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The water channel aquaporin-1 (AQP1) is the principal water pathway for isotonic water reabsorption in the kidney proximal tubule (PT). We investigated flow-mediated fluid (Jv) and HCO- 3 (JHCO3 ) reabsorption in PTs of the mouse kidney by microperfusion in wild-type (WT) and AQP1 knockout (KO) mice. Experiments were simulated in an adaptation of a mathematical model of the rat PT. An increase in perfusion rate from 5 to 20 nL/min increased Jv and JHCO3 in PTs of WT mice. AQP1 KO mice significantly decreased Jv at low and high flow rates compared with control. In contrast, JHCO3 was not reduced at either low or high flow rates. Cell volume showed no significant difference between WT and AQP1 KO mice. Renal clearance experiments showed significantly higher urine flow in AQP1 KO mice, but there was no significant difference in either Na+ and K+ or HCO-3 excretion. Acid-base parameters of blood pH, PCO2, HCO-3, and urine pH were the same in both WT and KO mice. In model calculations, tubules whose tight junction (TJ) water permeability (Pf) was that assigned to the rat TJ, showed no difference in Jv between WT and KO, whereas TJ Pf set to 25% of the rat predicted Jv concordant with our observations from AQP1 KO. These results affirm the dominance of AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrate that flowstimulated HCO3 reabsorption is intact and independent of AQP1. With reference to the model, the findings also suggest that TJ water flux in the Pf is less prominent in the mouse than in the rat kidney. NEW & NOTEWORTHY We found an absence of flow-dependent modulation of fluid absorption but no effect on either proximal tubule (Pf) HCO-3 absorption or acid-base parameters in the aquaporin 1 (AQP1) knockout mouse. We affirmed the dominance of the water channel AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrated that flow-stimulated HCO-3 reabsorption is independent of AQP1. With reference to the model, the findings also suggest that tight junctional water flux in the PT is less prominent in the mouse than rat kidney. [ABSTRACT FROM AUTHOR]

We studied sex differences in response to high K+ (HK) intake on thiazide-sensitive cation (Na+ and K+) excretion in wild-type (WT) and ANG II receptor subtype 1a (AT1aR) knockout (KO) mice. Renal clearance experiments were performed to examine Na+Cl- cotransporter (NCC) activity on mice fed with control and HK (5% KCl, 7 days) diets. Hydrochlorothiazide (HCTZ)-induced changes in urine volume, glomerular filtration rate, absolute Na and K excretion, and fractional excretion were compared. HK-induced changes in NCC, Na/H exchanger isoform 3 (NHE3), and ENaC expression were examined by Western blot analysis. In WT animals under the control diet, HCTZ-induced cation excretion was greater in female animals, reflecting larger increases in Na excretion, since there was little sex difference in HCTZ-induced K excretion. Under the HK diet, the sex difference in HCTZ-induced cation excretion was reduced because of larger increments in K excretion in male animals. The fraction of K excretion was 57 ±5% in male WT animals and 36 ± 4% in female WT animals (P ± 0.05), but this difference was absent in AT1aR KO mice. NCC abundance was higher in female animals than in male animals but decreased by similar fractions on HK diet. NHE3 abundance decreased, whereas cleaved forms of λ-ENaC increased, with HK in all groups; these changes were similar in male and female animals and were not significantly affected by AT1aR ablation. These results indicate that, with the HK diet, male animals display greater distal Na delivery and greater activation of K secretion mechanisms, all suggesting a more powerful male adaptation to HK intake. [ABSTRACT FROM AUTHOR]

To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 "knockdown" mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC. [ABSTRACT FROM AUTHOR]

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved α-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by **50% and **80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle's loop and distal convoluted tubule along with Cl-, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis. [ABSTRACT FROM AUTHOR]

