Distal Convoluted Tubule Cells Research Articles

Magnesium Excretion in C. elegans Requires the Activity of the GTL-2 TRPM Channel

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

Effect of Cadmium on the Renal Cortex of Adult Albino Rat and the Possible Protective Role of α- lipoic Acid

Background: Cadmium (Cd) toxicity is related to the tissue oxidative stress due to reactive oxygen species. Alpha-lipoic acid (α-LA) has been identified as an ideal antioxidant capable of regenerating endogenous antioxidants in the body. Aim of the Work: This study was conducted to elucidate the effect of administration of cadmium on the structure of the renal cortex of the albino rat and to find out the possible protective role of concomitant administration of α–LA. Material and Methods: This study was carried out on 60 male adult albino rats divided into four groups. Group I (normal control group), 10 rats. Group II (sham control group), 30 rats, they were divided into 3 equal subgroups IIa, IIb & IIc received saline, saline with NaOH solution and α-LA respectively. Group III, 10 rats, received Cd intraperitoneally. Group IV, 10 rats, received Cd and α-LA intraperitoneally. The animals of all groups were sacrificed after one and four weeks of the experiment and renal cortical specimens were subjected to light and electron microscopic studies. Results: After Cd administration, there were many changes in the renal cortex which were in line with the period of administration. The glomeruli appeared with congested blood capillaries, shrunken or severely degenerated. The glomerular capillary basement membrane was thickened and degenerated or disrupted in many areas. The major processes of some podocytes were degenerated with loss of minor processes. The Bowman's space was obliterated, widened, or contained hemorrhage or tissue debris. The proximal convoluted tubules (PCT) appeared with widened lumen with casts or debris. Some cells of the PCT showed disrupted brush border with few or absent microvilli. Other cells were of degenerated or vacuolated cytoplasm with few mitochondria or absence of organelles. The nuclei became pyknotic in some cells. The distal convoluted tubules (DCT) were less affected. Few of them showed tissue debris, casts or desquamated cells in their lumen. The cytoplasm of some cells of DCT was vacuolated. Concomitant administration of α–LA with Cd greatly minimized these changes. Conclusions: Cd administration resulted in marked degeneration in the renal cortex. These effects were in line with the period of exposure and that α –LA had a protective role against Cd induced toxicity

Functional Characterization of Mutations in Kir4.1 (KCNJ10) Associated with the SeSAME Syndrome

Kir4.1 channels are expressed in many brain cells, particularly astrocytes, and may be responsible for the K+ buffering action of the glia (J Biol Chem 270: 16339-46, 1995). In addition, Kir4.1 channels are found in the basolateral membrane of distal convoluted tubule cells, where they contribute to renal electrolyte homeostasis. Mutations in KCNJ10, the gene encoding Kir4.1, have been associated to the newly described SeSAME syndrome (Proc Natl Acad Sci USA 106: 5842-47, 2009), a unique set of symptoms that include sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. To determine the functional significance of these mutations, we performed radiotracer efflux assays and inside-out membrane patch clamping in COSm6 cells expressing wild-type (WT) or mutant (R65P, C140R, T164I, A167V, R199Stop, and R297C) channels. All mutations lead to varying degrees of loss of Kir4.1 channel function. In untransfected cells, the 86Rb efflux rate constant was 0.008 min−1 ± 0.001 (n=3), and in cells transfected with WT, the rate of Kir4.1-mediated 86Rb efflux (proportional to K+ conductance) was 0.018 min−1 ± 0.001 (n=3). The mutant Kir4.1-mediated rate constants were 60% (A167V), 21% (R297C), 20% (R65P), 15% (C140R), 12% (T164I), and 1% (R199Stop), relative to WT. No measurable currents were recorded from cells expressing C140R, T164I, R199Stop or R297C. Some of these mutations (R297C, R199Stop) are away from the channel pore, and ongoing studies are examining the potential for altered trafficking. In R65P and A167V, on-cell inward rectification, as well as sensitivity to block by spermine and barium were normal. However, while the current amplitude was similar to WT immediately upon patch excision, it decreased 50-80% within the first 2 min, suggesting that these mutations, located in the potential PIP2 binding site or at the PIP2-dependent gate, reduce open state stability.

