fibroblasts from patients with IDDM classified as having either overt nephropathy or absence of nephropathy on the basis of urinary albumin excretion and kidney biopsy findings. In IDDM patients with overt nephropathy, Na+/H+ antiporter activity in serum stimulated cells was increased as compared to cells from control subjects (9.62 +/- 0.89 vs. 5.67 +/- 0.97 mmol H+/min, P < 0.005, respectively) and cells from IDDM patients without nephropathy (7.22 +/- 0.67 mmol H+/min, P < 0.025). By contrast, in cells made quiescent by serum deprivation Na+/H+ antiporter activity was lower than in serum-stimulated cells and there were no significant differences between the three groups. DNA synthesis assessed by [3H] thymidine incorporation was increased in the IDDM group with nephropathy as compared to the group without nephropathy (138 +/- 14 vs. 105 +/- 13 cpm/1000 cells, respectively, P < 0.05) and as compared to control subjects (65 +/- 11 cpm/1000 cells, P < 0.001). By contrast, protein synthesis assessed by [14C] L-leucine incorporation was not increased in fibroblasts from IDDM patients with nephropathy, suggesting that cellular hypertrophy is not a feature of their altered growth phenotype. After chronic inhibition of the Na+/H+ antiporter using EIPA (25 microM), [3H] thymidine incorporation was reduced by about 20% both in cells from IDDM patients and controls. This parameter therefore remained higher in cells from IDDM patients with nephropathy than in those from controls (81 +/- 16 vs. 40 +/- 6 cpm/1000 cells, P < 0.05), while in cells from IDDM patients without nephropathy [3H] thymidine incorporation after EIPA (56 +/- 7.0 cpm/1000 cells) was intermediate between cells from controls and IDDM patients with nephropathy. We argue that cultured skin fibroblasts from IDDM patients, with nephropathy display an abnormal growth phenotype characterized by cell hyperplasia. This growth phenotype is associated with overactivity of the Na+/H+ antiporter during serum stimulation but not when cells are made quiescent and persists after inhibition of the Na+/H+ antiporter. Our data, therefore, further shows that overactivity of the Na+/H+ antiporter is not required for the expression of the altered growth phenotype of cultured skin fibroblasts from IDDM patients with nephropathy.
Adult↗
Angiotensin II and angiotensin-(1-7) effects on free cytosolic sodium, intracellular pH, and the Na(+)-H+ antiporter in vascular smooth muscle.
The aim of the present study was to define the effects of angiotensin II (Ang II) and Ang-(1-7) on free cytosolic Na+ (Na+i), intracellular pH (pHi), and the Na(+)-H+ antiporter in cultured vascular smooth muscle cells from rat aorta. Cells were loaded with either BCECF-AM or SBFI-AM for measurement of pHi and Na+i, respectively. Ang II (10(-6) mol/L) caused a rapid rise in Na+i followed by a progressive increase that peaked at about 10 minutes (from 11 +/- 1.5 to 16 +/- 1.5 mmol/L, P < .001), whereas Ang-(1-7) (10(-6) mol/L) did not affect Na+i significantly (from 11.5 +/- 1.1 to 11.8 +/- 0.07 mmol/L). The effect of Ang II on Na+i was concentration dependent (delta Na+i, 5.1 +/- 0.9, 3.8 +/- 0.6, 1.6 +/- 0.6, and 0.14 +/- 0.18 mmol/L with decreasing concentrations of 10(-6), 10(-7), 10(-8), and 10(-9) mol/L, respectively). Ang II caused a brief acidification followed by an increase in pHi (from 7.34 +/- 0.03 to 7.43 +/- 0.03 after 10 minutes, P < .005), and Ang-(1-7) had no significant effect on pHi (from 7.23 +/- 0.03 to 7.23 +/- 0.03). To investigate whether pHi and Na+i changes induced by Ang II were due to cell Na+ entry via stimulation of the Na(+)-H+ antiporter, we pretreated cells with EIPA (25 mumol/L) or ouabain (2.0 mmol/L). Ang II in the presence of ouabain caused a greater increase than that seen with ouabain alone (delta Na+i, 13 +/- 1.5 versus 6.3 +/- 1.2 mmol/L, P < .0025). EIPA by itself decreased Na+i and pHi. After EIPA, Ang II failed to increase both Na+i and pHi, demonstrating that the Na(+)-H+ antiporter is responsible for the rises in Na+i and pHi during stimulation with Ang II. To further characterize the mechanism of Ang II action, we exposed cells to an Ang II type I receptor antagonist (L-158,809, 10(-6) mol/L) or two different type 2 receptor antagonists (PD 123177 and CGP 421112A, 10(-6) mol/L). L-158,809 completely blocked the rise in pHi caused by Ang II, whereas PD 123177 and CGP 421112A did not. We conclude that Ang II increases both Na+i and pHi, and both effects are mediated by stimulation of the Na(+)-H+ antiporter. Ang-(1-7), by contrast, has no significant effect on Na+i, pHi, or the Na(+)-H+ antiporter. Stimulation of this antiporter by Ang II is exerted through the type 1 receptor.
Amiloride↗