We studied gender differences in Na+-Cl - cotransporter (NCC) activity and expression in wild-type (WT) and AT1a receptor knockout (KO) mice. In renal clearance experiments, urine volume (UV), glomerular filtration rate, absolute Na+(ENa) and K (EK), and fractional Na+ (FENa) and K excretion were measured and compared at peak changes after bolus intravenous injection of hydrochlorothiazide (HCTZ; 30 mg/kg). In WT, females responded more strongly than males to HCTZ, with larger fractional increases of UV (7.8- vs. 3.4-fold), ENa (11.7- vs. 5.7-fold), FENa(7.9- vs. 4.9-fold), and EK (2.8- vs. 1.4-fold). In contrast, there were no gender differences in the responses to the diuretic in KO mice; HCTZ produced greater effects on male KO than on WT but similar effects on females. In WT, total (tNCC) and phosphorylated (pNCC) NCC protein expressions were 1.8- and 4.6-fold higher in females compared with males (P < 0.05), consistent with the larger response to HCTZ. In KO mice, tNCC and pNCC increased significantly in males to levels not different from those in females. There were no gender differences in the expression of the Na+/H+ exchanger (NHE3) in WT; NHE3 protein decreased to similar extents in male and female KO animals, suggesting AT1a-mediated NHE3 expression in proximal tubules. The resulting increase in delivery of Na+-Cl - to the distal nephron may underlie increased NCC expression and activity in mice lacking the AT1a receptor. [ABSTRACT FROM AUTHOR]

In the proximal tubule, axial flow (drag on brush-border microvilli) stimulates Na+ and HCO3- reabsorption by modulating both Na/H exchanger 3 (NHE3) and HATPase activity, a process critical to glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes intact, but abrogates the flow effect. In the current work, we provide evidence implicating cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO3- (JHCO3) were measured. We examined the effect of high luminal Ca2+ (5 mM), 0 mM Ca2+, the Ca2+ chelator BAPTA-AM, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the Ca-ATPase inhibitor thapsigargin. In control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by 104%. With respect to Na+ reabsorption, high luminal Ca2+ decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca2+ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline flow, leaving the flow effect intact. With respect to HCO3- reabsorption, high luminal Ca2+ decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca2+ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect. These data implicate IP3 receptor-mediated intracellular Ca2+ signaling as a critical step in transduction of microvillous drag to modulate Na+ and HCO3- transport. [ABSTRACT FROM AUTHOR]

Underlying glomerulotubular balance (GTB) is the impact of axial flow to regulate Na+ and HCO3- transport by modulating Na+-H+ exchanger 3 (NHE3) and H-ATPase activity. It is not known whether the cascade of events following a change in flow relies on local angiotensin (ANG II) generation or receptor availability. Mouse tubules were microperfused in vitro at flows of 5 and 20 nl/min, and net fluid (Jv) and HCO3- (J HCO3) absorption and cell height were measured. Na+ (JNa) and Cl- (JCl) absorption and changes in microvillous torque were estimated. Raising flow increased Na+ and HCO3- reabsorption but did not change either Cl- transport or cell volume. Losartan reduced absolute Na+ and HCO3- absorption at both low and high flows but did not affect fractional flow-stimulated transport. Compared with controls, in AT1a knockout (KO) mouse tubules, 53% of flow-stimulated Na+ absorption was abolished, but flow-stimulated HCO3- absorption was retained at similar levels. The remaining flow-stimulated JHCO3 was eliminated by the H-ATPase inhibitor bafilomycin. Inhibition of the AT2 receptor by PD123319 increased both JNa and JHCO3 but did not affect flow-mediated fractional changes. NHE3 expression at the protein level was reduced in AT1a KO mice kidneys. We conclude that 1) although the AT1a receptor is necessary for flow to impact NHE3, the effect on H+-ATPase is independent of AT1a; 2) the small flow-mediated changes in cell volume suggest a coordinate flow effect on both luminal and basolateral transporters; and 3) there is no evidence of flow-dependent Cl- transport, and thus no evidence for convective paracellular Cl- transport in mouse tubules. [ABSTRACT FROM AUTHOR]

In response to volume expansion, locally generated dopamine decreases proximal tubule reabsorption by reducing both Na/H-exchanger 3 (NHE3) and Na-K-ATPase activity. We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ and HCO3- reabsorption and have suggested that this observation underlies glomerulotubular balance. In the present work, we investigate the impact of dopamine on the sensitivity of reabsorptive fluxes to changes in luminal flow. Mouse proximal tubules were microperfused in vitro at low and high flow rates, and volume and HCO3 - reabsorption (Jv and JHCO3) were measured, while Na+ and Cl- reabsorption (JNa and JCl) were estimated. Raising luminal flow increased Jv, JNa, and JHCO3 but did not change JCl. Luminal dopamine did not change Jv, JNa, and JHCO3 at low flow rates but completely abolished the increments of Na+ absorption by flow and partially inhibited the flow-stimulated HCO3 - absorption. The remaining flow-stimulated HCO3 - absorption was completely abolished by bafilomycin. The DA1 receptor blocker SCH23390 and the PKA inhibitor H89 blocked the effect of exogenous dopamine and produced a two to threefold increase in the sensitivity of proximal Na+ reabsorption to luminal flow rate. Under the variety of perfusion conditions, changes in cell volume were small and did not always parallel changes in Na+ transport. We conclude that 1) dopamine inhibits flow-stimulated NHE3 activity by activation of the DA1 receptor via a PKA-mediated mechanism; 2) dopamine has no effect on flow-stimulated H-ATPase activity; 3) there is no evidence of flow stimulation of Cl- reabsorption; and 4) the impact of dopamine is a coordinated modulation of both luminal and peritubular Na+ transporters. [ABSTRACT FROM AUTHOR]