Rare but Relevant Kidney Disorders

Occurrence of hypomagnesemia (serum Mg2+ levels <0.7 mmol/L) in the general population has been estimated to be approximately 2%. The kidney is central for the maintenance of the Mg+ balance. A large body of knowledge has been generated from the study of inherited defects of renal Mg2+ handling. The majority of filtered Mg+ is reabsorbed along the proximal tubule and thick ascending limb of Henle's loop via a passive paracellular pathway. Claudin 16, formerly paracellin 1, and claudin 19 are renal tight junction proteins that are important for paracellular reabsorption of calcium and magnesium in thick ascending limb. Mutations of the corresponding genes, CLDN 16 and 19, result in a rare autosomal recessive tubular disorder that is characterized by familial hypomagnesemia, hypercalciuria, and nephrocalcinosis (FHHNC). This disease is frequently associated with progressive renal failure as a result of both intrarenal calcium deposition and additional roles of claudins in the maintenance of tight junction integrity. Children with a complete loss of function of CLDN16 have a more rapid progression of renal failure than those with residual function (1). The thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule (DCT) is mutated in Gitelman syndrome. Hypokalemic metabolic alkalosis and hypocalciuria are associated with hypomagnesemia. At the apical membrane of DCT cells is also present the Mg2+-permeable transient receptor potential cation channel, subfamily M (TRPM6). Mg2+ uptake from the tubular fluid via TRPM6 is primarily driven by the negative potential across the luminal membrane. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6. Mutations in FXYD2, encoding the γ-subunit of the basolateral Na+-K+-ATPase, is responsible for dominant isolated renal Mg2+ wasting, as a result of misrouting of this subunit. This defect has been described so far in a single family (but read this summary to the end). Finally, the recently discovered magnesiotropic hormone EGF causes isolated recessive renal hypomagnesemia (see references in Glaudemans et al.). Glaudemans et al. identified a new mechanism for autosomal dominant hypomagnesemia. A large Brazilian family was studied. Twenty-one affected members had serum Mg2+ levels <0.4 mmol/L, which was responsible for muscle symptoms and tetany from infancy. The locus was identified on chromosome 12. The mutation was found in KCNA1, a gene encoding the voltage-gated K+ channel, Kv1.1. This channel co-localized with TRPM6 along the luminal membrane of DCT. Upon overexpression in a human kidney cell line, patch-clamp analysis revealed that the mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. The negative potential across the apical membrane is normally maintained by an apical K+ efflux via Kv1.1 energized by the action of Na+-K+-ATPase. When Kv1.1 function is lost, Mg2+ reabsorption in DCT is impaired. Recently, an unexpected mechanism of renal magnesium wasting and hypomagnesemia was discovered by Adalat et al. (2). They first identified a teenager who had hepatocyte nuclear factor 1B (HNF 1B) mutation and presented with tetany and hypomagnesemia (0.620 mmol/L). Then they retrospectively reviewed 91 pediatric cases of renal malformation screened for HNF 1B mutation. Twenty-one had heterozygous mutations. Eight (44%) of 18 mutation carriers had hypomagnesemia (<0.65 mmol/L) compared with one (2%) of 48 of those without mutations. HNF 1B is a transcription factor that is expressed in developing kidneys, whereas, postnatally, proximal and distal tubules express the gene. Hnf 1b in mouse upregulates transcription of various genes whose human homologues are involved in inherited cystic kidney diseases. Adalat et al. (2) detected a highly conserved HNF 1B recognition site in FXYD2. They demonstrated HNF 1B–mediated transactivation of FXYD2. Thus, HNF 1B regulates transcription of FXYD2, which participates in the tubular handling of Mg2+. These results extend the phenotype of HNF 1B mutations to include hypomagnesemia. Serum Mg2+ levels should be measured in patients with HNF 1B mutations (2).

