An in vivo protein landscape of the mouse DCT during high dietary K+ or low dietary Na+ intake.

The hormone aldosterone is essential for maintaining K+ and Na+ balance and controlling blood pressure. Aldosterone has different effects if it is secreted due to hypovolemia or hyperkalemia. The kidney distal convoluted tubule (DCT) is believed to play a central role in mediating the differential responses to aldosterone. To determine the alterations in the DCT that may be responsible for these effects, male mice with green fluorescent protein expression specifically in the DCT were maintained on diets containing low NaCl (hypovolemic state) or high potassium citrate (hyperkalemic state) for 4 days, and DCT cells were isolated using fluorescence-activated cell sorting. This pure population of DCT cells was subjected to analysis by liquid chromatography-coupled tandem mass spectrometry. Over 3,000 proteins were identified in the DCT, creating the first proteome of the mouse DCT. Of the identified proteins, 210 proteins were altered in abundance following a low-NaCl diet and 625 proteins following the high-K+ diet. Many of these changes were not detectable by analyzing whole kidney samples from the same animals. When comparing responses to high-K+ versus low-Na+ diets, protein translation, chaperone-mediated protein folding, and protein ubiquitylation were likely to be significantly altered in the DCT subsequent to a high-K+ diet. In conclusion, this study defines an in vivo protein landscape of the DCT in male mice following either a low-NaCl or a high-K+ diet and acts as an essential resource for the kidney research community.NEW & NOTEWORTHY The mineralocorticoid aldosterone, essential for maintaining body K+ and Na+ balance, has different effects if secreted due to hypovolemia or hyperkalemia. Here, we used proteomics to profile kidney distal convoluted tubule (DCT) cells isolated by a novel FACS approach from mice fed a low-Na+ diet (mimicking hypovolemia) or a high-K+ diet (mimicking hyperkalemia). The study provides the first in-depth proteome of the mouse DCT and insights into how it is physiologically regulated.

Distal convoluted tubule Cl- concentration is modulated via K+ channels and transporters.

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-K2 or K+-Cl- cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl-]i was ~7 mM. Stimulation of Cl- exit with low-Cl- hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl-]i . Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i, whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl-]i in the DCT.

Release and spread of Wingless is required to pattern the proximo-distal axis of Drosophila renal tubules.

Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. Here we demonstrate that release and spread of Wingless is required to pattern the proximo-distal (P-D) axis of Drosophila Malpighian tubules. Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending our understanding about the functional modes by which Wnts shape animal development, we anticipate this mechanism to be relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.

Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the sodium chloride cotransporter NCC.

Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent in situ hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones.

Potassium Sensing by Renal Distal Tubules Requires Kir4.1.

The mammalian distal convoluted tubule (DCT) makes an important contribution to potassium homeostasis by modulating NaCl transport. The thiazide-sensitive Na+/Cl- cotransporter (NCC) is activated by low potassium intake and by hypokalemia. Coupled with suppression of aldosterone secretion, activation of NCC helps to retain potassium by increasing electroneutral NaCl reabsorption, therefore reducing Na+/K+ exchange. Yet the mechanisms by which DCT cells sense plasma potassium concentration and transmit the information to the apical membrane are not clear. Here, we tested the hypothesis that the potassium channel Kir4.1 is the potassium sensor of DCT cells. We generated mice in which Kir4.1 could be deleted in the kidney after the mice are fully developed. Deletion of Kir4.1 in these mice led to moderate salt wasting, low BP, and profound potassium wasting. Basolateral membranes of DCT cells were depolarized, nearly devoid of conductive potassium transport, and unresponsive to plasma potassium concentration. Although renal WNK4 abundance increased after Kir4.1 deletion, NCC abundance and function decreased, suggesting that membrane depolarization uncouples WNK kinases from NCC. Together, these results indicate that Kir4.1 mediates potassium sensing by DCT cells and couples this signal to apical transport processes.

Genetic causes of hypomagnesemia, a clinical overview.