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant road- blocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron. [ABSTRACT FROM AUTHOR]

The role of mechanical forces in the regulation of glomerulotubular balance in the proximal tubule (PT) and Ca2+ signaling in the distal nephron was first recognized a decade ago, when it was proposed that the microvilli in the PT and the primary cilium in the cortical collecting duct (CCD) acted as sensors of local tubular flow. In this review, we present a summary of the theoretical models and experiments that have been conducted to elucidate the structure and function of these unique apical structures in the modulation of Na+, HCO3-, and water reabsorption in the PT and Ca2+ signaling in the CCD. We also contrast the mechanotransduction mechanisms in renal epithelium with those in other cells in which fluid shear stresses have been recognized to play a key role in initiating intracellular signaling, most notably endothelial cells, hair cells in the inner ear, and bone cells. In each case, small hydrodynamic forces need to be greatly amplified before they can be sensed by the cell's intracellular cytoskeleton to enable the cell to regulate its membrane transporters or stretch-activated ion channels in maintaining homeostasis in response to changing flow conditions. [ABSTRACT FROM AUTHOR]

Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of Na+-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient (Romk-/-) mice. Plasma aldosterone was 40% higher and urinary PGE2 excretion was 1.5-fold higher in Romk-/- compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule Na+/H+ exchanger (NHE3) and Na+-Pi cotransporter (NaPi-IIa) and TAL-specific Na+-K+-2Cl--cotransporter (NKCC2/BSC1) in Romk-/- mice, while the distal convoluted tubule (DCT)-specific Na+-Cl- cotransporter (NCC/TSC) was markedly increased. The abundance of the α-,β-, and -γ-subunits of the epithelial Na+ channel (ENaC) was slightly increased, although only differences for γ-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of Romk-/- mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent Na+ absorption in the TAL is compensated predominately by upregulation of Na+ transport in downstream DCT cells. These adaptive changes in Romk-/- mice may help to limit renal Na+ loss, and thereby, contribute to survival of these mice. [ABSTRACT FROM AUTHOR]

ROMK-deficient (Romk-/-) mice exhibit polyuria, natriuresis, and kaliuresis similar to individuals with type II Bartter's form of hyper-prostaglandin E syndrome (HPS; antenatal Bartter's syndrome). In the present study, we utilized both metabolic and clearance studies to define the contributions of specific distal nephron segments to the renal salt wasting in these mice. The effects of furosemide, hydrochlorothiazide, and benzamil on urinary Na+ and K+ excretion in both wild-type (RomK+/+) and Romk-/- mice were used to assess and compare salt transport by the Na+-K+-2Cl- cotransporter (NKCC2)-expressing thick ascending limb (TAL), the Na+-Cl- cotransporter (NCC)-expressing distal convoluted tubule (DCT1/DCT2), and the epithelial Na+ channel (ENaC)-expressing connecting segment (CNT) and collecting duct (CD), respectively. Whole kidney glomerular filtration rate was reduced by 47% in Romk-/- mice. Furosemide-induced increments in the fractional excretion rate of Na+ and K+ and absolute excretion of Na+ and K+ were significantly blunted in Romk-/- mice, consistent with a major salt transport defect in the TAL. In contrast, hydrochlorothiazide produced an exaggerated natriuresis in RomK-/- mice, indicating upregulation of salt absorption by the DCT. Benzamil resulted in a similar increment in absolute Na excretion in both RomK-/- and RomK+/+, indicating no significant upregulation of Na+ transport by ENaC in ROMK null mice. Moreover, hydrochlorothiazide increased the fractional K+ excretion rate in Romk-/- mice, confirming our recent observation that maxi-K channels contribute to distal K+ secretion in the absence of ROMK. [ABSTRACT FROM AUTHOR]

