Functional role of a novel cis-acting element (GAGA box) in human type-1 angiotensin II receptor gene transcription.

GH/growth factors have been shown to increase angiotensin type 1 receptor expression. In the present study we determined the cis-acting regulatory region controlling GH-induced transcription of the human type-1 angiotensin receptor (hAT(1)). In human proximal tubule cells transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid under the control of the hAT(1) promoter, GH induced CAT activity. Serial deletions of the hAT(1) promoter region indicated that an area between -314 bp and -70 bp upstream of the 5'-end of the cDNA sequence was essential for this activation to occur. Although sequence analysis identified putative multiple nuclear protein binding sites in this region, we determined that a 12 bp sequence (5'-GAGAGGGAGGAG-3', GAGA box) located between -161 bp and -149 bp was important for GH-mediated activation. Using mobility shift assays we demonstrated increased DNA binding activity to the labeled GAGA box in nuclear extracts treated with GH, suggesting this sequence is a GH response element. Southwestern analysis identified an 18 kDa GAGA box-binding protein (GAGA-BP). GH-induced activity of the GAGA-BP occurred within 2.5 min and reached a maximum at 5 min. Activation did not require de novo protein synthesis. Removal of the GAGA box abolished GH-induced transcription as well as basal transcription of the hAT(1) gene. Additional studies demonstrated that epidermal growth factor, platelet-derived growth factor and insulin activate the GAGA-BP, suggesting these growth factors can also regulate the transcription of the hAT(1) gene through the GAGA box. Our data show that the GAGA-BP acts as a trans-acting factor binding to the cis-acting regulatory element in the hAT(1) promoter, which is necessary for the basal and growth factor(s)-mediated transcriptional activation of the hAT(1) gene.

Endothelial cells regulate proximal tubule epithelial cell sodium transport.

BACKGROUND: Proximal tubule epithelial cells are in close contact with the renal microvasculature, but the effect of endothelial cells (ECs) on proximal tubule epithelial cell (PTEC) function is not known. METHODS: To determine if ECs regulate PTECs, we coincubated ECs with PTECs in a system that permitted cross-talk between the two cell types and the vectorial transport of sodium. RESULTS: In the presence (but not absence) of ECs, adding bradykinin or acetylcholine increased cGMP and decreased sodium transport, as well as Na,K-ATPase in PTECs. Interleukin (IL)1B preconditioning of ECs also increased cGMP and decreased sodium transport and Na,K-ATPase in PTECs. Bradykinin, acetylcholine, and IL1B EC-dependent effects were reversed with the nitric oxide (NO) synthase inhibitor L-NNA. In the absence of ECs, the addition of NO donors to PTECs increased cGMP and decreased sodium transport and Na,K-ATPase. 8Br-cGMP also decreased PTEC sodium transport and Na,K-ATPase. CONCLUSION: Endothelial cells regulate PTEC function. This effect is mediated by NO synthase-dependent up-regulation of cGMP in PTECs.

The role of neutrophils in acute renal failure.

The role of neutrophils in acute renal failure is controversial. Acute renal failure can clearly occur in the absence of neutrophils. However, recent studies using specific neutrophil markers indicate that neutrophils accumulate in postischemic kidneys. Moreover, reperfusion of ischemic kidneys with neutrophils worsens ischemic injury and causes kidney neutrophil retention. Neutrophil retention is dependent on the state of neutrophil activation and the duration of renal ischemia. This interaction could account for the high frequency of acute renal failure in conditions associated with prolonged prerenal asotemia and neutrophil priming such as the adult respiratory distress syndrome, or sepsis. Neutrophil retention is mediated by interaction of neutrophil integrins and endothelial cell ICAM-1 because maneuvers reducing the expression and/or function of these adhesion molecules is protective in experimental models of ischemia. Nitric oxide is a key modulator of neutrophil worsening of ischemic injury because maneuvers that decrease nitric oxide production worsen and those which increase nitric oxide protect ischemic kidneys from neutrophil effects. The clinical significance of neutrophils may relate to the observation that bioincompatible membranes activate complement, and retard recovery from acute renal failure. In conclusion, neutrophils are an important contributor to ischemic acute renal failure. It remains to be determined whether decreasing neutrophil function accelerates recovery in acute renal failure.

Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells.

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A (Kd = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP (Kd = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL (Kd = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1A receptors underwent agonist-dependent endocytosis (AP receptor: t1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial 22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.

Role of protein kinase C in beta 2-adrenoceptor function in cultured rat proximal tubule epithelial cells.

Renal sodium excretion is regulated by the adrenergic system. We recently demonstrated the presence of functional beta 2-adrenoceptors (beta 2-AR) in cultured rat proximal tubule epithelial cells beta 2-AR activation resulted in increases in Na-K-adenosinetriphosphatase (Na-K-ATPase) activity and transcellular sodium transport as a consequence of increased apical sodium entry. The purpose of this study was to determine the role of protein kinase C (PKC) on beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport in proximal tubules. To determine the effect of PKC on basal function, cultured rat proximal tubule cells were exposed to phorbol 12-myristate 13-acetate (PMA). PMA increased apical Na entry (+/-80%), decreased Na-K-ATPase activity (+/-25%), and prevented increases in Na-K-ATPase activity after sodium entry facilitation with monensin. Decreases in Na-K-ATPase activity were associated with decreases in sodium transport (+/-30%). To determine whether beta 2-AR function was transduced by PKC, PKC activity was measured in cells exposed to the selective beta 2-AR agonist metaproterenol. Metaproterenol caused increases in PKC activity, which were blocked by a beta 2-AR but not by a beta 1-AR-receptor antagonist. beta 2-AR-dependent increases in apical Na entry, Na-K-ATPase activity, and sodium transport were blocked by calphostin C or staurosporine. To determine whether PKC had additional effects on beta 2-AR function, cells were exposed to metaproterenol and PMA. Metaproterenol-induced increases in Na-K-ATPase activity and sodium transport were blocked by PMA. In conclusion, beta 2-AR-mediated increases in Na-K-ATPase activity and sodium flux are transduced by PKC acting through increases in apical Na entry. However, activation of PKC by phorbol esters inhibits beta 2-AR-dependent increases in Na-K-ATPase activity and sodium transport.

Endothelial cells protect vascular smooth muscle cells from H2O2 attack.

Endothelial-dependent vascular responses are altered in ischemic acute renal failure. Oxidants formed during reperfusion of ischemic kidneys injure the renal microvasculature and prevent recovery of renal function. To determine whether endothelial cells (EC) modulate oxidant attack on vascular smooth muscle cells (VSMC), rat mesenteric artery VSMC were grown on coverslips and then coincubated with bovine pulmonary artery EC grown in wells. In the absence of EC, H2O2 caused time- and concentration-dependent increases in VSMC injury as indicated by release of [3H]adenine. In contrast, addition of EC reduced H2O2-mediated (5 mM, 1 h) VSMC adenine release from 63.8 +/- 4.5% to 28.6 +/- 2.9% (P < 0.001). The protective effect of EC did not occur when H2O2 was added to the surface of VSMC unopposed to EC and was partially reversed when EC were treated with aminotriazole to inactivate catalase (41.7 +/- 2.7%). To determine whether EC nitric oxide (NO) modified H2O2 attack on VSMC, EC were treated with N omega-nitro-L-arginine (L-NNA). The protective effect of EC was partially abrogated with L-NNA (53.8 +/- 4.3%). Treatment of EC with interleukin-1 beta (IL-1 beta) for 24 h prior to coincubation with VSMC enhanced the protective effect of EC. IL-1 beta-induced protection was reversed with L-NNA. No protection was observed when VSMC were treated with 8-bromoguanosine 3',5'-cyclic monophosphate, forskolin, or phorbol 12-myristate 13-acetate. Our conclusions are as follows. VSMC are protected by EC from luminal but not contraluminal oxidant attack. The protective effect of EC is mediated by catalase- and NO-dependent inactivation of oxidants. EC dysfunction could account for renal injury caused by oxidants formed during reperfusion of ischemic kidneys.