Thiazide Effects and Adverse Effects

One of the longest-running debates in clinical medicine shows no sign of disappearing; just when it seems that thiazides have reassumed their role as front-line drugs to treat hypertension,1 new concerns emerge,2–4 leading some to question their role once again.5 Thiazides are effective antihypertensives with long track records and low cost. The major concerns about their use arise from their tendency to cause hypokalemia, impair glucose tolerance, increase serum cholesterol, and increase serum uric acid. Few medical controversies have generated as much heat, with well-established camps staking out positions that appear resistant to change.6–9 The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial, the largest study of antihypertensive monotherapy ever performed,10 was intended to identify the best first-line treatment for high-risk hypertensive individuals; however, despite its size and the numerous resulting publications, its implications and authority continue to be disputed. The goal of this review is not take sides in this debate but rather to inject a distinct, and sometimes neglected, perspective; during the past 15 years, remarkable developments in molecular biology and human genetics have provided substantial insights into the pathogenesis of hypertension and mechanisms and adverse effects of diuretics. Diuretic proponents and antagonists alike often neglect these developments when addressing the topic; it is the purpose of this Brief Review to integrate these developments into the debate with the goal of generating questions that can be addressed scientifically. Thiazide diuretics were developed during the 1950s, when chemists and physiologists at Merck Sharpe and Dohme tested derivatives of sulfonamide-based carbonic anhydrase inhibitors, with the goal of discovering drugs that enhance the excretion of sodium with chloride rather than sodium bicarbonate.* Although these drugs lower arterial pressure effectively, the mechanisms have long perplexed investigators.11 Thiazides reduce cardiac output acutely by reducing extracellular …

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na(+) and Cl(-) from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na(+) and/or Cl(-) uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H(+)-ATPase-rich (HR) cells and Na(+)-K(+)-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl(-) influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na(+) and Cl(-) influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl(-) influx and Cl(-) content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl(-) in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na(+) influx and content that resulted from upregulation of mRNA expressions of Na(+)-H(+) exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl(-) uptake mechanism in zebrafish skin/gills.

Initial FGF23-Mediated Signaling Occurs in the Distal Convoluted Tubule

Fibroblast growth factor-23 (FGF23), a hormone central to phosphate and vitamin D metabolism, reduces renal absorption of phosphate by downregulating the sodium-phosphate cotransporter Npt2a. However, the mechanisms of FGF23 action in the kidney are unclear, as Npt2a localizes to the proximal tubule (PT) and the FGF23 coreceptor alpha-Klotho (KL) localizes to the distal convoluted tubule (DCT). Immunofluorescent analyses following FGF23 injection in mice showed robust staining for phospho-ERK1/2, a marker of FGF23 bioactivity, only within the DCT in a subset of KL-positive cells. This activity colocalized with the FGF23 receptor FGFR1 and was present in DCT cells that were adjacent to Npt2a-expressing PT segments. Although KL is expressed as both secreted and membrane-bound isoforms, only the membrane-bound isoform was capable of mediating FGF23 bioactivity. These findings provide novel insight into the mechanisms of hormone-regulated phosphate metabolism by identifying an intrarenal signaling axis for FGF23.

A missense mutation in the Kv1.1 voltage-gated potassium channel–encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia

Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane. Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.

High osmolality induces the kidney-specific chloride channel CLC-K1 by a serum and glucocorticoid-inducible kinase 1 MAPK pathway

The kidney-specific chloride channels CLC-K1/2 and their functionally important subunit barttin, by mediating solute transport in medulla, contribute to the osmotic gradient. We sought to determine whether they themselves are regulated by variations of osmolality. The expression of CLC-K1 and barttin mRNA and protein was significantly increased in a distal convoluted tubule cell line after a shift to high osmolar medium. This upregulation paralleled that of serum and glucocorticoid-inducible kinase 1 (SGK1), a gene known to be upregulated by cell shrinkage. Specific knockdown of SGK1 or addition of the p38 MAPK pathway inhibitor SB203580 abolished the induction of SGK1, CLC-K1 and barttin by high osmolarity suggesting that a functional MAPK pathway is required to mediate osmotic-driven induction of all three genes. The physiological relevance of our in vitro data was confirmed by water deprivation of male C57BL6 mice, which caused a significant increase in serum osmolality along with induction of CLC-K1, barttin and SGK1. Our study shows that change in intracellular volume, because of high osmolality, result in SGK1 upregulation and the subsequent increase of CLC-K1/barttin expression in distal renal tubular cells in vivo and in vitro.

