A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.     

Potassium Depletion and Regulation of Angiotensin II Receptors in Vascular Smooth Muscle Cells

Abstract

Mesenteric artery smooth muscle cells were grown in culture media containing high, normal, or low concentrations of potassium to study the effects on angiotensin II (Ang II) receptor regulation. Cell growth was similar among cells grown in the different culture media. Cells grown in high potassium media (K=5.8 mEq/L) had an equilibrium dissociation constant, K_d, of 1.59 ± 0.2 nM, whereas those grown in normal potassium media (K=4.1 mEq/L) had a K_d of 1.79 ± 0.2 nM and those grown in a low potassium media (K=2.9 mEq/L) had a K_d of 1.19 ± 0.12 nM (not significantly different, NS). Binding capacity of smooth muscle cells grown in high potassium media was 81 ± 16.7 fmol/mg prot, 95.1 ± 12.4 fmol/mg prot in those grown in normal potassium media and those grown in low potassium media 86.4 ± 24.1 fmol/mg prot (NS). Binding of radiolabelled Ang II was reduced by approximately 70% in cells exposed to unlabelled Ang II for 30 or 60 minutes. However, this effect of exposure to Ang II to reduce subsequent binding of Ang II was identical in cells grown in high and low potassium medium. Therefore, we were unable to identify a direct effect of low potassium to induce changes in Ang II receptor binding affinity or binding capacity. Previously observed changes in these Ang II binding parameters in potassium-depleted rats was probably a consequence of other factors which were simultaneously altered by potassium deficiency.     

Combined effects of glucose and oxygen concentrations upon the ultrastructure of cultured islet cells and insulin accumulation in culture media

Rat pancreatic islets cultured for 6 days at 100 or 500 mg/dl glucose and 20 or 7% O2 were examined electron-microscopically, and insulin accumulation in the culture media was assayed immunologically. In the islets cultured at 500 mg/dl glucose and 20% O2, B cells exhibited hypertrophy of granular endoplasmic reticulum and Golgi apparatus, an abundance of free ribosomes, degranulation and the margination of secretory granules. In islets cultured at 500 mg/dl glucose and 7% O2, B cells exhibited dilatation of endoplasmic reticulum cisternae and dominance of Golgi vesicles in addition to the above-mentioned changes. These changes, together with the correlated data on insulin accumulation, are discussed with special reference to the effects of glucose and oxygen upon the synthesis and release of insulin in B cells.     

内皮素对心血管系统内血管紧张素Ⅱ释放的影响

采用放免测定及免疫组化技术证明大鼠心血管系统内有血管紧张素Ⅱ免疫活性物质的存在,其心房、主动脉组织及培养的主动脉平滑肌细胞内的含量分别为:7.2±2.7pg/mg 蛋白、152±59.2pg/mg 蛋白和3.5±0.8pg/2×10~5细胞。内皮素是由内皮细胞产生的一个具有强大缩血管功能的多肽,可显著增强培养的鼠主动脉及鼠主动脉细胞释放血管紧张素Ⅱ。这一结果表明,很可能存在一个区域化的内皮素——血管紧张素相互作用,这个相互作用可能参与局部血流及血管紧张度的调节,也还可能参与心肌肥厚,血管肥厚等疾病过程。     

Glucocorticoid metabolism in proximal tubules modulates angiotensin II-induced electrolyte transport.

The hormonal interactions that regulate electrolyte transport in the proximal tubule are complex and incompletely understood. Since endogenous glucocorticoids and angiotensin II each can affect electrolyte transport in this renal segment, we hypothesized that local metabolism of glucocorticoids by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) might alter the response to angiotensin II. Studies were conducted in cultured origin defective SV-40 transformed immortalized renal proximal tubule cells (IRPTC) derived from weanling Wistar rat kidney. The 11beta-HSD contained in these cells uses NADP+, has an apparent Km for corticosterone of 1.6 microM, but functions only as a dehydrogenase (corticosterone --> 11-dehydro-corticosterone). When mounted in modified Ussing chambers, IRPTC generate a transmembrane current, and angiotensin II (10 pM to 10 microM) increases this sodium-dependent current. Cells incubated with corticosterone (100 nM) and the 11beta-HSD inhibitor carbenoxolone (CBX) (1 microM) for 24 hr and then acutely stimulated with angiotensin (10 nM) show a greater rise in current than do cells exposed to corticosterone alone and stimulated with angiotensin (corticosterone + CBX: 64.2% +/- 20.5% vs. corticosterone: 18.8% +/- 5.9%; P < 0.02 at 180 min)[mean +/- SE percentage above baseline, n = 8/group]. Cells exposed to corticosterone (100 nM) or CBX (1 microM) alone for 24 hr and then stimulated with angiotensin II (10 nM) had responses similar to controls. Thus glucocorticoids can enhance angiotensin II-induced electrolyte transport in proximal tubule epithelial cells when local 11beta-HSD is inhibited.     