Magnesium is essential to the proper functioning of numerous cellular processes. Magnesium ion (Mg2+) deficits, as reflected in hypomagnesemia, can cause neuromuscular irritability, seizures and cardiac arrhythmias. With normal Mg2+ intake, homeostasis is maintained primarily through the regulated reabsorption of Mg2+ by the thick ascending limb of Henle's loop and distal convoluted tubule of the kidney. Inadequate reabsorption results in renal Mg2+ wasting, as evidenced by an inappropriately high fractional Mg2+ excretion. Familial renal Mg2+ wasting is suggestive of a genetic cause, and subsequent studies in these hypomagnesemic families have revealed over a dozen genes directly or indirectly involved in Mg2+ transport. Those can be classified into four groups: hypercalciuric hypomagnesemias (encompassing mutations in CLDN16, CLDN19, CASR, CLCNKB), Gitelman-like hypomagnesemias (CLCNKB, SLC12A3, BSND, KCNJ10, FYXD2, HNF1B, PCBD1), mitochondrial hypomagnesemias (SARS2, MT-TI, Kearns-Sayre syndrome) and other hypomagnesemias (TRPM6, CNMM2, EGF, EGFR, KCNA1, FAM111A). Although identification of these genes has not yet changed treatment, which remains Mg2+ supplementation, it has contributed enormously to our understanding of Mg2+ transport and renal function. In this review, we discuss general mechanisms and symptoms of genetic causes of hypomagnesemia as well as the specific molecular mechanisms and clinical phenotypes associated with each syndrome.

SPAK and OSR1 play essential roles in potassium homeostasis through actions on the distal convoluted tubule.

KEY POINTS: STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) phosphorylate and activate the renal Na(+) -K(+) -2Cl(-) cotransporter 2 (NKCC2) and Na(+) Cl(-) cotransporter (NCC). Mouse models suggest that OSR1 mainly activates NKCC2-mediated sodium transport along the thick ascending limb, while SPAK mainly activates NCC along the distal convoluted tubule, but the kinases may compensate for each other. We hypothesized that disruption of both kinases would lead to polyuria and severe salt-wasting, and generated SPAK/OSR1 double knockout mice to test this. Despite a lack of SPAK and OSR1, phosphorylated NKCC2 abundance was still high, suggesting the existence of an alternative activating kinase. Compensatory changes in SPAK/OSR1-independent phosphorylation sites on both NKCC2 and NCC and changes in sodium transport along the collecting duct were also observed. Potassium restriction revealed that SPAK and OSR1 play essential roles in the emerging model that NCC activation is central to sensing changes in plasma [K(+) ]. ABSTRACT: STE20 (Sterile 20)/SPS-1 related proline/alanine-rich kinase (SPAK) and oxidative stress-response kinase-1 (OSR1) activate the renal cation cotransporters Na(+) -K(+) -2Cl(-) cotransporter (NKCC2) and Na(+) -Cl(-) cotransporter (NCC) via phosphorylation. Knockout mouse models suggest that OSR1 mainly activates NKCC2, while SPAK mainly activates NCC, with possible cross-compensation. We tested the hypothesis that disrupting both kinases causes severe polyuria and salt-wasting by generating SPAK/OSR1 double knockout (DKO) mice. DKO mice displayed lower systolic blood pressure compared with SPAK knockout (SPAK-KO) mice, but displayed no severe phenotype even after dietary salt restriction. Phosphorylation of NKCC2 at SPAK/OSR1-dependent sites was lower than in SPAK-KO mice, but still significantly greater than in wild type mice. In the renal medulla, there was significant phosphorylation of NKCC2 at SPAK/OSR1-dependent sites despite a complete absence of SPAK and OSR1, suggesting the existence of an alternative activating kinase. The distal convoluted tubule has been proposed to sense plasma [K(+) ], with NCC activation serving as the primary effector pathway that modulates K(+) secretion, by metering sodium delivery to the collecting duct. Abundance of phosphorylated NCC (pNCC) is dramatically lower in SPAK-KO mice than in wild type mice, and the additional disruption of OSR1 further reduced pNCC. SPAK-KO and kidney-specific OSR1 single knockout mice maintained plasma [K(+) ] following dietary potassium restriction, but DKO mice developed severe hypokalaemia. Unlike mice lacking SPAK or OSR1 alone, DKO mice displayed an inability to phosphorylate NCC under these conditions. These data suggest that SPAK and OSR1 are essential components of the effector pathway that maintains plasma [K(+) ].

Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.

Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low-potassium diet. Recent data suggest that plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the with-no-lysine kinase (WNK)-Ste20p-related proline- and alanine-rich kinase (SPAK) pathway. As previous studies used extreme dietary manipulations, we sought to determine whether the relationship between potassium and NaCl cotransporter (NCC) is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect NCC in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride concentration. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3, and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable it to mediate effects of potassium on NCC in vivo.

Roles and Regulation of Renal K Channels.

More than two dozen types of potassium channels, with different biophysical and regulatory properties, are expressed in the kidney, influencing renal function in many important ways. Recently, a confluence of discoveries in areas from human genetics to physiology, cell biology, and biophysics has cast light on the special function of five different potassium channels in the distal nephron, encoded by the genes KCNJ1, KCNJ10, KCNJ16, KCNMA1, and KCNN3. Research aimed at understanding how these channels work in health and go awry in disease has transformed our understanding of potassium balance and provided new insights into mechanisms of renal sodium handling and the maintenance of blood pressure. This review focuses on recent advances in this rapidly evolving field.

Distal convoluted tubule.

The distal convoluted tubule (DCT) is a short nephron segment, interposed between the macula densa and collecting duct. Even though it is short, it plays a key role in regulating extracellular fluid volume and electrolyte homeostasis. DCT cells are rich in mitochondria, and possess the highest density of Na+/K+-ATPase along the nephron, where it is expressed on the highly amplified basolateral membranes. DCT cells are largely water impermeable, and reabsorb sodium and chloride across the apical membrane via electroneurtral pathways. Prominent among this is the thiazide-sensitive sodium chloride cotransporter, target of widely used diuretic drugs. These cells also play a key role in magnesium reabsorption, which occurs predominantly, via a transient receptor potential channel (TRPM6). Human genetic diseases in which DCT function is perturbed have provided critical insights into the physiological role of the DCT, and how transport is regulated. These include Familial Hyperkalemic Hypertension, the salt-wasting diseases Gitelman syndrome and EAST syndrome, and hereditary hypomagnesemias. The DCT is also established as an important target for the hormones angiotensin II and aldosterone; it also appears to respond to sympathetic-nerve stimulation and changes in plasma potassium. Here, we discuss what is currently known about DCT physiology. Early studies that determined transport rates of ions by the DCT are described, as are the channels and transporters expressed along the DCT with the advent of molecular cloning. Regulation of expression and activity of these channels and transporters is also described; particular emphasis is placed on the contribution of genetic forms of DCT dysregulation to our understanding.

Distal convoluted tubule.

The distal convoluted tubule is the nephron segment that lies immediately downstream of the macula densa. Although short in length, the distal convoluted tubule plays a critical role in sodium, potassium, and divalent cation homeostasis. Recent genetic and physiologic studies have greatly expanded our understanding of how the distal convoluted tubule regulates these processes at the molecular level. This article provides an update on the distal convoluted tubule, highlighting concepts and pathophysiology relevant to clinical practice.

Urotensin II exerts antiapoptotic effect on NRK-52E cells through prostacyclin-mediated peroxisome proliferator-activated receptor alpha and Akt activation.