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by 22Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na+/H+ exchange activity. [ABSTRACT FROM AUTHOR]

The mathematical model of rat proximal tubule has been extended to include calculation of microvillous torque and to incorporate torque- dependent solute transport in a compliant tubule. The torque calculation follows that of Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, and Wang T (Am J Physiol 290: F289 -F296, 2006). In the model calculations, torque-dependent scaling of luminal membrane transporter density [either as an ensemble or just type 3 Na+/H+ exchanger (NHE3) alone] had a relatively small impact on overall Na+ reabsorption and could produce a lethal derangement of cell volume; coordinated regulation of luminal and peritubular transporters was required to represent the overall impact of luminal flow on Na+ reabsorption. When the magnitude of torque-dependent Na+ reabsorption in the model agrees with that observed in mouse proximal tubules, the model tubule shows nearly perfect perfusion-absorption balance at high luminal perfusion rates, but enhanced sensitivity of reabsorption at low flow. With a slightly lower coefficient for torque- sensitive transporter insertion, perfusion-absorption balance in the model tubule is closer to observations in the rat over a broader range of inlet flows. In simulation of hyperglycemia, torque-dependent transport attenuated the diuretic effect and brought the model tubule into closer agreement with experimental observation in the rat. The model was also extended to represent finite rates of hydration and dehydration of CO2 and H2CO3. With carbonic anhydrase inhibition, torque-dependent transport blunted the diuretic effect and enhanced the shift from paracellular to transcellular NaCl reabsorption. The new features of this model tubule are an important step toward simulation of glomerulotubular balance. [ABSTRACT FROM AUTHOR]

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)- positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11, 12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 µM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ absorption (Du Z, Duan Y, Yan Q, Weinstein AM, Weinbaum S, and Wang T. Proc Nat! Acad Sci USA 101: 13068-13073, 2004). It was hypothesized that brush-border microvilli function as a sensor to detect and amplify luminal hydrodynamic forces and transmit them to the actin cytoskeleton. In the present study we examine whether 1) flow-dependent HCO3- transport is proportional to flow-dependent variations in microvilbus torque (bending moment); 2) both luminal membrane Na+/H+ exchange (NHE3) and H+-ATPase activity are modulated by axial flow; and 3) paracellular permeabilities contribute to the flux perturbations. HCO3- absorption is examined by microperfusion of mouse S2 proximal tubules in vitro, with varying perfusion rates, and in the presence of the Na/H-exchange inhibitor EIPA, the H+-ATPase inhibitor bafilomycin, and the actin cytoskeleton inhibitor cytochalasin D. Paracellular perme- ability changes are assessed with measurements of epithelial HCO3- permeability and transepithelial potential difference (PD). It is found that 1) an increase in perfusion rate enhances HCO3- absorption and microvilbus torque, and the fractional changes of each are nearly identical; 2) inhibition of NHE3 by EIPA, or H+-ATPase by bafibomycin, produced only partial inhibition of flow-stimulated bicarbonate transport; 3) disruption of the actin cytoskeleton by cytochalasin D blocked the increment of HCO3- absorption by high flow; and 4) HCO3- permeability and transepithelial PD are not modulated by flow. We conclude that flow-dependent modulation of proximal tubule HCO3- reabsorption is due to changes in both NHE3 and H+-ATPase activity within the luminal cell membrane and this requires an intact actin cytoskeleton. Paracellular permeability changes do not contribute to this flow dependence. Perfusion-absorption balance in the proximal tubule is a direct effect of flow-induced torque on brush-border microvilli to regulate luminal cell membrane transporter activity. [ABSTRACT FROM AUTHOR]

Apical potassium recycling is crucial for salt transport by the thick ascending limb (TAL). Loss-of-function mutations in the K channel, ROMK (Kir1.1; KCNJ1), cause Bartter syndrome, a genetically heterogeneous disorder characterized by severe reduction in salt absorption by the TAL, Na wasting, polyuria, and hypokalemic alkalosis. ROMK(-/-) null mice exhibit a Bartter phenotype and lack the small-conductance (30-pS) apical K channel (SK) in the TAL. However, a distinct 70-pS K channel can also significantly contribute to the apical conductance of TAL. We now examine the effect of ROMK deletion on the functional expression of the 70-pS K channel in the TAL. Functional expression of the 70-pS K channel was low [average channel acitivty (NPo) = 0.02] in ROMK(+/+) mice on a control K diet but increased to 0.27 by high-K intake for 2 wk. In contrast, the high-K diet decreased NPo of SK by ∼30%, from 2.04 to 1.44. In ROMK heterozygous (+/-) mice on a control K diet, SK activity was about one-half of that observed in ROMK(+/+) mice (0.95 vs. 2.04). The high-K diet also reduced SK activity in ROMK(+/-) mice by ∼40% (from 0.95 to 0.55) but increased NPo of the 70-pS K channel from 0 to 0.09 in ROMK(+/-) mice. This corresponds to ∼30% of channel activity (NPo = 0.27) observed in ROMK(+/+) mice. Neither the 70-pS nor the 30-pS K channels were observed in TAL cells from ROMK(-/-) mice on either the normal or high-K diets. Thus functional expression of the 70-pS K channel is enhanced by increasing dietary K and requires expression of ROMK. It is likely that ROMK forms a critical subunit of the 70-pS K channel, accounting for the loss of apical K secretory channel activity in ROMK Bartter syndrome. [ABSTRACT FROM AUTHOR]

We previously demonstrated that carbon monoxide (CO) stimulates the apical 70-pS K[sup +] channel in the thick ascending limb (TAL) of the rat kidney (Liu HJ, Mount DB, Nasjletti A, and Wang WH. J Clin Invest 103: 963-970, 1999). Because the apical K[sup +] channel plays a key role in K[sup +] recycling, we tested the hypothesis that heme oxygenase (HO)-dependent metabolites of heme may affect Na[sup +] transport in the TAL. We used in vivo microperfusion to study the effect of chromium mesoporphyrin (CrMP), an inhibitor of HO, on fluid absorption (J[sub v]) and Na[sup +] absorption (J[sub Na]) in the loop of Henle and renal clearance methods to examine the effect of CrMP on renal sodium excretion. Microperfusion experiments demonstrated that addition of CrMP to the loop of Henle decreased J[sub v] by 13% and J[sub Na] by 20% in animals on normal rat chow and caused a decrease in J[sub v] (39%) and J[sub Na] (40%) in rats on a high-K[sup +] (HK) diet. The effect of CrMP is the result of inhibition of HO because addition of MgPP, an analog of CrMP that does not inhibit HO, had no effect on J[sub v]. Western blot analysis showed that HO-2 is expressed in the kidney and that the level of HO-2 was significantly elevated in animals on a HK diet. Renal clearance studies demonstrated that the infusion of CrMP increased the excretion of urinary Na[sup +] (E[sub Na]) and volume (UV) without changes in glomerular filtration rate. The effect of CrMP on E[sub Na] and UV was larger in HK rats than those kept on normal chow. We conclude that HK intake increases HO-2 expression in the kidney and that HO-dependent metabolites of berne, presumably CO, play a significant role in the regulation of Na[sup +] transport in the loop of Henle. [ABSTRACT FROM AUTHOR]

It has been well documented that low concentrations of ANG II (10[sup -11] to 10[sup -10] M) stimulate, whereas high concentrations of ANG II (10[sup -8] to 10[sup -5] M) inhibit Na[sup +] transport in proximal tubules of rat and rabbit kidneys. Measured ANG II concentration in proximal tubular fluid is in the nanomolar range. In the present study, we investigated the role of PKC, intracellular Ca[sup 2+], and cAMP in modulating the effects of luminal ANG II on Na[sup +] absorption by microperfusion techniques in rabbit superficial segment of proximal tubules in vitro. We confirmed that ANG II (10[sup -9] M) had no change on fluid absorption (J[sub v]); however, fluid absorption increased significantly when 10[sup -9] M ANG II and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular calcium mobilization, were added together. In contrast, ANG II significantly decreased J[sub v] when PKC was inhibited. When 10[sup -9] M ANG II was present together with 1-(5-isoquinolinesulfonyl)-2-mehtylpiperazine and TMB-8, no significant change of J[sub v] occurred. Inhibition of endogenous cAMP activity by a PKA inhibitor did not change either basal or ANG II-stimulated fluid absorption. Our results indicate that ANG II regulates Na[sup +] absorption by a cAMP-independent mechanism and that PKC and intracellular calcium both play a critical role in modulating the effects of physiological concentration of ANG II on proximal tubule transport. Balance between these two cytosolic messengers modulates the effects of ANG II on fluid absorption in the proximal tubule. [ABSTRACT FROM AUTHOR]

Examines the formate-stimulated NaCl absorption in the proximal tubule of pendrin protein. Isolation of microvillus membrane vesicles; Determination on the concentration of protein; Identification of pendrin in the proximal tubules using immunoblots of membrane vesicles.

