[Figure: see text].

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

Background Angiotensin II stimulates epithelial Na+ channel (ENaC) by aldosterone-independent mechanism. We now test the effect of angiotensin II on ENaC in the distal convoluted tubule (DCT) and cortical collecting duct (CCD) of wild-type (WT) and kidney-specific mineralocorticoid receptor knockout mice (KS-MR-KO). Methods and Results We used electrophysiological, immunoblotting and renal-clearance methods to examine the effect of angiotensin II on ENaC in KS-MR-KO and wild-type mice. High K+ intake stimulated ENaC in the late DCT/early connecting tubule (DCT2/CNT) and in the CCD whereas low sodium intake stimulated ENaC in the CCD but not in the DCT2/CNT. The deletion of MR abolished the stimulatory effect of high K+ and low sodium intake on ENaC, partially inhibited ENaC in DCT2/CNT but almost abolished ENaC activity in the CCD. Application of losartan inhibited ENaC only in DCT2/CNT of both wild-type and KS-MR-KO mice but not in the CCD. Angiotensin II infusion for 3 days has a larger stimulatory effect on ENaC in the DCT2/CNT than in the CCD. Three lines of evidence indicate that angiotensin II can stimulate ENaC by MR-independent mechanism: (1) angiotensin II perfusion augmented ENaC expression in KS-MR-KO mice; (2) angiotensin II stimulated ENaC in the DCT2/CNT but to a lesser degree in the CCD in KS-MR-KO mice; (3) angiotensin II infusion augmented benzamil-induced natriuresis, increased the renal K+ excretion and corrected hyperkalemia of KS-MR-KO mice. Conclusions Angiotensin II-induced stimulation of ENaC occurs mainly in the DCT2/CNT and to a lesser degree in the CCD and MR plays a dominant role in determining ENaC activity in the CCD but to a lesser degree in the DCT2/CNT.

The prevalence of hypertension is about twofold higher in diabetic than in nondiabetic subjects. Hypertension aggravates the progression of diabetic complications, especially diabetic nephropathy. However, the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that enhanced constrictive responsiveness of renal afferent arterioles (Af-Art) to angiotensin II (ANG II) mediated by ANG II type 1 (AT1) receptors contributes to the development of hypertension in diabetes. In response to an acute bolus intravenous injection of ANG II, alloxan-induced diabetic mice exhibited a higher mean arterial pressure (MAP) (119.1 ± 3.8 vs. 106.2 ± 3.5 mmHg) and a lower renal blood flow (0.25 ± 0.07 vs. 0.52 ± 0.14 ml/min) compared with nondiabetic mice. In response to chronic ANG II infusion, the MAP measured with telemetry increased by 55.8 ± 6.5 mmHg in diabetic mice, but only by 32.3 ± 3.8 mmHg in nondiabetic mice. The mRNA level of AT1 receptor increased by ~10-fold in isolated Af-Art of diabetic mice compared with nondiabetic mice, whereas ANG II type 2 (AT2) receptor expression did not change. The ANG II dose-response curve of the Af-Art was significantly enhanced in diabetic mice. Moreover, the AT1 receptor antagonist, losartan, blocked the ANG II-induced vasoconstriction in both diabetic mice and nondiabetic mice. In conclusion, we found enhanced expression of the AT1 receptor and exaggerated response to ANG II of the Af-Art in diabetes, which may contribute to the increased prevalence of hypertension in diabetes.

Angiotensin II is a vaso-constrictive peptide that regulates blood pressure homeostasis. Even though the inflammatory effects of AngII in renal pathophysiology have been studied, there still exists a paucity of data with regard to the mechanism of action of AngII-mediated kidney injury. The objective of this study was to elucidate the mechanistic role of HMGB1-TLR4 signaling in AngII-induced inflammation in the kidney.

Rat tubular epithelial cells (NRK52E) were treated with AngII over a preset time-course. In another set of experiments, HMGB1 was neutralized and TLR4 was knocked down using small interfering RNA targeting TLR4. Cell extracts were subjected to RT-PCR, immunoblotting, flow cytometry, and ELISA.

AngII-induced inflammation in NRK52E cells increased gene and protein expression of TLR4, HMGB1 and key proinflammatory cytokines (TNFα and IL1β). Pretreatment with Losartan (an AT1 receptor blocker) attenuated the AngII-induced expression of TLR4 and inflammatory cytokines. TLR4 silencing was used to elucidate the specific role played by TLR4 in AngII-induced inflammation. TLR4siRNA treatment in these cells significantly decreased the AngII-induced inflammatory effect. Consistent observations were made when the Ang II treated cells were pretreated with anti-HMGB1. Downstream activation of NFκB and rate of generation of ROS was also decreased on gene silencing of TLR4 and exposure to anti-HMGB1.

These results indicate a key role for HMGB1-TLR4 signaling in AngII-mediated inflammation in the renal epithelial cells. Our data also reveal that AngII-induced effects could be alleviated by HMGB1-TLR4 inhibition, suggesting this pathway as a potential therapeutic target for hypertensive renal dysfunctions.

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K(+) reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K(+) secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)--components of an E3 ubiquitin ligase complex that targets WNKs for degradation--cause constitutively increased renal salt reabsorption and impaired K(+) secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3(S433) phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K(+) secretion in the setting of volume depletion.

UT-A and UT-B families of urea transporters consist of multiple isoforms that are subject to regulation of both acutely and by long-term measures. This chapter provides a brief overview of the expression of the urea transporter forms and their locations in the kidney. Rapid regulation of UT-A1 results from the combination of phosphorylation and membrane accumulation. Phosphorylation of UT-A1 has been linked to vasopressin and hyperosmolality, although through different kinases. Other acute influences on urea transporter activity are ubiquitination and glycosylation, both of which influence the membrane association of the urea transporter, again through different mechanisms. Long-term regulation of urea transport is most closely associated with the environment that the kidney experiences. Low-protein diets may influence the amount of urea transporter available. Conditions of osmotic diuresis, where urea concentrations are low, will prompt an increase in urea transporter abundance. Although adrenal steroids affect urea transporter abundance, conflicting reports make conclusions tenuous. Urea transporters are upregulated when P2Y2 purinergic receptors are decreased, suggesting a role for these receptors in UT regulation. Hypercalcemia and hypokalemia both cause urine concentration deficiencies. Urea transporter abundances are reduced in aging animals and animals with angiotensin-converting enzyme deficiencies. This chapter will provide information about both rapid and long-term regulation of urea transporters and provide an introduction into the literature.