High glucose and angiotensin II increase β1 integrin and integrin-linked kinase synthesis in cultured mouse podocytes

Alterations of integrin α3β1 may play a role in the development of diabetic nephropathy. We have investigated the effects of high glucose and angiotensin II on the expression of integrin α3 and β1, and whether these changes are associated with integrin-linked kinase (ILK) in cultured mouse podocytes. Integrin β1 and ILK mRNA expression and protein production were rapidly up-regulated in a dose-dependent manner by high glucose and angiotensin II stimulation. ILK mRNA levels in the mouse podocytes exposed to 30 mmol/l glucose were 1.66, 1.89, and 1.28 times higher than those in control cells at 6, 24, and 72 h exposure, respectively. ILK mRNA levels in mouse podocytes exposed to 1 nM, 10 nM, and 100 nM angiotensin II for 6 h were 1.38, 1.55, and 1.93 times higher, respectively, than those in control cells. Angiotensin-II-induced integrin β1 and ILK mRNA expression was significantly inhibited by treatment with losartan (100 μM). In addition, the up-regulation of ILK synthesis induced by these stimuli was related to β1 integrin synthesis and increased ILK kinase activity. Cell adhesion assay displayed inhibitory effects when podocytes were exposed to high concentrations of angiotensin II. Interestingly, glucose and angiotensin II stimulation induced shrinkage of the cell body and elongation of the podocyte processes, a phenotype similar to that of immature podocytes. In addition, β1 integrin showed higher levels of staining on both the cell membranes and the cell-cell contact areas. Thus, high glucose and angiotensin II may affect the regulation of the integrin-ILK system in podocytes; this system may therefore play a role in the pathogenesis of diabetic nephropathy and other renal diseases affecting podocytes. The results presented in this paper have not been published previously in whole or part, except in abstract form. This work was supported by grant R01–2002–000–00139–0 from the Basic Research Program of the Korea Science & Engineering Foundation.     

Effect of glucose on Na, K-ATPase activity in cultured bovine aortic endothelial cells.

The effect of high concentrations of glucose on Na, K-ATPase activity and the polyol pathway was studied using cultured bovine aortic endothelial cells. Na, K-ATPase activity was expressed as ouabain-sensitive K+ uptake. A significant decrease in Na, K-ATPase activity with an intracellular accumulation of sorbitol was found in confluent endothelial cells incubated with 400 mg/dl glucose for 96 h. However, there was no significant change in the Na, K-ATPase activity or sorbitol content of the cells incubated with 100 mg/dl glucose plus 300 mg/dl mannitol. The decrease in Na, K-ATPase induced by the high glucose concentration was restored by the simultaneous addition of 10(-4) M ponalrestat (ICI 128,436; Statil), an aldose reductase inhibitor. The addition of this agent also significantly reduced the increase in sorbitol induced by high glucose levels. These results suggest that the decrease in Na, K-ATPase activity induced in cultured aortic endothelial cells by high concentrations of glucose may be caused in part by the accumulation of sorbitol.     

Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.     

20-HETE and EETs in Diabetic Nephropathy: A Novel Mechanistic Pathway

Diabetic nephropathy (DN), a major complication of diabetes, is characterized by hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. Hypertrophy is a major factor inducing proximal tubular epithelial cells injury. However, the mechanisms leading to tubular injury is not well defined. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. In conclusion, we show that hyperglycemia in diabetes has a significant effect on the expression of Arachidonic Acid (AA)-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites.     

High glucose increases extracellular matrix production in pancreatic stellate cells by activating the renin-angiotensin system

Pancreatic stellate cells (PSCs) are involved in pancreatic inflammation and fibrosis. Recent studies have shown that blocking the renin-angiotensin system (RAS) attenuates pancreatic inflammation and fibrosis. However, there are few data about the direct effects of high glucose on extracellular matrix (ECM) protein synthesis and angiotensin II (Ang II) induction in PSCs. PSCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5.5 mM (LG group) or 27 mM D-glucose (HG group). Levels of Ang II and transforming growth factor-beta (TGF-beta) in culture media were measured and Ang II-positive cells were counted. We used real-time polymerase chain reaction (PCR) to detect Ang II receptor expression and Western blot analysis for the expression of ECM proteins such as connective-tissue growth factor (CTGF) and collagen type IV. Cells were also treated with an Ang II-receptor antagonist (candesartan, 10 microM) or angiotensin-converting enzyme (ACE) inhibitor (ramiprilat, 100 nM). Thymidine uptake by PSCs increased fourfold with high glucose treatment. Ang II levels and the proportion of Ang II-positive PSCs were significantly increased after 6 h under high-glucose conditions. TGF-beta concentrations also increased significantly with high glucose. After 72 h, the expression of CTGF and collagen type IV proteins in high-glucose cultures increased significantly and this increase was effectively attenuated by the candesartan or the ramiprilat. All together, high glucose induced PSCs proliferation and ECM protein synthesis, and these effects were attenuated by an Ang II-receptor antagonist. The data suggest that pancreatic inflammation and fibrosis aggravated by hyperglycemia, and Ang II play an important role in this pathogenesis.     

Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.     

Characterization of Angiotensin-Converting Enzyme 2 Ectodomain Shedding from Mouse Proximal Tubular Cells

Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼90 kDa and ∼70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met⁷⁰⁶ is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM) (and to a lesser extent Ang II) for 48–72 h increased ACE2 activity in the media (p<0.001), an effect blocked by inhibition of a disintegrin and metalloproteinase (ADAM)17. High D-glucose increased ADAM17 activity in cell lysates (p<0.05). These data indicate that two glycosylated ACE2 fragments are constitutively shed from mouse proximal tubular cells. ACE2 shedding is stimulated by high D-glucose, at least partly via an ADAM17-mediated pathway. The results suggest that proximal tubular shedding of ACE2 may increase in diabetes, which could enhance degradation of Ang II in the tubular lumen, and increase levels of Ang-(1-7).     

Aldosterone antagonists in monotherapy are protective against streptozotocin-induced diabetic nephropathy in rats

Angiotensin converting enzyme inhibitors (ACEi) and angiotensin II receptor blockers (ARB) are the standard clinical therapy of diabetic nephropathy (DN), while aldosterone antagonists are only used as adjuncts. Previously in experimental DN we showed that Na/K ATPase (NKA) is mislocated and angiotensin II leads to superimposed renal progression. Here we investigated the monotherapeutic effect of aldosterone blockers on the progression of DN and renal NKA alteration in comparison to ACEi and ARBs. Streptozotocin-diabetic rats developing DN were treated with aldosterone antagonists; ACEi and ARB. Renal function, morphology, protein level and tubular localization of NKA were analyzed. To evaluate the effect of high glucose per se; HK-2 proximal tubular cells were cultured in normal or high concentration of glucose and treated with the same agents. Aldosterone antagonists were the most effective in ameliorating functional and structural kidney damage and they normalized diabetes induced bradycardia and weight loss. Aldosterone blockers also prevented hyperglycemia and diabetes induced increase in NKA protein level and enzyme mislocation. A monotherapy with aldosterone antagonists might be as, or more effective than ACEi or ARBs in the prevention of STZ-induced DN. Furthermore the alteration of the NKA could represent a novel pathophysiological feature of DN and might serve as an additional target of aldosterone blockers.     

Insulin binding in cultured Chinese hamster kidney epithelial cells the effects of glucose concentration in the medium and tunicamycin

An epithelial cell line established from a Chinese hamster kidney, CHK-AC_E, was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-AC_E-100 and CHK-AC_E-400 cells to ¹²⁵I-labeled insulin showed similar pH and time dependency; ¹²⁵I-labeled insulin binding as a function of insulin concentration differed in the two sublines, however. Degradation of ¹²⁵I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK_AC_E-400 cells than with CHK-AC_E-100 cells. When CHK-AC_E-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-AC_E-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 μg/ml, showed no direct effect on the assay of ¹²⁵I-labeled insulin binding to CHK-AC_E-100 cells; exposure of CHK-AC_E-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 μg/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.     

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells

BACKGROUND: GLP-1 is secreted into the circulation after food intake. The main biological effects of GLP-1 include stimulation of glucose dependent insulin secretion and induction of satiety feelings. Recently, it was demonstrated in rats and humans that GLP-1 can stimulate renal excretion of sodium. Based on these data, the existence of a renal GLP-1 receptor (GLP-1R) was postulated. However, the exact localization of the GLP-1R and the mechanism of this GLP-1 action have not yet been investigated. METHODS: Primary porcine proximal tubular cells were isolated from porcine kidneys. Expression of GLP-1R was measured at the mRNA level by quantitative RT-PCR. Protein expression of GLP-1R was verified with immunocytochemistry, immunohistochemistry and Western blot analysis. Functional studies included transport assessments of sodium and glucose using three different GLP-1 concentrations (200 pM, 2 nM and 20 nM), 200 pM exendin-4 (GLP-1 analogue) and an inhibitor of the dipeptidylpeptidase IV (DPPIV) enzyme (P32/98 at 10 microM). Finally, the expression of NHE3, the predominant Na(+)/H(+) exchanger in proximal tubular cells, was also investigated. RESULTS: GLP-1R, NHE3 and DPPIV were expressed at the mRNA level in porcine proximal tubular kidney cells. GLP-1R expression was confirmed at the protein level. Staining of human and pig kidney cortex revealed that GLP-1R was predominantly expressed in proximal tubular cells. Functional assays demonstrated an inhibition of sodium re-absorption with GLP-1 after 3 h of incubation. Exendin-4 and GLP-1 in combination with P32/98 co-administration had no clear influence on glucose and sodium uptake and transport. CONCLUSION: GLP-1R is functionally expressed in porcine proximal tubular kidney cells. Addition of GLP-1 to these cells resulted in a reduced sodium re-absorption. GLP-1 had no effect on glucose re-absorption. We conclude that GLP-1 modulates sodium homeostasis in the kidney most likely through a direct action via its GLP-1R in proximal tubular cells.