Ischemia increases neutrophil retention and worsens acute renal failure: role of oxygen metabolites and ICAM 1.

UNLABELLED: The role of neutrophils in acute renal failure (ARF) is controversial. Although ARF occurs in neutropenic subjects, we found that ischemic kidneys activated neutrophils to cause ARF in isolated perfused rat kidneys. To further define the interaction between neutrophils and renal ischemia, we performed quantitative assessment of neutrophil accumulation during renal ischemia. Non-ischemic and ischemic rat kidneys were perfused by the isolated kidney technique with unstimulated, primed, or fully activated, indium-labeled neutrophils. Neutrophil accumulation was quantitated by measuring indium retention after 60 minutes of perfusion. In non-ischemic kidneys, only activated neutrophils were retained while after 20 minutes of renal ischemia, unstimulated as well as primed neutrophils were retained. Following 10 minutes of ischemia, primed neutrophils (but not unstimulated neutrophils) were retained. In the presence of neutrophil retention, there were decreases in GFR and tubular sodium reabsorption. To determine the role of ICAM 1 in ischemic injury, rats were treated with anti-ICAM 1 prior to ischemia and ischemic kidneys were reperfused with unstimulated neutrophils and anti-ICAM 1. After ischemia, the neutrophil component of reperfusion injury in isolated kidneys was prevented with anti-ICAM 1. Oxygen metabolites have been shown to induce EC expression of ICAM 1. To determine the role of ICAM 1 in oxidant-mediated renal injury, ischemic isolated kidneys were reperfused with catalase (CAT) and non-ischemic kidneys were perfused with hydrogen peroxide. Following ischemia, reperfusion with CAT prevented neutrophil retention and injury. In non-ischemic kidneys, hydrogen peroxide caused primed neutrophil retention, activation and renal injury which were completely prevented with anti-ICAM 1. IN CONCLUSION: (1) Ischemic kidneys cause neutrophil retention, activation, and worsening of renal injury in isolated kidneys; and 2) neutrophil retention is dependent on the state of neutrophil activation, duration of renal ischemia and is mediated by oxygen metabolites and ICAM 1. This synergism could account for the high frequency of ARF in conditions such as sepsis where there is both renal hypoperfusion and neutrophil priming.

Compulsive water drinking in the setting of anticholinergic drug use: an unrecognized cause of chronic renal failure.

Compulsive water drinking (psychogenic polydipsia) is a well-recognized clinical entity that is often seen in individuals with psychiatric disorders, especially schizophrenia. Although urinary tract abnormalities including enlarged bladders and hydronephrosis have been reported, the presence of chronic renal failure is rarely reported in this disorder. We report four patients with psychogenic polydipsia who presented with chronic renal failure due to obstructive uropathy in the absence of demonstrable anatomic causes of obstruction. The likely mechanism of functional obstructive uropathy is bladder failure due to a combination of excessive water ingestion, enlarged bladder volumes, and use of anticholinergic medications.

Beta-adrenergic receptor function in rat proximal tubule epithelial cells in culture.

