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      High Glucose Levels Alter Angiotensin II-Induced Ca 2+ Uptake via PKC and cAMP Pathways in Renal Proximal Tubular Cells

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          Although a dysfunction of the calcium metabolism occurs in diabetes mellitus, alterations of Ca<sup>2+</sup> uptake induced by angiotensin II (ANG II) in renal proximal tubular cells (PTCs) grown in high-glucose medium are not fully elucidated. Thus, we examined whether high glucose concentrations can induce an alteration of the ANG II effect on the Ca<sup>2+</sup> uptake and its action mechanism in primary cultured renal PTCs. PTCs were exposed to different glucose concentrations (5–100 m M) and time intervals (0–48 h). There was a sustained increase of Ca<sup>2+</sup> uptake at glucose concentrations >15 m M. Thus, we selected 25 m M glucose and incubation for 48 h to maintain a hyperglycemic condition in vitro, unlike short-time regulatin. ANG II significantly inhibited the Ca<sup>2+</sup> uptake in a dose-dependent manner in a 5-m M glucose medium. In addition, downregulation of ANG II receptors appeared in a glucose dose dependent manner. However, PTCs treated with 25 m M glucose for 48 h, not 12 h, did not exhibit the inhibitory effect of ANG II (10<sup>–7</sup>  M) on Ca<sup>2+</sup> uptake, although the inhibitory effect of ANG II on Ca<sup>2+</sup> uptake occurred in the presence of 25 m M mannitol or L-glucose. Staurosporine, bisindolylmaleimide I (protein kinase C, PKC, inhibitors), 12- o-tetradecanoylphorbol 13-acetate pretreatment, SQ 22536 (an adenylate cyclase inhibitor), and myristoylated protein kinase A inhibitor amide 14–22 (a protein kinase A inhibitor) blocked the 25-m M-glucose-induced alteration of ANG II effect on Ca<sup>2+</sup> uptake. These results suggest that both PKC and cyclic adenosine monophosphate (cAMP) pathways are involved in the high-glucose-induced alteration of ANG II effect on Ca<sup>2+</sup> uptake. Indeed, 25 m M glucose increased PKC activity and cAMP contents. In conclusion, a high glucose concentration altered ANG II induced inhibition of Ca<sup>2+</sup> uptake via PKC and cAMP pathways in the PTCs.

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          Most cited references 10

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          Diabetes mellitus: a disease of abnormal cellular calcium metabolism?

          Although the pathogenesis of the diabetes mellitus syndrome remains poorly understood, both insulin-dependent diabetes mellitus and non-insulin-dependent diabetes mellitus predispose the individual to a similar spectrum of complications, including hypertension, macrovascular and microvascular disease, cataracts cardiomyopathy, neuropathy, and premature aging, suggesting that these complications develop along a pathway common to both diabetic conditions. Yet not all diabetic persons are affected by all of these complications or to the same degree. What causes this marked variability in the clinical manifestations of the diabetes syndrome remains an enigma. Accumulating data from animal models of diabetes and from studying patients with diabetes reveal that intracellular calcium levels are increased in most tissues. The activities of the membrane, adenosine triphosphatase (ATPase) associated cation pumps, which determine intracellular calcium level (i.e., calcium-ATPase and [sodium + potassium]-ATPase), are also altered. The nature of the alteration is often tissue specific and may depend on the level of blood glucose or insulin, or both. In this review we discuss the potential contribution of these changes in intracellular calcium regulation, whether acquired or genetically determined, to the pathogenesis of the diabetes syndrome, to the abnormalities in insulin secretion and action (mainly in non-insulin-dependent diabetes), and to the complications of both diabetes syndromes. Altered intracellular calcium metabolism may represent a common, underlying abnormality linking the metabolic, cardiovascular, ocular, and neural manifestations of the diabetic disease process.
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            Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

            Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.
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              Effect of glucose on the expression of the angiotensinogen gene in opossum kidney cells.

              To investigate whether D(+)-glucose has a stimulatory effect on the expression of the angiotensinogen (Ang) gene in opossum kidney (OK) cells, we used OK cells with a fusion gene containing various lengths of the 5'-flanking regulatory sequence of the rat Ang gene fused with the human growth hormone (hGH) gene as a reporter, stably integrated into their genomes. The level of expression of the fusion gene was quantified by the amount of immunoreactive-human growth hormone (IR-hGH) secreted into the medium. The addition of D(+)-glucose stimulated the expression of pOGH (Ang N-1498/+18) in OK 27 cells in a dose-dependent manner (5 to 25 mM), whereas the addition of D-mannitol, L-glucose and 2-deoxy-D-glucose (25 mM) had no effect. The stimulatory effect of D(+)-glucose (25 mM) was blocked by the presence of staurosporine or H7 (an inhibitor of protein kinase C) or U73122 (an inhibitor of phospholipase C and A2) but not blocked by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A). The addition of D(+)-glucose (25 mM) also stimulated the expression of pOGH (Ang N-960/+18) and pOGH (Ang N-688/+18) in OK 960 and OK 688 cells, respectively. It had no stimulatory effect, however, on the expression of pOGH (Ang N-280/+18) and pOGH (Ang N-35/+18) in OK 280 and OK 35 cells, respectively. The addition of D(+)-glucose also had no effect on the expression of pTKGH in OK 13 cells, an OK cell line, into which had been stably integrated a fusion gene, pTKGH containing the promoter/enhancer DNA sequence of the viral thymidine-kinase (TK) gene fused with a human growth hormone gene as a reporter. These studies demonstrate that the stimulatory effect of high D(+)-glucose concentration (25 mM) on the expression of the angiotensinogen-growth hormone fusion genes in OK cells is mediated via the 5'-flanking region of the angiotensinogen gene and the protein kinase C signal transduction pathway. Our data indicate that a high glucose concentration may activate the renin-angiotensin system in the renal proximal tubular cells.

                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                29 June 2001
                : 24
                : 2
                : 84-91
                College of Veterinary Physiology, Hormone Research Center, Chonnam National University, Kwangju, Korea
                54212 Kidney Blood Press Res 2001;24:84–91
                © 2001 S. Karger AG, Basel

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                Figures: 5, Tables: 2, References: 42, Pages: 8
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