Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms.

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+ and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway.

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4-/- or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF)-/- mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs.

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Ovariectomy causes overexpression of renal Na(+),K(+)-ATPase and sodium-sensitive hypertension in adult Wistar rats.

We investigated the effect of ovariectomy(oVx) on renal and systemic hemodynamic, electrolyte excretion and total and dephosphorylated Na(+),K(+)-ATPase α1 subunit (t-d-NKA) in normotensive Wistar rats under a normal sodium (NS, 0.24%) or high sodium (HS, 1%) intake versus intact female (IF). On NS intake, t-d-NKA was higher in oVx rats and overexpressed in the thick ascending limbs (P < .01 vs. IF) and renal plasma flow was increased. On HS intake, oVx rats maintained a greater dephosphorylated NKA, excreted less sodium, and developed arterial hypertension (134 ± 4 vs. IF 112 ± 5 mm Hg, P < .05). Sodium load caused salt-sensitive hypertension in oVx Wistar rats.

Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4.

Angiotensin II (ANG II) stimulates production of superoxide (O(2)(-)) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O(2)(-) production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O(2)(-) production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O(2)(-) production in wild-type and NOX2 knockout mice (KO). ANG II increased O(2)(-) production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O(2)(-) production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O(2)(-) production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O(2)(-) production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O(2)(-) production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O(2)(-) production by TALs.

Eicosanoids and tumor necrosis factor-alpha in the kidney.

The thick ascending limb of Henle's loop (TAL) is capable of metabolizing arachidonic acid (AA) by cytochrome P450 (CYP450) and cyclooxygenase (COX) pathways and has been identified as a nephron segment that contributes to salt-sensitive hypertension. Previous studies demonstrated a prominent role for CYP450-dependent metabolism of AA to products that inhibited ion transport pathways in the TAL. However, COX-2 is constitutively expressed along all segments of the TAL and is increased in response to diverse stimuli. The ability of Tamm-Horsfall glycoprotein, a selective marker of cortical TAL (cTAL) and medullary (mTAL), to bind TNF and localize it to this nephron segment prompted studies to determine the capacity of mTAL cells to produce TNF and determine its effects on mTAL function. The colocalization of calcium-sensing receptor (CaR) and COX-2 in the TAL supports the notion that activation of CaR induces TNF-dependent COX-2 expression and PGE2 synthesis in mTAL cells. Additional studies showed that TNF produced by mTAL cells inhibits 86Rb uptake, an in vitro correlate of natriuresis, in an autocrine- and COX-2-dependent manner. The molecular mechanism for these effects likely includes inhibition of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) expression and trafficking.

Akt1 mediates purinergic-dependent NOS3 activation in thick ascending limbs.

Extracellular ATP regulates many physiological processes via release of nitric oxide (NO). ATP stimulates NO in thick ascending limbs (TALs), but the signaling cascade involved in the cells of this nephron segment, as well as many other types of cells, is poorly understood. We hypothesized that ATP enhances NO synthase (NOS) activity by stimulating PI3 kinase and Akt. We measured 1) NO in TALs using the NO-sensitive dye DAF-2 DA and 2) Akt activity by fluorescence resonance energy transfer and phosphorylation of Akt isoforms. ATP (100 microM) stimulated NO in wild-type mice [26 +/- 4 arbitrary units (AU)], but not in NOS3 -/- mice (2 +/- 2 AU; P < 0.04). In the presence of the NOS1- and NOS2-selective inhibitors 7-NI and 1400W, ATP stimulated NO by 30 +/- 2 and 33 +/- 3 AU, respectively (not significant vs. control). In the presence of the PI3 kinase inhibitor LY294002, ATP-increased NO was reduced by 85% (5 +/- 2 vs. 28 +/- 4 AU; P < 0.02). ATP alone increased Akt activity and this effect was significantly blocked by suramin, a P2 receptor antagonist. In the presence of an Akt-selective inhibitor, ATP-induced NO was blocked by 90 +/- 4%. ATP significantly stimulated Akt1 phosphorylation at Ser(473) by 91 +/- 13%, whereas Akt2 phosphorylation remained unchanged and Akt3 phosphorylation decreased. In vivo transduction of TALs with a dominant-negative Akt1 significantly decreased ATP-induced NO by 88 +/- 6%. We concluded that ATP increases NOS3-derived NO via Akt1 activation in the TAL.

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance.