The adrenergic system is important in regulating proximal tubule sodium reabsorption. Although alpha-adrenergic receptors have been identified in proximal tubules, the presence and function of beta-adrenergic receptors (BAR) in proximal tubules is less certain. The purpose of our study was to determine whether functional BAR are present on apical or basolateral surfaces of proximal tubule epithelial cells (PTEC) of rat kidney. We specifically focused on BAR coupling to adenylate cyclase and on differences between requirements for apical and basolateral receptor coupling to adenylate cyclase. To determine BAR expression and function, primary cultures of rat PTECs were grown on permeable supports. Scatchard analysis of 125I-labeled cyanopindolol binding revealed a single class of receptors on both apical and basolateral surfaces. Apical isoproterenol (ISO) resulted in time- and concentration-dependent increases in adenosine 3',5'-cyclic monophosphate (cAMP) that were 50% of responses after basolateral ISO. Apical BAR-cAMP coupling was mediated by B1-adrenergic receptors (B1AR), since apical cAMP responses were abrogated with apical (but not basolateral) B1 but not B2 antagonists. Apical B1AR required endocytosis prior to adenylate cyclase activation, since increases in cAMP were prevented by phenylarsine oxide or colchicine. B1AR-adenylate cyclase coupling was independent of intra- or extracellular calcium, cyclooxygenase metabolites, and protein kinase C (PKC) and dependent on Gs guanine nucleotide regulatory protein. Prolonged exposure to ISO resulted in time- and concentration-dependent homologous desensitization of cAMP responses. Desensitization was independent of receptor sequestration, PKA, or PKC. We conclude the following: B1AR are present on both apical and basolateral surfaces of rat PTECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C.

Angiotensin II (ANG II) stimulates proximal tubule sodium transport by decreasing adenylyl cyclase activity. The role of ANG II-dependent phospholipase C is less certain. To determine the contribution of phospholipase C and adenylyl cyclase to apical (AP) ANG II-dependent sodium transport, unidirectional (AP to basolateral) 22Na flux was measured in rat proximal tubule cells cultured on permeable supports. AP ANG II (100 nM)-dependent sodium flux was prevented by preincubation with concentrations of the phospholipase C inhibitor U-73122 (1 microM) that blocked ANG II-dependent inositol phosphate formation. AP ANG II-dependent sodium flux was also abolished by preincubation with the intracellular calcium mobilization inhibitor 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), further suggesting that ANG II-dependent sodium transport was mediated by inositol phosphates. Neither U-73122 nor TMB-8 prevented ANG II-dependent adenosine 3',5'-cyclic monophosphate (cAMP) decreases. Incubation with dibutyryl cAMP (10 microM) or forskolin (10 microM) prevented ANG II-dependent sodium flux as well as ANG II-dependent inositol phosphate formation. In conclusion, ANG II-dependent proximal tubule sodium transport in cultured cells was transduced by phospholipase C and adenylyl cyclase. The adenylyl cyclase effect on ANG II-dependent sodium transport was mediated by phospholipase C.

Interleukin-1 treatment increases neutrophils but not antioxidant enzyme activity or resistance to ischemia-reperfusion injury in rat kidneys.

Hearts from rats treated with interleukin-1 (IL-1) intraperitoneally developed a rapid (6 h after IL-1), transient increase in neutrophils, tissue hydrogen peroxide (H2O2), and oxidized glutathione (GSSG) levels, and a subsequent (36 h after IL-1) increase in myocardial glucose-6-phosphate dehydrogenase (G6PD) activity and tolerance to ischemia-reperfusion. In the present investigation, we found that rats treated similarly with IL-1 had increased numbers of neutrophils in their kidneys, which were comparable to myocardial neutrophil increases, but did not develop increased renal tissue H2O2 or GSSG levels acutely (6 h after IL-1) or increased G6PD activity or resistance to ischemia-reperfusion injury later (36 h after IL-1). Our findings indicate that IL-1 treatment increased neutrophil accumulation in rat kidneys but did not increase oxidative stress, antioxidant enzyme activity, or resistance to ischemia-reperfusion injury. We conclude that organ-to-organ differences exist with respect to IL-1-induced tolerance.

Glucocorticoid uncoupling of antiogensin II-dependent phospholipase C activation in rat vascular smooth muscle cells.

Vascular tone is maintained by both angiotensin II (Ang II) and glucocorticoids, but the effect of glucocorticoids on Ang II function in vascular smooth muscle cells (VSMC) is unclear. To determine the direct influence of glucocorticoids on VSMC Ang II receptor function, the effects of dexamethasone on Ang II receptor binding, Ang II-induced phospholipase C (PLC) activation, and Ang II-dependent cell growth were studied in cultured rat VSMC. Dexamethasone caused concentration- and time-dependent increases in Ang II binding which were prevented by glucocorticoid receptor inhibition with RU 38486. Dexamethasone-induced enhancement of Ang II binding resulted from increased AT1 receptors, as indicated by Northern blot analysis and competitive binding assays. Despite causing increased Ang II receptor number, dexamethasone preincubation prevented Ang II-induced PLC activation, as indicated by phosphatidylinositol 4,5-bisphosphate degradation and inositol trisphosphate formation. When PLC activity was directly measured in VSMC soluble and membrane fractions, Ang II receptor activation caused decreased soluble and increased membrane PLC activity, consistent with the interpretation that Ang II caused cytosol-to-membrane PLC translocation. The effect of Ang II on PLC translocation was prevented by dexamethasone preincubation. Finally, prolonged incubation with dexamethasone and Ang II had additive effects on VSMC hypertrophy. In conclusion, glucocorticoids directly altered Ang II function in VSMC by causing increased Ang II receptor number, Ang II receptor/PLC uncoupling, and enhanced Ang II-dependent hypertrophy.

Parathyroid hormone/adenylate cyclase coupling in vascular smooth muscle cells.

Parathyroid hormone (PTH) has been implicated in hypertension, but PTH infusion results in vasodilation. PTH activates adenylate cyclase in vascular smooth muscle, but little is known about the factors that regulate PTH receptor/adenylate cyclase coupling in vascular cells. To characterize hormone-receptor signaling, we measured cyclic AMP levels in rat arterial smooth muscle cells in culture exposed to PTH (bovine 1-34). PTH yielded time- and concentration-dependent increases in cyclic AMP levels. Compared with isoproterenol, PTH was more potent, with a threshold at 2 x 10(-9) versus 5 x 10(-8) mol/L and half maximal responses at 10(-8) versus 2.4 x 10(-7) mol/L. PTH-induced increases in cyclic AMP were independent of extracellular calcium, cyclooxygenase metabolites, phospholipase C, and protein kinase C because PTH-induced increases in cyclic AMP were not prevented by variations in extracellular calcium, indomethacin, angiotensin II, vasopressin, and protein kinase C activators or inhibitors. PTH/adenylate cyclase coupling was G protein-dependent because increases in cyclic AMP were prevented by preincubation with cholera toxin but not with pertussis toxin. Prolonged exposure to PTH resulted in time- and concentration-dependent homologous desensitization of cyclic AMP responses. Desensitization occurred proximal to G protein/adenylate cyclase because after prolonged PTH, responses to forskolin and cholera toxin remained intact. Desensitization was independent of protein kinase A and receptor sequestration because cyclic AMP responses remained after prolonged exposure to forskolin and pretreatment with phenylarsine oxide, colchicine, and cytochalasin D. We conclude that in vascular smooth muscle cells, PTH is coupled to adenylate cyclase through a cholera toxin-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis.

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.

Potassium therapy of hypertension.

Hypertension is an important contributor to cerebrovascular and cardiovascular morbidity and mortality. Nonpharmacological treatment has been an important first-line therapy for mild hypertension. In the last decade, there is increasing evidence from epidemiological, interventional, and animal studies for the role of potassium supplementation in the management of hypertension. The antihypertensive effect of potassium is mediated predominantly via natriuresis. Hence, it is especially effective in the management of salt-sensitive essential hypertensive patients. In this review, we will summarize the available data and discuss the role of potassium supplementation in the management of essential hypertension.

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.

Mild renal ischemia activates primed neutrophils to cause acute renal failure.

The role of neutrophils (PMN) in acute renal failure (ARF) is controversial. Although the development of acute renal failure (ARF) frequently occurs in situations where there is partial activation of PMN (primed PMN) and mild renal ischemia, the interaction between primed PMN and ischemic organs has not been studied in any biological system. To define the interaction between primed PMN and mild renal ischemia, kidneys were made ischemic for 10 minutes in situ and reperfused by the isolated kidney technique with untreated PMN or PMN primed with low concentrations of lipopolysaccharide (LPS) or phorbol myristate acetate (PMA). We found that primed PMN had no effect on control (non-ischemic) kidneys and that untreated PMN did not cause injury to kidneys previously subjected to mild ischemia. However, addition of primed PMN to mildly ischemic kidneys caused severe injury. To determine the nature of renal injury, ischemic kidneys were reperfused with primed PMN and catalase (CAT) or the elastase inhibitor, Eglin C. In ischemic kidneys reperfused with LPS-primed PMN, Eglin C (but not CAT) was partially protective while in ischemic kidneys reperfused with PMA-primed PMN, CAT (but not Eglin C) was partially protective. Reperfusion with both CAT and Eglin C completely prevented the damaging effects of either LPS- or PMA-primed PMN. In conclusion, addition of primed but not untreated PMN causes ARF in mildly ischemic kidneys by PMN oxidant- and/or protease-mediated mechanisms. This synergism could account for the high frequency of ARF in conditions associated with prerenal azotemia and primed PMN.

Aldosterone enhances angiotensin II receptor binding and inositol phosphate responses.

Clinical states in which angiotensin II is increased are often associated with increases in mineralocorticoids. To determine the effects of mineralocorticoids on angiotensin II action, we examined the effects of aldosterone on angiotensin II receptor expression and function in cultured rat vascular smooth muscle cells. Incubation with aldosterone resulted in concentration- and time-dependent increases in angiotensin II receptor number, without changes in binding affinity. For example, incubation with 1 microM aldosterone for 40 hours resulted in 59% increases in angiotensin II receptor number. Increases in angiotensin II receptors were dependent on protein synthesis as evidenced by the time dependency of upregulation and inhibition by cycloheximide. Incubation with aldosterone resulted in enhanced angiotensin II-stimulated phospholipase C activation, as demonstrated by increases in angiotensin II-induced inositol phosphate responses in proportion to the increases in receptor number. In addition, aldosterone prevented angiotensin II-induced downregulation of angiotensin II surface receptors and angiotensin II desensitization of inositol phosphate formation. In summary, aldosterone 1) directly increased angiotensin II receptor number, 2) increased angiotensin II-stimulated inositol phosphate responses, and 3) prevented angiotensin II-induced downregulation and desensitization. In conclusion, aldosterone may potentiate the pressor responses of angiotensin II via effects on angiotensin II receptors.

Role of neutrophil derived oxidants and elastase in lipopolysaccharide-mediated renal injury.

Gram-negative bacterial sepsis is frequently associated with acute renal failure but the specific effects of lipopolysaccharide (LPS) and other bacterial products on kidney function are not known. Since either LPS or formyl-methionyl-leucyl-phenylalanine (FMLP)--a chemotactic peptide from bacterial cell walls--activate neutrophils (PMN) to release a number of potentially toxic factors in vitro, we determined the effect of adding PMN with LPS and/or FMLP to isolated perfused rat kidneys. Isolated rat kidneys perfused with LPS alone or LPS and normal PMN had normal glomerular filtration rates (GFR) and tubular Na reabsorption (TNa). Kidneys perfused with FMLP alone or FMLP and normal PMN also had normal GFR and TNa. In contrast, addition of PMN with both FMLP and LPS caused progressive renal dysfunction. For example, after 60 minutes of perfusion, GFR was reduced from 610 +/- 31 to 147 +/- 17 microliters/min/g and TNa from 97 +/- 1 to 72 +/- 2%, both P less than 0.01. Perfusion with the O2 metabolite scavengers catalase or dimethylthiourea afforded no protection while perfusion with the neutrophil elastase inhibitor Eglin C conferred substantial, but not complete, protection: GFR 492 +/- 34 microliters/min/g; TNa 91 +/- 3%. However, perfusion with both Eglin C and catalase completely prevented the toxic effects of LPS and FMLP-treated PMN on renal function. We conclude that in isolated kidneys, 1) the toxic effects of LPS requires FMLP-treated PMN and that 2) LPS and FMLP treated PMN cause progressive renal injury which is mediated by both O2 metabolites and neutrophil elastase.