We used the patch-clamp technique to examine the effect of angiotensin II (ANG II) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of ANG II increased the channel activity and the current amplitude of the basolateral 50-pS K channel. The stimulatory effect of ANG II on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of ANG II on the basolateral K channels in the TAL. This suggests that the stimulatory effect of ANG II on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that ANG II increased the phosphorylation of c-Src at tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of ANG II on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of ANG II on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of ANG II on the basolateral 50-pS K channel. We conclude that ANG II increases basolateral 50-pS K channel activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.

The uriniferous tubule is divided into the proximal tubule, the intermediate (thin) tubule, the distal tubule and the collecting duct. The present chapter is based on the chapters by Maunsbach and Christensen on the proximal tubule, and by Kaissling and Kriz on the distal tubule and collecting duct in the 1992 edition of the Handbook of Physiology, Renal Physiology. It describes the fine structure (light and electron microscopy) of the entire mammalian uriniferous tubule, mainly in rats, mice, and rabbits. The structural data are complemented by recent data on the location of the major transport- and transport-regulating proteins, revealed by morphological means(immunohistochemistry, immunofluorescence, and/or mRNA in situ hybridization). The structural differences along the uriniferous tubule strictly coincide with the distribution of the major luminal and basolateral transport proteins and receptors and both together provide the basis for the subdivision of the uriniferous tubule into functional subunits. Data on structural adaptation to defined functional changes in vivo and to genetical alterations of specified proteins involved in transepithelial transport importantly deepen our comprehension of the correlation of structure and function in the kidney, of the role of each segment or cell type in the overall renal function,and our understanding of renal pathophysiology.

The aim is to review the recently reported effects of angiotensin II (Ang II) on sodium and potassium transport in the aldosterone-sensitive distal nephron, including the signaling pathways between receptor and transporter, and the (patho)physiological implications of these findings.

Ang II can activate the sodium chloride cotransporter (NCC) through phosphorylation by Ste20-related, proline-alanine rich kinase (SPAK), an effect that is independent of aldosterone but dependent on with no lysine kinase 4 (WNK4). A low-sodium diet (high Ang II) activates NCC, whereas a high-potassium diet (low Ang II) inhibits NCC. NCC activation also contributes to Ang-II-mediated hypertension. Ang II also activates the epithelial sodium channel (ENaC) additively to aldosterone, and this effect appears to be mediated through protein kinase C and superoxide generation by nicotinamide adenine dinucleotide phosphate oxidase. While aldosterone activates the renal outer medullary potassium channel (ROMK), this channel is inhibited by Ang II. The key kinase responsible for this effect is c-Src, which phosphorylates ROMK and leaves WNK4 unphosphorylated to further inhibit ROMK.

The effects of Ang II on NCC, ENaC, and ROMK help explain the renal response to hypovolemia which is to conserve both sodium and potassium. Pathophysiologically, Ang-II-induced activation of NCC appears to contribute to salt-sensitive hypertension.

Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.

The renin-angiotensin-aldosterone system (RAAS) was initially thought to be fairly simple. However, this idea has been challenged following the development of RAAS blockers, including renin inhibitors, angiotensin-converting-enzyme (ACE) inhibitors, type 1 angiotensin II (AT(1))-receptor blockers and mineralocorticoid-receptor antagonists. Consequently, new RAAS components and pathways that might contribute to the effectiveness of these drugs and/or their adverse effects have been identified. For example, an increase in renin levels during RAAS blockade might result in harmful effects via stimulation of the prorenin receptor (PRR), and prorenin-the inactive precursor of renin-might gain enzymatic activity on PRR binding. The increase in angiotensin II levels that occurs during AT(1)-receptor blockade might result in beneficial effects via stimulation of type 2 angiotensin II receptors. Moreover, angiotensin 1-7 levels increase during ACE inhibition and AT(1)-receptor blockade, resulting in Mas receptor activation and the induction of cardioprotective and renoprotective effects, including stimulation of tissue repair by stem cells. Finally, a role of angiotensin II in sodium and potassium handling in the distal nephron has been identified. This finding is likely to have important implications for understanding the effects of RAAS inhibition on whole body sodium and potassium balance.

Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

The distal convoluted tubule (DCT) plays a central role in blood pressure and potassium homeostasis, as evidenced by diseases that occur when its function is modified. The paper by van der Lubbe and colleagues makes clear that angiotensin II itself increases the activity and abundance of the thiazide-sensitive Na-Cl cotransporter (NCC), independent of changes in circulating aldosterone. This Commentary provides additional perspective on that work.

Urea transport proteins were initially proposed to exist in the kidney in the late 1980s when studies of urea permeability revealed values in excess of those predicted by simple lipid-phase diffusion and paracellular transport. Less than a decade later, the first urea transporter was cloned. Currently, the SLC14A family of urea transporters contains two major subgroups: SLC14A1, the UT-B urea transporter originally isolated from erythrocytes; and SLC14A2, the UT-A group with six distinct isoforms described to date. In the kidney, UT-A1 and UT-A3 are found in the inner medullary collecting duct; UT-A2 is located in the thin descending limb, and UT-B is located primarily in the descending vasa recta; all are glycoproteins. These transporters are crucial to the kidney's ability to concentrate urine. UT-A1 and UT-A3 are acutely regulated by vasopressin. UT-A1 has also been shown to be regulated by hypertonicity, angiotensin II, and oxytocin. Acute regulation of these transporters is through phosphorylation. Both UT-A1 and UT-A3 rapidly accumulate in the plasma membrane in response to stimulation by vasopressin or hypertonicity. Long-term regulation involves altering protein abundance in response to changes in hydration status, low protein diets, adrenal steroids, sustained diuresis, or antidiuresis. Urea transporters have been studied using animal models of disease including diabetes mellitus, lithium intoxication, hypertension, and nephrotoxic drug responses. Exciting new animal models are being developed to study these transporters and search for active urea transporters. Here we introduce urea and describe the current knowledge of the urea transporter proteins, their regulation, and their role in the kidney.