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Role of focal adhesion kinase (FAK) in renal ischaemia and reperfusion.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays important roles in cell migration, cell proliferation and cell survival. Because these processes participate in the restoration of tubular integrity in renal ischaemia and reperfusion, FAK expression and phosphorylation at Tyr-397, the latter indicative of its activity, were examined in the different kidney zones by Western blot analysis and immunohistochemistry. Expression and phosphorylation of FAK were also studied in Madin-Darby canine kidney (MDCK) and medullary thick ascending limb (mTAL) cells after ATP depletion and repletion. In control rat kidneys, FAK expression in outer and inner medulla exceeded that in cortex, and phosphorylation of FAK at Tyr-397 was most pronounced in the inner medulla. Although this expression pattern was not affected by 20 (40, 60)-min ischaemia and 20 (40, 60)-min ischaemia followed by 60-min or 24-h reperfusion, FAK phosphorylation was significantly reduced in all kidney zones immediately after ischaemia, but increased during reperfusion, exceeding control values in the outer and inner medulla. ATP depletion and repletion of MDCK and mTAL cells were associated with a decrease in FAK phosphorylation during ATP depletion, followed by an increase during repletion. Rephosphorylation of FAK after ATP repletion was enhanced by N-acetylcysteine, a reactive oxygen species scavenger. ATP depletion disrupted focal adhesions in MDCK cells. Their reformation after ATP repletion paralleled the increase in FAK phosphorylation. These findings suggest an essential role for FAK-signalling during renal ischaemia and early reperfusion.

Control of potassium excretion: a Paleolithic perspective.

PURPOSE OF REVIEW: Regulation of potassium (K) excretion was examined in an experimental setting that reflects the dietary conditions for humans in Paleolithic times (high, episodic intake of K with organic anions; low intake of NaCl), because this is when major control mechanisms were likely to have developed. RECENT FINDINGS: The major control of K secretion in this setting is to regulate the number of luminal K channels in the cortical collecting duct. Following a KCl load, the K concentration in the medullary interstitial compartment rose; the likely source of this medullary K was its absorption by the H/K-ATPase in the inner medullary collecting duct. As a result of the higher medullary K concentration, the absorption of Na and Cl was inhibited in the loop of Henle, and this led to an increased distal delivery of a sufficient quantity of Na to raise K excretion markedly, while avoiding a large natriuresis. In addition, because K in the diet was accompanied by 'future' bicarbonate, a role for bicarbonate in the control of K secretion via 'selecting' whether aldosterone would be a NaCl-conserving or a kaliuretic hormone is discussed. SUMMARY: This way of examining the control of K excretion provides new insights into clinical disorders with an abnormal plasma K concentration secondary to altered K excretion, and also into the pathophysiology of calcium-containing kidney stones.

Differential effects of hyperosmolality on Na-K-ATPase and vasopressin-dependent cAMP generation in the medullary thick ascending limb and outer medullary collecting duct.

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2- and is diminished by a high-salt diet.

A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 microM) reduced Na-K-ATPase activity from 0.44 +/- 0.03 to 0.30 +/- 0.04 nmol P(i).microg protein(-1).min(-1) in THALs from rats on a normal diet (P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity (P < 0.04). After 20 min, SPM had no effect (change -0.07 +/- 0.05 nmol P(i).microg protein(-1).min(-1)). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO(-) formed by NO reacting with O(2)(-) was involved, we measured O(2)(-) production. THALs from rats on normal and high salt produced 35.8 +/- 0.3 and 23.7 +/- 0.8 nmol O(2)(-).min(-1).mg protein(-1), respectively (P < 0.01). Because O(2)(-) production differed, we studied the effects of the O(2)(-) scavenger tempol. In the presence of 50 microM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 +/- 0.05 vs. 0.52 +/- 0.07 nmol P(i).microg protein(-1).min(-1)). Propyl gallate, another O(2)(-) scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O(2)(-) levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on O(2)(-). NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O(2)(-) production.

Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ.

ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26-180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.

TNFalpha regulates renal COX-2 in the rat thick ascending limb (TAL).

We have examined cyclooxygenase (COX)-2-dependent mechanisms in preglomerular microvessels and the thick ascending limb (TAL). These renal structures are linchpins in the regulation of the renal circulation and extracellular fluid volume. Cytochrome P450 monooxygenases are the principal oxygenases in the TAL segment; however, COX-2 can be expressed in the TAL, as when challenged by angiotensin II. Glucocorticoids also affect the expression and activity of oxygenases in the TAL. Before adrenalectomy, <2% TAL cells expressed COX-2; after, >30% of TAL cells expressed COX-2. Recruitment of COX-2 is initiated in the renal cortex and proceeds to the medulla associated with: (1) COX-2 mRNA accumulation; (2) increased COX-2 expression; and (3) a two-fold increase in PGE2 production by cortical microsomes. These changes were nullified by dexamethasone. COX-2 mRNA, protein expression and PGE2 synthesis in the TAL are also increased in response to increased extracellular Ca2+. The Ca2+ sensing receptor is G-protein coupled and responds to changes in extracellular Ca2+ concentration by increasing protein kinase C activity to produce expression of COX-2. Thus, multiple signaling pathways contribute to COX-2 expression in TAL cells.

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat.

Sodium transport correlates with varying Na+-K+-ATPase activity rates along the nephron. Whether differences in Na+-K+-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na+-K+-ATPase alpha1 subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na+-K+-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na+-K+-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43+/-1.99 versus 2.3+/-0.20, respectively, P<0.01) while Na+-K+-ATPase activity was higher in mTAL (3,402+/-83 vs 711+/-158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10(-6) M decreased phosphorylation in PCT to 125+/-10% ( P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na+-K+-ATPase. Both calcineurin inhibitor FK-506 10(-6) M and phorbol 12-myristate 13-acetate (PMA) 10(-6) M increased the degree of Na+-K+-ATPase phosphorylation ( P<0.05) and inhibited Na+-K+-ATPase activity to 657+/-152 and 1,448+/-347 pmol/mm tubule per hour, respectively, in mTAL ( P<0.01). Increase in [Na+]i to 30, 50 and 70 mM resulted in no changes in Na+-K+-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na+]i were paralleled by decreased phosphorylation (from 120+/-7 to 160+/-15% of controls, P<0.05) and increased Na+-K+-ATPase activity (from 850+/-139 to 1,874+/-203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10(-6) M decreased both Na+-K+-ATPase dephosphorylation to 41.85+/-9.58% ( P<0.05) and Na+-K+-ATPase activity to 2,405+/-176 pmol/mm tubule per hour in mTAL ( P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na+-K+-ATPase alpha1 subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.

Gene transfer of eNOS to the thick ascending limb of eNOS-KO mice restores the effects of L-arginine on NaCl absorption.

The thick ascending limb of the loop of Henle (THAL) plays an essential role in the regulation of sodium and water homeostasis by the kidney. l-Arginine, the substrate for nitric oxide synthase (NOS), decreases NaCl absorption by THALs. We hypothesized that eNOS produces the NO that regulates THAL NaCl transport and that selective expression of eNOS in the THAL of eNOS knockout(-/-) mice would restore the effects of l-arginine on NaCl absorption. eNOS-/- mice were anesthetized, the left kidney was exposed, and the renal interstitium was injected with recombinant adenoviral vectors that expressed green fluorescent protein (GFP) or eNOS driven by the promoter of the Na/K/2Cl cotransporter Ad-NKCC2GFP and Ad-NKCC2eNOS, respectively. In Ad-NKCC2eNOS-transduced kidneys, eNOS expression was detected 7 days after injection but was absent in Ad-NKCC2GFP-transduced kidneys. In THALs from eNOS-/- mice transduced with Ad-NKCC2eNOS, adding L-arginine increased DAF-2DA fluorescence, a measure of NO production, by 9.1+/-1.1% (P<0.05; n=5), but not in THALs transduced with Ad-NKCC2GFP. In THALs from eNOS-/- mice transduced with Ad-NKCC2eNOS, Cl absorption averaged 85.9+/-11.8 pmol/min per millimeter. Adding l-arginine (1 mmol/L) to the bath decreased Cl absorption to 59.7+/-11.0 pmol/min per millimeter (P<0.05; n=6). In THALs transduced with Ad-NKCC2GFP, Cl absorption averaged 96.0+/-21.0 pmol/min per millimeter. Adding L-arginine to the bath did not significantly affect Cl absorption (100.6+/-20.6 pmol/min per millimeter; n=4). We concluded that gene transfer of eNOS to the THAL of eNOS-/- mice restores L-arginine-induced inhibition of NaCl transport and NO production. These data indicate that eNOS is essential for the regulation of THAL NaCl transport by NO.

Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells.

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca(2+) (Ca(o)(2+)) concentration, resulting in increases of intracellular Ca(2+) concentration and PKC activity. Exposure of mTAL cells to either Ca(o)(2+) or the CaR-selective agonist poly-L-arginine increased TNF-alpha synthesis. Moreover, the response to Ca(o)(2+) was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca(o)(2+), suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca(2+)-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE(2) synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE(2) production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca(2+)-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE(2) synthesis also was observed when cells were challenged with Ca(o)(2+) in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.

The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities.

BACKGROUND: 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. METHODS: 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. RESULTS: 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+]. CONCLUSIONS: 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.

Segmental localization of mRNAs encoding Na(+)-K(+)-ATPase alpha(1)- and beta(1)-subunits in diabetic rat kidneys using RT-PCR.

The present study evaluated renal Na(+)-K(+)-ATPase activity and mRNA in rats with diabetes mellitus. To localize the segmental alpha(1)- and beta(1)-mRNAs of Na(+)-K(+)-ATPase 1 and 8 days after induction of diabetes, we used the polymerase chain reaction after reverse transcription of the mRNA in microdissected nephron segments. Na(+)-K(+)-ATPase activity in the proximal convoluted tubule (PCT) rose on days 1 and 8 by 42 and 23%, respectively. In the medullary thick ascending limb (MTAL), it remained unchanged on day 1 and increased on day 8 by 55%. In the cortical collecting duct (CCD), activity rose by 81 and 45% on days 1 and 8, respectively. In parallel, alpha(1)-mRNA in the PCT increased by 52 and 22% on days 1 and 8, respectively. In the MTAL, alpha(1)-mRNA remained unchanged on day 1 and rose by 47% on day 8. In the CCD, alpha(1)-mRNA increased by 140 and 110% on days 1 and 8, respectively. beta(1)-mRNA was unchanged in the PCT throughout the study and was elevated in the MTAL and CCD on days 1 and 8. Thus there was a temporal dissociation between alpha(1)- and beta(1)-subunit expression. There was a highly significant linear correlation between Na(+)-K(+)-ATPase activity and alpha(1)-mRNA in all nephron segments throughout the experiment. It appears that microdissection of nephron tubules combined with reverse transcription-polymerase chain reaction defines the molecular identity of the amplified gene product and its segmental distribution in the nephron. We propose that altered gene expression may be the mechanism underlying enhanced Na(+) pump activity along the nephron in diabetic rats.

Regulation of intracellular pH in rat renal inner medullary thin limbs of Henle's loop.

Regulation of intracellular pH (pHi) was studied in isolated rat renal inner medullary thin limbs of Henle's loop in bicarbonate/phosphate-buffered medium with high pCO2, high osmolality ( congruent with670 mosmol/kg H2O; 270 mM urea; 180 mM NaCl), organic osmolytes, and a pH of 6.8 to approximate the physiological in vivo environment. The pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi. Resting pHi was always acid and significantly more acid in descending thin limb (DTL) cells than in ascending thin limb (ATL) cells from pure or mixed-type thin limbs. Resting pHi was slightly but significantly higher in both DTLs and ATLs in high osmolality ( approximately 670 mosmol/kg H2O) than in low osmolality ( approximately 290 mosmol/kg H2O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pHi following additional acidification with an NH4Cl pulse was reduced by Na+ removal from the medium and by the addition of 60 microM HOE642 (an inhibitor of the Na+/H+ exchanger, NHE1), 55 microM S1611 (inhibitor of Na+/H+ exchanger, NHE3), 1 microM bafilomycin A1 (an inhibitor of vacuolar H+ -ATPase), or 20 microM Schering 28080 (an inhibitor of H+ -K+ -ATPase) to the medium. Resting pHi was also reduced by 60 microM HOE642, 55 microM S1611, and 20 microM Schering 28080. In both DTLs and ATLs, RT-PCR revealed message for NHE1, NHE3, and vacuolar H+ -ATPase; immunocytochemistry demonstrated the expression of the protein for NHE1 (basolateral membrane), NHE3 (luminal membrane), and H+ -K+ -ATPase (luminal membrane). These data suggest that pHi in rat inner medullary thin limbs is regulated by urea and by basolateral and luminal H+ extrusion via NHE1, NHE3, vacuolar H+ -ATPase, and H+ -K+ -ATPase.