The -glucuronidase klotho exclusively activates the epithelial Ca2+ channels TRPV5 and TRPV6

Active Ca(2+) reabsorption in the kidney is facilitated by the epithelial transient receptor potential vanilloid Ca(2+) channel subtype 5 (TRPV5). The complex-glycosylated TRPV5 is expressed at the apical membrane of the renal distal convoluted tubule (DCT) cells where the pro-urine hormone klotho can stimulate its activity by N-oligosaccharide hydrolysis. This study investigates whether klotho and its closely related analogue, beta-glucuronidase, can activate other renal ion channels than TRPV5 expressed by DCT cells. To determine the specificity of this stimulatory effect of klotho and beta-glucuronidase, a selection of ion channels and transporters expressed in the kidney (TRPV4, TRPV5, TRPV6 and TRPM6) was screened in transfected HEK293 cells by using Ca(2+)-influx measurements. Klotho and beta-glucuronidase have been found to significantly increase the activity of TRPV5 and TRPV6, but had no effect on TRPV4 and TRPM6. Furthermore, deglycosylation by endoglycosidase-F also stimulated the activity of TRPV4, TRPV5 and TRPV6, but not of TRPM6. These results suggest a modulating effect for klotho primarily restricted to the epithelial Ca(2+) channels TRPV5 and TRPV6.

Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension

We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

Mouse model of type II Bartter's syndrome. II. Altered expression of renal sodium- and water-transporting proteins

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 PGE(2) 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(+)-P(i) 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 alpha-,beta-, and gamma-subunits of the epithelial Na(+) channel (ENaC) was slightly increased, although only differences for gamma-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.

Renal magnification by EGF

A paper recently published by Groenestege and colleagues [8] used positional cloning to determine the cause of a rare inherited magnesium-wasting syndrome, autosomal recessive renal hypomagnesemia. The results showed that certain mutations in the epidermal growth factor (EGF) gene cause this disease. This suggests, perhaps surprisingly, that EGF and its receptor comprise a previously unrecognized signaling pathway in the human kidney that participates importantly in magnesium homeostasis. The EGF gene is highly expressed along the distal convoluted tubule (DCT), an important site for regulating urinary magnesium excretion. The product of the gene is a large membrane-bound molecule, expressed at both the apical and basolateral surfaces. A portion of the extracellular domain can be cleaved to form the 53-amino-acid hormone, EGF; it is believed that cleavage occurs preferentially at the basolateral surface, leading to increased apparent EGF abundance at the apical membrane. In the DCT, EGF binds to receptors at the basolateral surface, making basolateral EGF expression a necessary prerequisite for signaling. The gene defect that causes autosomal recessive renal hypomagnesemia appears to disrupt trafficking of pro-EGF to the basolateral membrane, thereby impeding its signaling capability. The results also showed that EGF stimulates magnesium transport by TRPM6 (transient receptor potential cation channel, subfamily M, member 6), a channel that is expressed at the apical membrane of DCT cells and appears to be a primary path for apical magnesium entry. Interestingly, the investigators also corroborated previous reports that cancer patients treated with an antagonist of the EGF receptor, cetuximab, develop renal magnesium wasting and hypomagnesemia, emphasizing the significance of EGF in maintaining magnesium balance in humans.

WNK Kinases and Renal Sodium Transport in Health and Disease

Hypertension affects 25% of the adult population in the developed world and is a major independent risk factor for stroke, myocardial infarction, and heart and kidney failure. Although many genetic and environmental contributors are involved, the kidney plays a dominant role, both in animal models,1 and in human essential hypertension.2 Most monogenic hypertensive syndromes result from increased Na+ transport along the aldosterone-sensitive distal nephron.3 The majority of these, however, are associated with hypokalemia, indicating that activation of the epithelial Na+ channel, ENaC, is a primary pathophysiologic process. In contrast, familial hyperkalemic hypertension (FHHt; also known as Gordon’s syndrome or type II pseudohypoaldosteronism) is characterized by hypertension with hyperkalemia, indicating that stimulated ENaC cannot be the primary event. FHHt was first described in 19644 and later shown to be inherited in an autosomal dominant manner.5,6 Patients with FHHt all exhibit hyperkalemia, which seems to be the most consistent feature of the disease. Hypertension, although commonly present and sometimes severe, often appears later in the natural history. Other characteristic features include mild metabolic acidosis, suppressed plasma renin activity, and aldosterone levels that are lower than would be expected, considering the hyperkalemia. Infusing the chloride salt of Na+ (NaCl) does not increase urinary potassium excretion in patients with FHHt, as it does in the normal individual, whereas infusing nonchloride salts of Na+ does increase K+ excretion in FHHt patients to normal levels.7,8 Patients are often remarkably sensitive to thiazide diuretics, which can correct both the hyperkalemia and hypertension, in many cases.9 In 2001, some cases of FHHt were shown to result from mutations in WNK1 and WNK4,10 identifying WNK kinases as previously undiscovered components of a novel electrolyte homeostasis pathway. Since that time, information about the physiological role of WNK …