Our laboratory has previously shown that mice lacking neuronal nitric oxide synthase (nNOS) are defective in fluid absorption (J[sub v]) and HCO[sup -, sub 3] absorption (J[sub HCO3]) in the proximal tubule and develop metabolic acidosis. The present study examined the transport of fluid and HCO[sup -, sub 3] in the proximal tubule and acid-base status in mice lacking two other isoforms of NOS, inducible NOS (iNOS) and endothelial NOS (eNOS). Proximal tubules were microperfused in situ in wild-type and NOS knockout mice by methods previously described (Wang T, Yang C-L, Abbiati T, Schultheis P J, Shull GE, Giebisch G, and Aronson PS. Am J Physiol Renal Physiol 277: F298F302, 1999). [³H]inulin and total CO[sub 2] concentrations were measured in the perfusate and collected fluid, and net J[sub v] and J[sub HCO3] were analyzed. These data show that J[sub HCO3] was 35% lower (71.7 ± 6.4 vs. 109.9 ± 7.3 pmol·min[sup -1]·mm[sup -1], n = 13, P < 0.01) and J[sub v] was 38% lower (0.95 ± 0.15 vs. 1.54 ± 0.17 nl·min[sup -1]·mm[sup -1], n = 13, P < 0.05) in iNOS knockout mice compared with their wild-type controls. Addition of the iNOS-selective inhibitor L-N[sup 6]-(1-iminoethyl) lysine, reduced both J[sub v] and J[sub HCO3] significantly in wild-type, but not in iNOS knockout, mice. In contrast, both J[sub HCO3] (93.3 ± 7.9 vs. 110.6 ± 6.18 pmol·min[sup -1]·mm[sup -1]) and J[sub v] (1.56 ± 0.17 vs. 1.55 ± 0.16 nl·min[sup -1]·mm[sup -1]) did not change significantly in eNOS knockout mice. These results indicated that iNOS upregulates Na[sup +] and HCO[sup -, sub 3] transport, whereas eNOS does not directly modulate Na[sup +] and HCO[sup -, sub 3] transport in the kidney proximal tubules. [ABSTRACT FROM AUTHOR]

Examines the role of sodium/hydrogen exchange (NHE) isoforms in the mediation of bicarbonate reabsorption along the nephron. Inhibition of transport in the proximal tubule; Predominance of NHE2 in apical membrane exchange in the distal convoluted tubule; Expression of NHE isoforms in the mammalian kidney.

The effects of nitric oxide (NO) on blood pressure and renal hemodynamics are well established, but those of NO on renal tubule HCO3- and Na+ transport are not fully understood. In this study, we combined renal clearance and in situ microperfusion techniques to investigate the effects of NO on the renal excretion of Na (FENa%) and the rates of renal tubule absorption of fluid (Jv) and bicarbonate (JHCo3) in the rat kidney. Administration of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 6 mg/kg iv bolus) did not change mean blood pressure and glomerular filtration rate significantly. However, L-NAME significantly increased urine flow rate and FE N&, and these effects were maintained over a 60-min period. Addition of L-NAME markedly decreased both Jv and JHCo3 3 in the proximal tubule. In contrast, addition of 1 uM sodium nitroprusside (SNP) or S-nitroso-W acetylpenicillamine (SNAP) significantly increased both Jv and JHCo3 . Similar stimulation was also observed when 8bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 1 pM) was added to the luminal perfusate. The stimulatory effects of SNP and 8BrcGMP on Jv and JHCo3 were not additive. The increments in Jv and JHCo3 due to 3SNP were abolished by the Na+/H+ exchange blocker ethylisopropylamiloride and the guanylate cyclase inhibitor methylene blue. These results indicate that NO stimulates proximal tubule Na+ and HCO3- transport through a cGMP-linked pathway in the kidney proximal tubule. guanosine 3',5'-cyclic monophosphate; sodium/proton exchange; bicarbonate concentration NITRIC OXIDE (NO) has a surprisingly [ABSTRACT FROM AUTHOR]