Increasing Na delivery to epithelial Na channels (ENaCs) in the connecting tubule (CNT) causes dilation of the afferent arteriole (Af-Art), a process we call CNT glomerular feedback (CTGF). Angiotensin II (Ang II) stimulates ENaC in the collecting duct via Ang II type 1 receptors. We hypothesized that Ang II in the CNT lumen enhances CTGF by activation of Ang II type 1 receptors, protein kinase C and ENaC. Rabbit afferent arterioles and their adherent CNT were microperfused and preconstricted with norepinephrine. Each experiment involved generating 2 consecutive concentration-response curves by increasing NaCl in the CNT lumen. During the control period, the maximum dilation of the afferent arteriole was 7.9±0.4 μm, and the concentration of NaCl in the CNT needed to achieve half maximal response (EC(50)) was 34.7±5.2 mmol/L. After adding Ang II (10(-9) mol/L) to the CNT lumen, the maximal response was 9.5±0.7 μm and the EC(50) was 11.6±1.3 mmol/L (P=0.01 versus control). Losartan, an Ang II type 1 antagonist (10(-6) mol/L) blocked the stimulatory effect of Ang II; PD123319, an Ang II type 2 antagonist (10(-6) mol/L), did not. The protein kinase C inhibitor staurosporine (10(-8) mol/L) added to the CNT inhibited the stimulatory effect of Ang II. The ENaC inhibitor benzamil (10(-6) mol/L) prevented both CTGF and its stimulation by Ang II. We concluded that Ang II in the CNT lumen enhances CTGF via activation of Ang II type 1 and that this effect requires activation of protein kinase C and ENaC. Potentiation of CTGF by Ang II could help preserve glomerular filtration rate in the presence of renal vasoconstriction.

The aldosterone-sensitive distal nephron (ASDN) includes the late distal convoluted tubule 2, the connecting tubule (CNT) and the collecting duct. The appropriate regulation of sodium (Na(+)) absorption in the ASDN is essential to precisely match urinary Na(+) excretion to dietary Na(+) intake whilst taking extra-renal Na(+) losses into account. There is increasing evidence that Na(+) transport in the CNT is of particular importance for the maintenance of body Na(+) balance and for the long-term control of extra-cellular fluid volume and arterial blood pressure. Na(+) transport in the CNT critically depends on the activity and abundance of the amiloride-sensitive epithelial sodium channel (ENaC) in the luminal membrane of the CNT cells. As a rate-limiting step for transepithelial Na(+) transport, ENaC is the main target of hormones (e.g. aldosterone, angiotensin II, vasopressin and insulin/insulin-like growth factor 1) to adjust transepithelial Na(+) transport in this tubular segment. In this review, we highlight the structural and functional properties of the CNT that contribute to the high Na(+) transport capacity of this segment. Moreover, we discuss some aspects of the complex pathways and molecular mechanisms involved in ENaC regulation by hormones, kinases, proteases and associated proteins that control its function. Whilst cultured cells and heterologous expression systems have greatly advanced our knowledge about some of these regulatory mechanisms, future studies will have to determine the relative importance of the various pathways in the native tubule and in particular in the CNT.

The obese Zucker rat reportedly has increased activity of the intrarenal renin-angiotensin-aldosterone system, which conceptually could contribute to elevated salt sensitivity and blood pressure (BP). Our aim was to determine whether there was increased angiotensin II type 1 receptor (AT(1)R)-mediated upregulation of expression or activity of the bumetanide-sensitive Na-K-2Cl cotransporter, the thiazide-sensitive Na-Cl cotransporter (NCC), and/or the epithelial sodium channel (ENaC) in obese vs. lean Zucker rats. Male obese and lean Zucker rats (10-wk old) were fed either 1) control chow (1% NaCl) or 2) chow with candesartan (CAN), an AT(1)R antagonist (25 mg/kg.diet) for 14 wk (n = 8/treatment/body type). BP measured by radiotelemetry, was markedly reduced by CAN ( approximately 20-25 mmHg) in both lean and obese rats with no body-type differences. Obese rats had significantly greater net natriuretic response to single injections of hydrochlorothiazide and benzamil, suggesting increased activity of NCC and ENaC, respectively; however, only the response to benzamil was reduced by CAN. CAN led to a significant reduction in whole kidney levels of NCC and gamma-ENaC (70-kDa band) in both lean and obese rats. However, it significantly increased alpha-ENaC and Na-K-2Cl cotransporter levels, and these increases were greater in obese rats. These studies suggest that relatively increased ENaC, but not NCC activity, in obese rats is due to enhanced AT(1)R activity. CAN attenuated the reduction of several renal transporters in the obese rat kidney. Finally, differences in intrarenal AT(1)R activity do not seem directly responsible for BP differences between lean and obese rats.

Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl-cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type-1 (AT(1)) blockade with losartan.

In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT(1) receptor blockade on this system.

CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM-operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day(-1) i.p.).

CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10(-6) m) stimulated cAMP levels were significantly increased in CHF rats (25.52 +/- 4.49 pmol cAMP microg(-1) protein, P < 0.05) compared to Sham rats (8.13 +/- 1.14 pmol cAMP microg(-1) protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 +/- 1.93 fmol microg(-1) protein vs. Los-CHF: 7.49 +/- 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 +/- 0.59 vs. Los-SHAM: 4.75 +/- 0.71). AVP-mediated cAMP accumulation was absent in both groups treated with losartan (Los-SHAM: 4.75 +/- 0.71 and Los-CHF: 7.49 +/- 1.08).

The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT(1) receptor blockade prevents AVP-mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption.

To explore the role of the kallikrein-kinin system in relation to ischemia/reperfusion injury in the kidney, we generated mice lacking both the bradykinin B1 and B2 receptor genes (B1RB2R-null, Bdkrb1-/-/Bdkrb2-/-) by deleting the genomic region encoding the two receptors. In 4-month-old mice, blood pressures were not significantly different among B1RB2R-null, B2R-null (Bdkrb2-/-), and WT mice. After 30 min of bilateral renal artery occlusion and 24 h of reperfusion, mortality rates, renal histological and functional changes, 8-hydroxy-2'-deoxyguanosine levels in total DNA, mtDNA deletions, and the number of TUNEL-positive cells in the kidneys increased progressively in the following order (from lowest to highest): WT, B2R-null, and B1RB2R-null mice. Increases in mRNA levels of TGF-beta1, connective tissue growth factor, and endothelin-1 after ischemia/reperfusion injury were also exaggerated in the same order (from lowest to highest): WT, B2R-null, and B1RB2R-null. Thus, both the B1 and B2 bradykinin receptors play an important role in reducing DNA damage, apoptosis, morphological and functional kidney changes, and mortality during renal ischemia/reperfusion injury.

The mechanisms that govern homeostasis of complex systems have been elusive but can be illuminated by mutations that disrupt system behavior. Mutations in the gene encoding the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and hyperkalemia. We show that physiology in mice transgenic for genomic segments harboring wild-type (TgWnk4(WT)) or PHAII mutant (TgWnk4(PHAII)) Wnk4 is changed in opposite directions: TgWnk4(PHAII) mice have higher blood pressure, hyperkalemia, hypercalciuria and marked hyperplasia of the distal convoluted tubule (DCT), whereas the opposite is true in TgWnk4(WT) mice. Genetic deficiency for the Na-Cl cotransporter of the DCT (NCC) reverses phenotypes seen in TgWnk4(PHAII) mice, demonstrating that the effects of the PHAII mutation are due to altered NCC activity. These findings establish that Wnk4 is a molecular switch that regulates the balance between NaCl reabsorption and K+ secretion by altering the mass and function of the DCT through its effect on NCC.

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.

Employing a purified lgG fraction of a polyclonal anti-AT1 receptor anti-body, raised against a synthetic octapeptide encompassing residues 14-21 of the first extracellular domain of the AT1 polypeptide, selective AT1 receptor expression was immunohistochemically demonstrable within renal structures in Sprague-Dawley (SD) rats and the desert rodent Meriones crassus. In both animal models, prominent AT1 receptor labelling was evident in renal vascular elements, particularly cortical inter-lobular arteries (IA) as well as vasa recta bundles in the inner stripe of the outer medulla. Less intense labelling was observed among peritubular capillary endothelia within the deep cortex, and at both the outer stripe and the inter-bundle regions of the inner stripe of the outer medulla. The binding of the anti-peptide anti-body was, however, lacking among glomeruli and, except for the intense labelling confined to basement membranes of Bowman's capsule of deep nephrons, was virtually absent in all renal tubular structures of both animal models. Structural assessment of the expressed AT1 receptors by two-dimensional Western blotting revealed that a spectrum of structurally distinct AT1 receptor isoforms is expressed in the renal tissues of both animal models. This spectrum was constituted by isoforms of equal size (70 kDa) but distinct pls in SD rats, and of both different sizes (67-73 kDa) and isoelectric points in M. crassus. In either species, the charge and/or size heterogeneity of AT1 receptor isoforms may be attributed in part to differential post-translational glycosylation mechanisms of the AT1 receptor polypeptide backbone. The potential for the differential glycosylation state of AT1 receptors to alter recognition properties may add another level of complexity to tissue-specific and/or species-specific mechanisms underlying angiotensin II interactions in the kidney.

For determination of the molecular mechanisms underlying the induction of epithelial cell hypertrophy by angiotensin II (Ang II), a well-characterized porcine renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptor subtypes, was transfected with cDNA encoding Ang II subtype 1 receptor (AT1R/Cl4). Ang II transactivated the EGF receptor (EGFR) in these AT1R/Cl4 cells, which was blocked by the selective AT1R antagonist losartan but not by the selective AT2R antagonist PD123319. Ang II did not transactivate EGFR in empty vector-transfected LLCPKcl4 cells (Vector/Cl4). Ang II elicited release of soluble heparin-binding EGF-like growth factor (HB-EGF) from AT1R/Cl4 cells, and Ang II-induced EGFR activation was prevented by pretreatment with the specific HB-EGF inhibitor CRM197 or the metalloproteinase inhibitors batimastat or phenanthroline, none of which had any effect on EGFR activation by exogenously administered EGF. Ang II stimulated protein synthesis and cell hypertrophy in AT1R/Cl4 cells without increasing cell number, and signaling studies revealed that Ang II stimulated phosphorylation of the 40S ribosomal protein S6 and the eukaryotic translation initiation factor 4E-binding protein 1, the two downstream target proteins of the mammalian target of rapamycin, which is a central regulator of protein synthesis and cell size. Ang II-induced mammalian target of rapamycin activation, [3H]leucine incorporation, and cellular hypertrophy were inhibited by pretreatment with either batimastat or CRM197 or by pretreatment with rapamycin or the EGFR tyrosine kinase inhibitor AG1478. Ang II also stimulated Smad 2/3 phosphorylation, which was blocked by a selective TGF-beta receptor I kinase inhibitor but not by CRM197. With blockade of TGF-beta receptor, Ang II-mediated hypertrophy was converted into cell proliferation, which was blocked by CRM197. In summary, this is the first demonstration that HB-EGF shedding-dependent EGFR transactivation, along with activation of TGF-beta signaling pathways, mediates Ang II-induced renal tubular epithelial cell hypertrophy.

1. The renin-angiotensin system may be involved in the compensatory adaptations occurring after the reduction of renal mass and during the consecutive changes leading to chronic renal failure. We therefore investigated the regulation of angiotensin II receptors in two models of renal hypertrophy in the rat: hypertrophy following uninephrectomy (UNx) or subtotal nephrectomy (STNx). The level of angiotensin type 1 (AT1A-R and AT1B-R) and type 2 (AT2-R) receptor mRNA was quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR) in specific renal zones and the intrarenal distribution of angiotensin II receptors was analysed by immunohistochemistry. 2. In the UNx rats, AT1-R mRNA expression was not modified in the cortex or in the inner stripe of the outer medulla of the residual kidney at any time after the surgery (1, 4 and 12 weeks). In contrast, AT1-R mRNA expression was significantly reduced in these zones in STNx rats (-33% and -40%, respectively). This downregulation was organ-specific, as AT1-R mRNA levels were not modified in the liver. The proportions of AT1-R subtype (AT1A and AT1B) mRNA were unchanged by UNx or STNx. Very low levels of AT2-R mRNA were found in the cortex of all groups. Immunostaining revealed a similar localization of AT1-R in mesangial cells, proximal tubule, basolateral membrane of thick ascending limb, in both models of hypertrophy. AT1-R labelling was also detected in the apical membrane of intercalated cells of cortical collecting ducts. 3. This differential mRNA expression of angiotensin II receptors during compensatory hypertrophy and renal injury suggests that the development of renal hypertrophy is independent of AT1-R and AT2-R gene expression levels.

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+ transporters. Rats were on a fixed intake of Na+ and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng x min(-1) x kg(-1) ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+ exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 +/- 28 (ANG II-50, n = 5), 166 +/- 23 (ANG II-100, n = 7), and 167 +/- 19% (ANG II-200, n = 4) of control levels (n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (BSC-1) in the ISOM was also increased to 187 +/- 28 (ANG II-50), 162 +/- 23 (ANG II-100), and 166 +/- 19% (ANG II-200) of control levels (P < 0.05), but there were no changes in the abundance of Na(+)-K(+)-ATPase and the electroneutral Na(+)-HCO3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng x min(-1) x kg(-1) for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+ and HCO3 reabsorption in response to ANG II.

Urea plays a critical role in the urine-concentrating mechanism in the inner medulla. Physiologic data provided evidence that urea transport in red blood cells and kidney inner medulla was mediated by specific urea transporter proteins. Molecular approaches during the past decade resulted in the cloning of two gene families for facilitated urea transporters, UT-A and UT-B, encoding several urea transporter cDNA isoforms in humans, rodents, and several nonmammalian species. Polyclonal antibodies have been generated to the cloned urea transporter proteins, and the use of these antibodies in integrative animal studies has resulted in several novel findings, including: (1) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; (2) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (3) UT-A protein abundance is upregulated in uremia in both liver and heart; and (4) UT-B is expressed in many nonrenal tissues and endothelial cells. This review will summarize the knowledge gained from using molecular approaches to perform integrative studies into urea transporter protein regulation, both in normal animals and in animal models of human diseases, including studies of uremic rats in which urea transporter protein is upregulated in liver and heart.

Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

Endogenous intratubular angiotensin II (Ang II) supports an autocrine tonic stimulation of NaCl absorption in the proximal tubule, and its production may be regulated independently of circulating Ang II. In addition, endogenous Ang II activity may be regulated at the brush border membrane (BBM), by the rate of aminopeptidase A and N (APA and APN) activities and the rate of Ca2+-independent phospholipase A2 (PLA2-dependent endocytosis and recycling of the complex Ang II subtype 1 (AT1) receptor (AT1-R). The aim of the present study was to look for subcellular localization of AT1-R, and APA and APN activities in the medullary thick ascending limb of Henle (mTAL), as well as search for an asymmetric coupling of AT1-R to signal transduction pathways.

Preparations of isolated basolateral membrane (BLMV) and luminal (LMV) membrane vesicles from rat mTAL were used to localize first, AT1-R by 125I-[Sar1, Ile8] Ang II binding studies and immunoblot experiments with a specific AT1-R antibody, and second, APA and APN activities. Microfluorometric monitoring of cytosolic Ca2+ with a Fura-2 probe was performed in mTAL microperfused in vitro, after apical or basolateral application of Ang II.

AT1-R were present in both LMV and BLMV, with a similar Kd (nmol/L range) and Bmax. Accordingly, BLMV and LMV preparations similarly stained specific AT1-R antibody. APA and APN activities were selectively localized in LMV, although to a lesser extent than those measured in BBM. In the in vitro microperfused mTAL, basolateral but not apical Ang II induced a transient increase in cytosolic [Ca2+].

Besides the presence of basolateral AT1-R in mTAL coupled to the classical Ca2+-dependent transduction pathways, AT1-R are present in LMV, not coupled with Ca2+ signaling, and co-localized with APA and APN activities. Thus, apical APA and APN may play an important role in modulating endogenous Ang II activity on NaCl reabsorption in mTAL.

Urea plays a key role in the urine-concentrating mechanism. Physiologic and molecular data demonstrate that urea transport in kidney and red blood cells occurs by specific urea transporter proteins. Two gene families for facilitated urea transporters, UT-A and UT-B, and several urea transporter cDNA isoforms have been cloned from human, rat, mouse, and several non-mammalian species. Polyclonal antibodies have been generated to many of the urea transporter proteins, and several novel findings have resulted from their use in integrative animal studies. For example, (a) vasopressin increases the phosphorylation of UT-A1 in rat inner medullary collecting duct; (b) UT-A1 protein abundance is increased in the rat inner medulla during conditions in which urine-concentrating ability is reduced; and (c) urea transporters are expressed in non-renal tissues, and UT-A protein abundance is up-regulated in uremia in both liver and heart. In addition to the facilitated urea transporters, functional evidence exists for active urea transport in the kidney collecting duct. This review summarizes the physiologic evidence for the existence of facilitated and active urea transporters, the molecular biology of the facilitated urea transporter gene families and cDNAs, and integrative studies into urea transporter protein regulation, both in the kidney and in other organs.

Differences involving serine residues in the sequence of the carboxyl-terminal tail of type 1 angiotensin II (Ang II) receptor subtypes AT(1A) and AT(1B) suggest differences in desensitization ability. We examined the Ang II-induced homologous desensitization patterns of both receptor subtypes in freshly isolated renal structures: glomerulus (Glom), afferent arteriole, and cortical thick ascending limb (CTAL), whose content in each subtype mRNA is different, by measuring variations in intracellular calcium concentration. A preexposure to a maximal dose of Ang II, followed by a second application of the same concentration, induced: 1) a complete desensitization in Glom, where AT(1A) and AT(1B) mRNAs were expressed in similar proportions, and 2) no or partial desensitization in afferent arteriole and CTAL, where AT(1A) mRNA was predominant. In the absence of nephron structure containing only AT(1B) mRNA, we studied rat anterior pituitary cells that exhibit high content in this subtype and observed that desensitization was not complete. In Glom, CTAL, and pituitary cells, desensitization proceeded in a dose-dependent manner. In Glom and CTAL, desensitization occurred via a PKC-independent mechanism. These results suggest that desensitization does not depend on the nature of Ang II receptor subtype but either on the proportion of each subtype in a given cell and/or on cell specific type. This could allow adaptive biological responses to Ang II appropriate to the specific function of a given cell type.

For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption.

In the rat and the rabbit, a number of studies have reported the effects of angiotensin II (ANG II) on Na(+) reabsorption by the proximal (PT) and distal (DT) convoluted tubules of the kidney. The aim of the present study was to examine the effect of ANG II on Ca(2+) uptake by the luminal membranes of the PT and DT of the rabbit. Incubation of PT and DT with 10(-12) M ANG II enhanced the initial Ca(2+) uptake in the two segments. Dose-response experiments revealed, for Ca(2+) as well as for Na(+) transport, a biphasic action with a maximal effect at 10(-12) M. Ca(2+) transport by the DT luminal membrane presents a dual kinetic. ANG II action influenced the high-affinity Ca(2+) channel, increasing maximal velocity from 0.72 +/- 0.03 to 0.90 +/- 0.05 pmol x microg(-1) x 10 s(-1) (P < 0.05, n = 3) and leaving the Michaelis-Menten constant unchanged. The effect of ANG II was abolished by losartan, suggesting that the hormone is acting through AT1 receptors. In the PT, calphostin C inhibited the effect of the hormone. It is therefore probable that protein kinase C is involved as a messenger. In the DT, however, neither Rp cAMP, calphostin C, nor econazole (a phospholipase A inhibitor) influenced the hormone action. Therefore, the mechanisms involved in the hormone action remain undetermined. Finally, we questioned whether ANG II acts in the same DT segment as does parathyroid hormone on Ca(2+) transport. The two hormones increased Ca(2+) transport, but their actions were not additive, suggesting that they both influence the same channels in the same segment of the distal nephron, i.e., the segment responsible for the high-affinity calcium channel.

1. Obese Zucker rats (OZR) were shown to be salt-sensitive in that they develop hypertension when placed on a high-salt diet. Because angiotensin (Ang) II is a major antinatriuretic factor, the present studies were undertaken to determine whether the characteristic of salt-sensitivity of OZR is associated with an enhanced antinatriuretic function of endogenous AngII. 2. The extent of AngII-mediated antinatriuresis was investigated in OZR and lean Zucker rats (LZR) using candesartan (100 microg/kg, i.v.), a selective angiotensin AT1 receptor antagonist, and ramipril (1 mg/kg, i.v.), an angiotensin-converting enzyme (ACE) inhibitor. The total number of AngII binding sites and their affinity were also assessed in renal cortical tubular membrane preparations of OZR and LZR using a specific radioligand-binding assay. Plasma renin activity was determined using a standard radioimmunoassay. 3. Both candesartan and ramipril produced substantially greater increases in urinary sodium excretion and urine flow in OZR and these effects were significantly greater than those observed in LZR. These observations suggest that basal antinatriuretic function of endogenous AngII is exaggerated in OZR. 4. The functional overexpression of AngII was not due to any alterations in the affinity or the total number of AngII binding sites in renal cortical tubular membranes. Higher plasma renin values in the OZR could have contributed to the phenomenon. 5. In conclusion, marked diuresis and natriuresis after AT1 receptor blockade and/or ACE inhibition suggest that the extent of endogenous AngII-mediated sodium transport under basal conditions is greatly augmented in OZR. It is proposed this phenomenon may be a contributing factor for the salt- sensitivity in the OZR.

Tubular reabsorption of filtered sodium is quantitatively the main contribution of kidneys to salt and water homeostasis. The transcellular reabsorption of sodium proceeds by a two-step mechanism: Na(+)-K(+)-ATPase-energized basolateral active extrusion of sodium permits passive apical entry through various sodium transport systems. In the past 15 years, most of the renal sodium transport systems (Na(+)-K(+)-ATPase, channels, cotransporters, and exchangers) have been characterized at a molecular level. Coupled to the methods developed during the 1965-1985 decades to circumvent kidney heterogeneity and analyze sodium transport at the level of single nephron segments, cloning of the transporters allowed us to move our understanding of hormone regulation of sodium transport from a cellular to a molecular level. The main purpose of this review is to analyze how molecular events at the transporter level account for the physiological changes in tubular handling of sodium promoted by hormones. In recent years, it also became obvious that intracellular signaling pathways interacted with each other, leading to synergisms or antagonisms. A second aim of this review is therefore to analyze the integrated network of signaling pathways underlying hormone action. Given the central role of Na(+)-K(+)-ATPase in sodium reabsorption, the first part of this review focuses on its structural and functional properties, with a special mention of the specificity of Na(+)-K(+)-ATPase expressed in renal tubule. In a second part, the general mechanisms of hormone signaling are briefly introduced before a more detailed discussion of the nephron segment-specific expression of hormone receptors and signaling pathways. The three following parts integrate the molecular and physiological aspects of the hormonal regulation of sodium transport processes in three nephron segments: the proximal tubule, the thick ascending limb of Henle's loop, and the collecting duct.

Angiotensin II receptors are present along the rat inner medullary collecting duct (IMCD), although their physiological role is unknown. Because urea is one of the major solutes transported across the terminal IMCD, we measured angiotensin II's effect on urea permeability. In the perfused rat terminal IMCD, angiotensin II had no effect on basal urea permeability but significantly increased vasopressin-stimulated urea permeability by 55%. Angiotensin II, both without and with vasopressin, also increased the amount of (32)P incorporated into urea transporter (UT)-A1 in inner medullary tissue exposed to these hormones ex vivo. Because angiotensin II activates protein kinase C, we tested the effect of staurosporine (SSP). In the absence of angiotensin II, SSP had no effect on vasopressin-stimulated urea permeability in the perfused terminal IMCD. However, SSP completely and reversibly blocked the angiotensin II-mediated increase in vasopressin-stimulated urea permeability. SSP and chelerythrine reduced the angiotensin II-stimulated (32)P incorporation into UT-A1 in inner medullary tissue exposed ex vivo. We conclude that angiotensin II increases vasopressin-stimulated facilitated urea permeability and (32)P incorporation into the 97- and 117-kDa UT-A1 proteins via a protein kinase C-mediated signaling pathway. These data suggest that angiotensin II augments vasopressin-stimulated facilitated urea transport in the rat terminal IMCD and may play a physiological role in the urinary concentrating mechanism by augmenting the maximal response to vasopressin.

To study the relationship between the sensitivity to sodium content of the diet in terms of development of hypertension and the regulation of the expression of type 1 angiotensin II receptor subtypes by such a diet.

The expression of angiotensin II receptor subtype (AT1A and AT1B) mRNAs was studied by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the four zones of the kidneys of Sabra rats, sensitive or resistant to DOCA salt-induced hypertension (SBH/y and SBN/y, respectively). Rats were fed a high (8%) or normal (0.4%) NaCl diet. As vasopressin is known to be elevated in SBH/y rats and to be involved in DOCA-salt hypertension, we studied an additional group of SBH/y rats, fed a high sodium diet, enriched in water.

With the absence of DOCA, SBH/y rats did not develop hypertension. The high sodium diet induced a greater fall in the plasma renin activity in the SBH/y (-95%) than in the SBN/y (-63%). In the cortex (C) and inner stripe (IS), the high sodium diet decreased AT1A and AT1B mRNAs in SBH/y and SBN/y, with a higher magnitude for SBH/y, than for SBN/y (C, -28 versus -20%; IS, -42 versus -20%). The addition of water to the high sodium diet lessened the effect of sodium in the C and IS, although the plasma renin activity (PRA) was not altered.

A high sodium diet significantly decreases both AT1A and AT1B gene expression in two specific zones of the rat kidney containing the target cells of angiotensin II (C and IS). This down-regulation is organ-specific since it was observed in the kidney and adrenals, but not in the liver. Finally, SBH/y and SBN/y rats differ in the basal level of AT1 mRNA expression in the IS, and in the ability to modulate AT1 mRNA level under sodium intake.

Angiotensin II (Ang II) has a dose-dependent, biphasic effect on the activity of the Na+/H+ antiport system in the renal proximal tubule (RPT). The aim of the present study was to further delineate the signaling pathways involved in Ang II action.

To examine Ang II signaling, 22Na+ uptake studies were conducted with a primary rabbit RPT cell culture system. The activation of phospholipase A2 (PLA2) was assessed by measuring the release of [3H]-arachidonic acid (AA), and changes in intracellular calcium levels were determined by means of confocal microscopy.

Low dosages of Ang II (<10-10 mol/L) stimulated Na+ uptake, whereas high dosages of Ang II (>10-10 mol/L) inhibited Na+ uptake. Ang II (>10-10 mol/L) also caused an increase in AA release associated with an increase in intracellular calcium. Not only did exogenous AA inhibit Na+ uptake, but two PLA2 inhibitors (mepacrine and AACOCF3) blocked the Ang II-mediated inhibition of Na+ uptake. However, the cytochrome P450-dependent epoxygenase inhibitor econazole also blocked the Ang II-induced inhibition of Na+ uptake. Inhibition of Na+ uptake was obtained by the metabolic product of the epoxygenase 5,6-EET. In turn, the inhibitory effect of 5,6-EET was blocked by indomethacin.

The results indicate the involvement of a calcium-dependent PLA2 in mediating the inhibitory effect of Ang II on Na+ uptake. The AA, which is released following PLA2 activation, acts indirectly, through its own metabolism, via a cytochrome P450 epoxygenase pathway and ultimately cyclooxygenase itself.

Mice lacking AT(1A) receptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a -/- mice. Plasma vasopressin levels increase normally in the AT(1A) receptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT(1A) receptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a -/- mice since the impairment is largely reproduced in wild-type mice by treatment with an AT(1)-receptor antagonist. These studies demonstrate a critical role for the AT(1A) receptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.

ANG II contributes importantly to the regulation of renal vascular resistance, glomerular filtration, and tubular epithelial transport, yet there remains a paucity of information regarding the localization of the ANG II type 1 and 2 (AT1 and AT2) receptors within the rat kidney particularly within the vasculature. The present study was designed to localize the transcriptional and translational site(s) of AT1 and AT2 receptor (AT1R and AT2R, respectively) expression within the rat kidney. Using immunohistochemistry, we detected the AT(1)R translational sites throughout the kidney, with the strongest labeling found in the vasculature of the renal cortex and the proximal tubules of the outer medulla. The AT2R protein expression was found throughout the rat kidney, although there was little to no expression found in the glomerulus and medullary thick ascending limbs of Henle (TAL). Gene-specific primers were then designed to distinguish between the receptor subtypes within microdissected renal tubular and vascular segments using RT-PCR. AT1AR, AT1BR, and AT2R mRNA were found within the renal vasculature (afferent arterioles, arcuate artery, and outer medullary descending vasa recta). The mRNA for both the AT1R isoforms was also detected in the glomeruli and the renal tubules (proximal tubules, TAL, and collecting ducts); however, no AT2R mRNA was detected within the glomerulus and was inconsistently found within the medullary TAL (MTAL). Taken together, these data show that mRNA for the AT1R subtypes was located in all of the renal tubular and vascular segments. Evidence for AT2R mRNA was also found in all but two of the vascular and tubular segments, the MTAL, and the glomeruli. These results are consistent with the whole tissue immunohistochemically localized receptors.

The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10(-8) M in the bath) inhibits HCO-3 absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 microM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 microM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M), the major P-450 metabolite in the MTAL, inhibited HCO-3 absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10(-8) M) to the bath had no effect on HCO-3 absorption. The inhibition of HCO-3 absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 microM) or herbimycin A (1 microM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10 microM) or the NO synthase (NOS) substrate L-arginine (1 mM) to the bath stimulated HCO-3 absorption by 35%, suggesting that NO directly regulates MTAL HCO-3 absorption. Addition of 10(-11) to 10(-10) M ANG II to the bath did not affect HCO-3 absorption. We conclude that ANG II inhibits HCO-3 absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of HCO-3 absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.

The proximal tubule can endogenously synthesize and secrete luminal angiotensin II at a concentration approximately 100- to 1000-fold higher than that in the systemic circulation. We have recently shown that this endogenously produced and luminally secreted angiotensin II regulates proximal tubule volume reabsorption, which is a reflection of sodium transport within this segment. In this study, we use in vivo microperfusion of angiotensin II receptor antagonists into the lumen of the proximal tubule to examine the role of the luminal AT1 and AT2 receptor in the regulation of volume reabsorption. Systemically administered (intravenous) AT1 and AT2 receptor antagonists, acting through basolateral angiotensin II receptors, have previously been shown to inhibit proximal tubule transport. Luminal perfusion of 10(-6) mol/L Dup 753 (AT1 antagonist) and 10(-6) mol/L PD 123319 (AT2 antagonist) decreased proximal tubule volume reabsorption from 2.94 +/- 0.18 to 1.65 +/- 0.18 and 1.64 +/- 0.19 nL/mm x min, respectively, P < .01. Luminal perfusion of 10(-4) mol/L CGP 42112A, another AT2 antagonist, similarly decreased volume reabsorption to 1.32 +/- 0.36 nL/nm x min, P < .01. The inhibition of transport with AT1 and AT2 antagonist was additive, as luminal perfusion of 10(-6) mol/L Dup 753 plus 10(-6) mol/L 123319 resulted in a decrease in volume reabsorption to 0.41 +/- 0.31 nL/mm x min, P < .001 v control, P < .05 v Dup 753, and P < .01 v PD 123319. These results show that endogenously produced angiotensin II regulates proximal tubule volume transport via both luminal AT1 and AT2 receptors.

The cellular site of erythropoietin (epo) production within the mammalian kidney is still not completely understood. In the present study, we examined the expression of epo mRNA in microdissected rat nephron segments by RT-PCR after induction of epo expression with cobalt chloride. Erythropoietin mRNA was not detected in nephron segments from saline injected rats. In cobalt chloride injected animals, epo mRNA was found in the majority of samples from the cortical region of the nephron, PCT, and CAL. Medullary tubule preparations (MCT and MAL) were mostly negative for epo mRNA, and glomeruli were uniformly negative. The induction of epo transcripts in tubular cells by cobalt chloride was paralleled by stimulation of the major transport enzyme in the kidney, namely, Na-K ATPase in a tubular profile similar to that of induction of epo transcripts. These results support some earlier findings that epo gene expression in response to cobalt salt stimulation of rat kidney occurs in transporting tubular epithelial cells.

Angiotensin II (AG II) stimulates the ouabain-insensitive, furosemide- sensitive Na+-ATPase present in the basolateral membrane of pig renal proximal tubules in a dose dependent manner. Maximum effect was obtained with 10-8 M AG II, which corresponded to an activity 134% higher than control. Half of the maximum effect was observed between 10-11 M and 10-10 M, corresponding to physiological hormone levels. Saralasin, an AG II peptide analogue receptor antagonist, abolished the phenomenon, demonstrating that AG II interacts with specific sites in pig proximal tubules. The AG II stimulatory effect was also prevented by dithiothreitol (DTT), a reducing compound, and by 10 nM losartan, a non-peptide antagonist highly specific for AT1 receptors, characterizing AG II binding to AT1 receptors. GTPgammaS, a non-hydrolysable GTP analogue, increased by 159% the enzyme activity as compared to the control values. The simultaneous addition of 10-5 M GTPgammaS and 10-8 M AG II did not have additive effects. Furthermore, the stimulatory action of AG II was completely abolished by 0.1 microM GDPbetaS, a non-hydrolysable GDP analogue. Two microgram ml-1 pertussis toxin, an inhibitor of Gi-protein, did not modulate the AG II stimulatory effect. On the other hand, the Na+-ATPase activity was enhanced 100% in the presence of cholera toxin and 85% in the presence of both AG II and cholera toxin. Taken together, these data suggest that AG II activates the Na+-ATPase activity through AT1 receptors coupled to a pertussis-insensitive and cholera-sensitive G-protein.