Stanniocalcin-1 secretion and receptor regulation in kidney cells

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.

Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive (22)Na(+) uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.

Renal expression of parvalbumin is critical for NaCl handling and response to diuretics

The distal convoluted tubule (DCT) plays an essential role in the reabsorption of NaCl by the kidney, a process that can be inhibited by thiazide diuretics. Parvalbumin (PV), a Ca(2+)-binding protein that plays a role in muscle fibers and neurons, is selectively expressed in the DCT, where its role remains unknown. We therefore investigated the renal phenotype of PV knockout mice (Pvalb(-/-)) vs. wild-type (Pvalb(+/+)) littermates. PV colocalized with the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in the early DCT. The Pvalb(-/-) mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Acute furosemide administration increased diuresis and natriuresis/kaliuresis, but, surprisingly, did not increase calciuria in Pvalb(-/-) mice. NaCl supplementation of Pvalb(-/-) mice increased calciuria at baseline and after furosemide. The Pvalb(-/-) mice showed no significant diuretic response to hydrochlorothiazide, but an accentuated hypocalciuria. A decreased expression of NCC was detected in the early DCT of Pvalb(-/-) kidneys in the absence of ultrastructural and apoptotic changes. The PV-deficient mice had a positive Ca(2+) balance and increased bone mineral density. Studies in mouse DCT cells showed that endogenous NCC expression is Ca(2+)-dependent and can be modulated by the levels of PV expression. These results suggest that PV regulates the expression of NCC by modulating intracellular Ca(2+) signaling in response to ATP in DCT cells. They also provide insights into the Ca(2+)-sparing action of thiazides and the pathophysiology of distal tubulopathies.

When EGF is offside, magnesium is wasted

Our understanding of magnesium (Mg(2+)) regulation has recently been catapulted forward by the discovery of several disease loci for monogenic disorders of Mg(2+) homeostasis. In this issue of the JCI, Groenestege et al. report that their study of a rare inherited Mg(2+) wasting disorder in consanguineous kindred shows that EGF acts as an autocrine/paracrine magnesiotropic hormone (see the related article beginning on page 2260). EGF stimulates Mg(2+) reabsorption in the renal distal convoluted tubule (DCT) via engagement of its receptor on the basolateral membrane of DCT cells and activation of the Mg(2+) channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6) in the apical membrane. These authors show that a point mutation in pro-EGF retains EGF secretion to the apical but not the basolateral membrane, disrupting this cascade and causing renal Mg(2+) wasting. This work is another seminal example of the power of the study of monogenic disorders in the quest to understand human physiology.

β-Arrestin-Dependent Parathyroid Hormone-Stimulated Extracellular Signal-Regulated Kinase Activation and Parathyroid Hormone Type 1 Receptor Internalization

PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by beta-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is beta-arrestin (betaAr) independent, whereas prolonged activation is betaAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and betaAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. betaAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from betaAr1- and -2-null mice. In wild-type MEFs, PTH(1-34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for betaAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in betaAr2-null MEFs. In betaAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of betaAr1 [Ar(319-418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319-418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve betaAr1. Furthermore, the betaAr-dependent late phase of ERK activation by PTH requires the participation of betaAr2 and PTH1R internalization.

Cellular distribution of the potassium channel KCNQ1 in normal mouse kidney

Mechanisms of K(+) secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in K(+) secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a approximately 75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ1 has cell-specific roles in renal ion transport and may participate in K(+) secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD.