In 1977, Wyngaarden expressed concern about the declining interest of physicians in clinical research, helping to initiate a national dialogue about the state of clinical research in the United States ( 1 Goldstein J.L. Brown M.S. The clinical investigator bewitched, bothered, and bewildered—but still beloved. J Clin Invest. 1997; 99: 2803-2812 Crossref PubMed Scopus (263) Google Scholar , 2 Wyngaarden J.B. The clinical investigator as an endangered species. New Engl J Med. 1977; 301: 1254-1259 Crossref Scopus (411) Google Scholar ). Nearly three decades later, concern remains throughout the medical research community that discoveries in basic science are not translating into the delivery of better health care. These concerns focus primarily on the need to enhance public participation in clinical trials, develop research-friendly information technology systems, foster an adequately trained and sufficiently large workforce, and ensure sufficient funding for clinical research ( 3 Institute of MedicineExploring challenges, progress, and new models for engaging the public in the clinical research enterprise. The National Academies Press, Washington, DC2003 Google Scholar , 4 Sung N.S. Crowley W.F. Genel M. et al. Central challenges facing the national clinical research enterprise. JAMA. 2003; 289: 1278-1287 Crossref PubMed Scopus (978) Google Scholar ).

The mRNA 3'-untranslated region (3'-UTR) of many genes has been identified as an important regulator of the mRNA transcript itself as well as the translated product. Previously, we demonstrated that Chinese-hamster ovary-K1 cells stably expressing angiotensin receptor subtypes (AT(1A)) with and without 3'-UTR differed in AT(1A) mRNA content and its coupling with intracellular signalling pathways. Moreover, RNA mobility-shift assay and UV cross-linking studies using the AT(1A) 3'-UTR probe identified a major mRNA-binding protein complex of 55 kDa in Chinese-hamster ovary-K1 cells. In the present study, we have determined the functional significance of the native AT(1A) receptor 3'-UTR in rat liver epithelial (WB) cell lines by co-expressing the AT(1A) 3'-UTR sequence 'decoy' to compete with the native receptor 3'-UTR for its mRNA-binding proteins. PCR analysis using specific primers for the AT(1A) receptor and [(125)I]angiotensin II (AngII)-binding studies demonstrated the expression of the native AT(1A) receptors in WB (B(max)=2.7 pmol/mg of protein, K(d)=0.56 nM). Northern-blot analysis showed a significant increase in native receptor mRNA expression in 3'-UTR decoy-expressing cells, confirming the role of 3'-UTR in mRNA destabilization. Compared with vehicle control, AngII induced DNA and protein synthesis in wild-type WB as measured by [(3)H]thymidine and [(3)H]leucine incorporation respectively. Activation of [(3)H]thymidine and [(3)H]leucine correlated with a significant increase in cell number (cellular hyperplasia). In these cells, AngII stimulated GTPase activity by AT(1) receptor coupling with G-protein alpha i. We also delineated that functional coupling of AT(1A) receptor with G-protein alpha i is an essential mechanism for AngII-mediated cellular hyperplasia in WB by specifically blocking G-protein alpha i activation. In contrast with wild-type cells, stable expression of the 3'-UTR 'decoy' produced AngII-stimulated protein synthesis and cellular hypertrophy as demonstrated by a significant increase in [(3)H]leucine incorporation and no increase in [(3)H]thymidine incorporation and cell number. Furthermore, [(125)I]AngII cross-linking and immunoprecipitation studies using specific G-protein alpha antibodies showed that in wild-type cells, the AT(1A) receptor coupled with G-protein alpha i, whereas in cells expressing the 3'-UTR 'decoy', the AT(1A) receptor coupled with G-protein alpha q. These findings indicate that the 3'-UTR-mediated changes in receptor function may be mediated in part by a switch from G-protein alpha i to G-protein alpha q coupling of the receptor. Our results suggest that the 3'-UTR-mediated post-transcriptional modification of the AT(1A) receptor is critical for regulating tissue-specific receptor functions.

Angiotensin II (ANG II), acting through angiotensin type I (AT(1)) receptors on apical and basolateral surfaces of proximal tubule epithelial cells, increases sodium reabsorption in proximal tubules. Apical and basolateral receptors internalize after exposure to ANG II, but the role of internalization in receptor signaling and transport is not well defined. To determine the role of receptor internalization in ANG II-mediated receptor signaling and sodium transport, we stably expressed full-length and truncated AT(1A) receptors in opossum kidney cells. After stimulation with ANG II, wild-type receptors on apical and basolateral surfaces rapidly internalized, inhibited adenylate cyclase, and increased transcellular sodium transport. Truncation of the cytoplasmic tail of the AT(1A) receptor (TL314) resulted in receptors that were expressed on apical and basolateral surfaces but did not internalize, inhibit adenylate cyclase, or increase sodium transport. Because the cytoplasmic tail contains putative G protein coupling sites, mutant receptors that leave G protein interaction sites intact were designed. Cells expressing the truncation (TK333) or deletion (Del 315-329) also failed to internalize. When ANG II was added to basolateral surfaces of TK333 or Del 315-329, adenylate cyclase activity was inhibited and sodium transport was increased. In contrast, apical addition of ANG II was not associated with decreases in adenylate cyclase or increases in sodium transport. In conclusion, internalization pathways are important for AT(1A) receptor function in polarized proximal tubule epithelial cells. Apical AT(1A) receptors internalize before they interact with G proteins and signal cAMP. In contrast, basolateral AT(1A) receptors interact with G proteins and signal cAMP without internalizing.

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.

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1Aare present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1Afunction 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 AT1AmRNA (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 AT1Aexpression 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. AT1Awere 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 AT1Areceptors 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 transepithelial22Na 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 AT1Aon 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 AT1Aexpression and function in PTEC.

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-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.

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.

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.

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)

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.

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.

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.