Urotensin II (UII) is a cyclic vasoactive peptide which is mainly expressed in kidneys. Although elevated plasma UII levels are associated with renal impairment, the influence of UII on renal injury is unclear. In this study, we monitored the influence of UII on gentamicin-induced apoptosis in rat tubular cells (NRK-52E). We found that UII significantly reduced gentamicin-induced apoptosis and apoptotic signals. Blocking endogenous UII secretion caused cells to be more susceptible to gentamicin. In gentamicin-treated mice, UII also expressed protective effect on renal tubular cells. UII was also found to induce prostacyclin (PGI2) production, which caused peroxisomal proliferator-activated receptor α (PPARα) activation as revealed by both PGI2 synthase siRNA transfection and piroxicam treatment. Blockage of PPARα by siRNA transfection inhibited UII-induced Akt phosphorylation and the antiapoptotic effect of UII. Our results suggest that UII can protect renal tubular cells from gentamicin-induced apoptosis through PGI2-mediated PPARα and Akt activation.

Evolving concepts on regulation and function of renin in distal nephron.

Sustained stimulation of the intrarenal/intratubular renin-angiotensin system in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis, and eventual renal injury. Activation of luminal AT(1) receptors in proximal and distal nephron segments by local Ang II formation stimulates various transport systems. Augmented angiotensinogen (AGT) production by proximal tubule cells increases AGT secretion contributing to increased proximal Ang II levels and leading to spillover of AGT into the distal nephron segments, as reflected by increased urinary AGT excretion. The increased distal delivery of AGT provides substrate for renin, which is expressed in principal cells of the collecting tubule and collecting ducts, and is also stimulated by AT(1) receptor activation. Renin and prorenin are secreted into the tubular lumen and act on the AGT delivered from the proximal tubule to form more Ang I. The catalytic actions of renin and or prorenin may be enhanced by binding to prorenin receptors on the intercalated cells or soluble prorenin receptor secreted into the tubular fluid. There is also increased luminal angiotensin converting enzyme in collecting ducts facilitating Ang II formation leading to stimulation of sodium reabsorption via sodium channel and sodium/chloride co-transporter. Thus, increased collecting duct renin contributes to Ang II-dependent hypertension by augmenting distal nephron intratubular Ang II formation leading to sustained stimulation of sodium reabsorption and progression of hypertension.

Regulation of the renal Na+-Cl- cotransporter by phosphorylation and ubiquitylation.

The activity of the renal thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule plays a key role in defining arterial blood pressure levels. Increased or decreased activity of the NCC is associated with arterial hypertension or hypotension, respectively. Thus it is of major interest to understand the activity of NCC using in vivo models. Phosphorylation of certain residues of the amino-terminal domain of NCC has been shown to be associated with its activation. The development of phospho-specific antibodies against these sites provides a powerful tool that is helping to increase our understanding of the molecular physiology of NCC. Additionally, NCC expression in the plasma membrane is modulated by ubiquitylation, which represents another major mechanism for regulating protein activity. This work presents a review of our current knowledge of the regulation of NCC activity by phosphorylation and ubiquitylation.

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron.

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na(+) channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (ΔI(ami)) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased ΔI(ami) in CNT/CCD but had little effect on ΔI(ami) in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS(-/-)) mice that lack aldosterone. In CNT/CCD of AS(-/-) mice, ΔI(ami) was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS(-/-) mice, ΔI(ami) was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

[Magnesium homeostasis and its disturbances].

Magnesium homeostasis is maintained through normal functions of the kidney, intestine, and bone. In the kidney, approximately 80% magnesium is filtered by the glomeruli. In general, 95% filtered magnesium is collectively reabsorbed in the proximal tubule (15%-20%) , thick ascending limb of Henle (TAL, 65%-75%) , and the distal convoluted tubule (DCT, 5%-10%) . In the TAL, magnesium reabsorption regulated by the paracellular pathway via claudin-16 is driven by electrochemical voltage. Chloride channel Kb and renal outer medullary potassium channels control this lumen-positive voltage. In the DCT, the transcellular pathway via transient receptor potential melastatin 6 (TRPM6) plays a fundamental role in the final 5%-10% magnesium reabsorption. The functions of TRPM6 depend on Na-Cl co-transporters and Na( + )-K( + )-ATPase. Defects in these regulatory proteins may cause inherited or drug-induced disorders of magnesium metabolism. Recently, some proteins have been confirmed to be responsible for magnesium homeostasis ; however, further research is required to elucidate the mechanisms underlying the maintenance of magnesium homeostasis.

A new model of the distal convoluted tubule.

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Mechanisms of carbon monoxide attenuation of tubuloglomerular feedback.

Carbon monoxide (CO) is a physiological messenger with diverse functions in the kidney, including controlling afferent arteriole tone both directly and via tubuloglomerular feedback (TGF). We have reported that CO attenuates TGF, but the mechanisms underlying this effect remain unknown. We hypothesized that CO, acting via cGMP, cGMP-dependent protein kinase, and cGMP-stimulated phosphodiesterase 2, reduces cAMP in the macula densa, leading to TGF attenuation. In vitro, microdissected rabbit afferent arterioles and their attached macula densa were simultaneously perfused. TGF was measured as the decrease in afferent arteriole diameter elicited by switching macula densa NaCl from 10 to 80 mmol/L. Adding a CO-releasing molecule (CORM-3, 5 × 10(-5) mol/L) to the macula densa blunted TGF from 3.3 ± 0.3 to 2.0 ± 0.3 µm (P<0.001). The guanylate cyclase inhibitor LY-83583 (10(-6) mol/L) enhanced TGF (5.8 ± 0.6 µm; P<0.001 versus control) and prevented the effect of CORM-3 on TGF (LY-83583+CORM-3, 5.5 ± 0.3 µm). Similarly, the cGMP-dependent protein kinase inhibitor KT-5823 (2 × 10(-6) mol/L) enhanced TGF and prevented the effect of CORM-3 on TGF (KT-5823, 6.0 ± 0.7 µm; KT-5823+CORM-3, 5.9 ± 0.8 µm). However, the phosphodiesterase 2 inhibitor BAY-60-7550 (10(-6) mol/L) did not prevent the effect of CORM-3 on TGF (BAY-60-7550, 4.07 ± 0.31 µm; BAY-60-7550+CORM-3, 1.84 ± 0.31 µm; P<0.001). Finally, the degradation-resistant cAMP analog dibutyryl-cAMP (10(-3) mol/L) prevented the attenuation of TGF by CORM-3 (dibutyryl-cAMP, 4.6 ± 0.5 µm; dibutyryl-cAMP+CORM-3, 5.0 ± 0.6 µm). We conclude that CO attenuates TGF by reducing cAMP via a cGMP-dependent pathway mediated by cGMP-dependent protein kinase rather than phosphodiesterase 2. Our results will lead to a better understanding of the mechanisms that control the renal microcirculation.

Renal outer medullary potassium channel knockout models reveal thick ascending limb function and dysfunction.

The renal outer medullary potassium channel (ROMK) is an adenosine triphosphate-sensitive inward-rectifier potassium channel (Kir1.1 or KCNJ1) highly expressed in the cortical and medullary thick ascending limbs (TAL), connecting segment (CNT) and cortical collecting duct (CCD) in the mammalian kidney, where it serves to recycle potassium (K(+)) across the apical membrane in TAL and to secrete K(+) in the CNT and CCD. ROMK channel mutations cause type II Bartter's syndrome with salt wasting and dehydration, and ROMK knockout mice display a similar phenotype of Bartter's syndrome in humans. Studies from ROMK null mice indicate that ROMK is required to form both the small-conductance (30pS, SK) K channels and the 70pS (IK) K channels in the TAL. The availability of ROMK(-/-) mice has made it possible to study electrolyte transport along the nephron in order to understand the TAL function under physiological conditions and the compensatory mechanisms of salt and water transport under the conditions of TAL dysfunction. This review summarizes previous progress in the study of K(+) channel activity in the TAL and CCD, ion transporter expression and activities along the nephron, and renal functions under physiological and pathophysiological conditions using ROMK(-/-) mice.