GDF15 mediates renal cell plasticity in response to potassium depletion in mice.

OBJECTIVE: To understand the mechanisms involved in the response to a low-K+ diet (LK), we investigated the role of the growth factor GDF15 and the ion pump H,K-ATPase type 2 (HKA2) in this process. METHODS: Male mice of different genotypes (WT, GDF15-KO, and HKA2-KO) were fed an LK diet for different periods of time. We analyzed GDF15 levels, metabolic and physiological parameters, and the cellular composition of collecting ducts. RESULTS: Mice fed an LK diet showed a 2-4-fold increase in plasma and urine GDF15 levels. Compared to WT mice, GDF15-KO mice rapidly developed hypokalemia due to impaired renal adaptation. This is related to their 1/ inability to increase the number of type A intercalated cells (AIC) and 2/ absence of upregulation of H,K-ATPase type 2 (HKA2), the two processes responsible for K+ retention. Interestingly, we showed that the GDF15-mediated proliferative effect on AIC was dependent on the ErbB2 receptor and required the presence of HKA2. Finally, renal leakage of K+ induced a reduction in muscle mass in GDF15-KO mice fed LK diet. CONCLUSIONS: In this study, we showed that GDF15 and HKA2 are linked and play a central role in the response to K+ restriction by orchestrating the modification of the cellular composition of the collecting duct.

Hypomagnesemia, Hypocalcemia, and Tubulointerstitial Nephropathy Caused by Claudin-16 Autoantibodies.

BACKGROUND: Chronic hypomagnesemia is commonly due to diarrhea, alcoholism, and drugs. More rarely, it is caused by genetic defects in the effectors of renal magnesium reabsorption. METHODS: In an adult patient with acquired severe hypomagnesemia, hypocalcemia, tubulointerstitial nephropathy, and rapidly progressing kidney injury, similarities between the patient's presentation and features of genetic disorders of renal magnesium transport prompted us to investigate whether the patient had an acquired autoimmune cause of renal magnesium wasting. To determine if the patient's condition might be explained by autoantibodies directed against claudin-16 or claudin-19, transmembrane paracellular proteins involved in renal magnesium absorption, we conducted experiments with claudin knockout mice and transfected mouse kidney cells expressing human claudin-16 or claudin-19. We also examined effects on renal magnesium handling in rats given intravenous injections of IgG purified from sera from the patient or controls. RESULTS: Experiments with the knockout mice and in vitro transfected cells demonstrated that hypomagnesemia in the patient was causally linked to autoantibodies directed against claudin-16, which controls paracellular magnesium reabsorption in the thick ascending limb of Henle's loop. Intravenous injection of IgG purified from the patient's serum induced a marked urinary waste of magnesium in rats. Immunosuppressive treatment combining plasma exchange and rituximab was associated with improvement in the patient's GFR, but hypomagnesemia persisted. The patient was subsequently diagnosed with a renal carcinoma that expressed a high level of claudin-16 mRNA. CONCLUSIONS: Pathogenic claudin-16 autoantibodies represent a novel autoimmune cause of specific renal tubular transport disturbances and tubulointerstitial nephropathy. Screening for autoantibodies targeting claudin-16, and potentially other magnesium transporters or channels in the kidney, may be warranted in patients with acquired unexplained hypomagnesemia.

Acidosis-induced activation of distal nephron principal cells triggers Gdf15 secretion and adaptive proliferation of intercalated cells.

AIM: Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. METHODS: We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. RESULTS: Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. CONCLUSION: Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status.

SIRT7 modulates the stability and activity of the renal K-Cl cotransporter KCC4 through deacetylation.

SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.

The serine-threonine kinase PIM3 is an aldosterone-regulated protein in the distal nephron.

The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na+ and K+ balance, blood volume, and arterial blood pressure, by acting in the aldosterone-sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto-oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin-regulated group of kinases, is stimulated by aldosterone in vitro (mCCDcl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3-/- mouse model, we found that these mice have an upregulated Renin-Angiotensin-Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na+ -deficient diet. Surprisingly, we did not observe any obvious salt-losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na+ transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3-/- mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone-induced protein, but its precise role in aldosterone-dependent renal homeostasis remains to be determined.

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Endothelin-1 mediates natriuresis but not polyuria during vitamin D-induced acute hypercalcaemia.

KEY POINTS: Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism. A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)-1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia-induced effects. In the present study, we demonstrate that, during vitamin D-induced hypercalcaemia, the activation of ET system by increased ET-1 is responsible for natriuresis but not for polyuria. Vitamin D-treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited. We have identified an original pathway that specifically mediates the effects of vitamin D-induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. ABSTRACT: Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D-induced hypercalcaemia increases the renal expression of endothelin (ET)-1, we hypothesized that ET-1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8-week-old, parathyroid hormone-supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT-treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET-1 and the transcription factors CCAAT-enhancer binding protein ß and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT-treated mice. To examine the role of the ET system in hypercalcaemia-induced natriuresis and polyuria, mice were treated with the ET-1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT-treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT-treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D-induced hypercalcaemia increases the renal production of ET-1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria.

PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor.

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

Expression profile of nuclear receptors along male mouse nephron segments reveals a link between ERRß and thick ascending limb function.

The nuclear receptor family orchestrates many functions related to reproduction, development, metabolism, and adaptation to the circadian cycle. The majority of these receptors are expressed in the kidney, but their exact quantitative localization in this ultrastructured organ remains poorly described, making it difficult to elucidate the renal function of these receptors. In this report, using quantitative PCR on microdissected mouse renal tubules, we established a detailed quantitative expression map of nuclear receptors along the nephron. This map can serve to identify nuclear receptors with specific localization. Thus, we unexpectedly found that the estrogen-related receptor ß (ERRß) is expressed predominantly in the thick ascending limb (TAL) and, to a much lesser extent, in the distal convoluted tubules. In vivo treatment with an ERR inverse agonist (diethylstilbestrol) showed a link between this receptor family and the expression of the Na⁺,K⁺-2Cl⁻ cotransporter type 2 (NKCC2), and resulted in phenotype presenting some similarities with the Bartter syndrom (hypokalemia, urinary Na⁺ loss and volume contraction). Conversely, stimulation of ERRß with a selective agonist (GSK4716) in a TAL cell line stimulated NKCC2 expression. All together, these results provide broad information regarding the renal expression of all members of the nuclear receptor family and have allowed us to identify a new regulator of ion transport in the TAL segments.

Secretory carrier membrane protein 2 regulates exocytic insertion of NKCC2 into the cell membrane.

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.

Tissue kallikrein permits early renal adaptation to potassium load.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.

Tissue compartment analysis for biomarker discovery by gene expression profiling.

BACKGROUND: Although high throughput technologies for gene profiling are reliable tools, sample/tissue heterogeneity limits their outcomes when applied to identify molecular markers. Indeed, inter-sample differences in cell composition contribute to scatter the data, preventing detection of small but relevant changes in gene expression level. To date, attempts to circumvent this difficulty were based on isolation of the different cell structures constituting biological samples. As an alternate approach, we developed a tissue compartment analysis (TCA) method to assess the cell composition of tissue samples, and applied it to standardize data and to identify biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: TCA is based on the comparison of mRNA expression levels of specific markers of the different constitutive structures in pure isolated structures, on the one hand, and in the whole sample on the other. TCA method was here developed with human kidney samples, as an example of highly heterogeneous organ. It was validated by comparison of the data with those obtained by histo-morphometry. TCA demonstrated the extreme variety of composition of kidney samples, with abundance of specific structures varying from 5 to 95% of the whole sample. TCA permitted to accurately standardize gene expression level amongst >100 kidney biopsies, and to identify otherwise imperceptible molecular disease markers. CONCLUSIONS/SIGNIFICANCE: Because TCA does not require specific preparation of sample, it can be applied to all existing tissue or cDNA libraries or to published data sets, inasmuch specific operational compartments markers are available. In human, where the small size of tissue samples collected in clinical practice accounts for high structural diversity, TCA is well suited for the identification of molecular markers of diseases, and the follow up of identified markers in single patients for diagnosis/prognosis and evaluation of therapy efficiency. In laboratory animals, TCA will interestingly be applied to central nervous system where tissue heterogeneity is a limiting factor.

NKCC2 surface expression in mammalian cells: down-regulation by novel interaction with aldolase B.

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

Sodium retention and ascites formation in a cholestatic mice model: role of aldosterone and mineralocorticoid receptor?

UNLABELLED: Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. CONCLUSION: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids.

Kidney collecting duct acid-base "regulon".

Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was compared with that of both normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through an NH4Cl-supplemented diet for 3 days, not only induced acid secretion but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport, and cell proliferation. In particular, >25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase-2, aldolase) were co-regulated during acidosis. These transcripts, which cooperate to achieve a similar function and are co-regulated during acidosis, constitute a functional unit that we propose to call a "regulon".

Molecular identification of Sch28080-sensitive K-ATPase activities in the mouse kidney.

Rat collecting ducts display either an ouabain-insensitive or an ouabain-sensitive K-ATPase activity inhibited by Sch28080 according as animals are fed a normal or a potassium-depleted diet (types I and III K-ATPase, respectively). Two isoforms of H,K-ATPase have been cloned from rat gastric mucosa and colon, respectively. Gastric and colonic H,K-ATPase are expressed in the kidney, suggesting that they might account for types I and III K-ATPases. However, this hypothesis is not fully supported by segmental expression of gastric and colonic H,K-ATPase along the rat collecting duct, as well as by comparison of the pharmacological properties of gastric and colonic H,K-ATPase expressed in Xenopus ovocyte and types I and III K-ATPases in rat collecting ducts. The aim of the present work is to address directly the molecular origin of types I and III K-ATPases in the mouse collecting duct by measuring K-ATPase activities in collecting ducts of wild-type mice and mice genetically deficient in either gastric or colonic H,K-ATPase fed either a regular or a potassium-depleted diet. Like the rat, mouse collecting ducts display type I or III K-ATPase activity when fed a regular or a potassium-depleted diet, respectively. Type I K-ATPase activity is detected in colonic H,K-ATPase-deficient mice but not in gastric H,K-ATPase-deficient animals. Conversely, type III K-ATPase activity disappears in colonic H,K-ATPase-deficient but not in gastric H,K-ATPase-deficient mice. In conclusion, types I and III K-ATPases measured in collecting ducts of normal and potassium-depleted mice reflect the functional expression of gastric and colonic H,K-ATPase, respectively.

Differential regulation of collecting duct Na+, K+-ATPase and K+ excretion by furosemide and piretanide: role of bradykinin.

In response to chronic treatment with furosemide, collecting ducts adapt their function to the initial loss of Na+ to prevent further Na+ loss and extracellular volume decrease. This adaptation, which includes the overexpression of Na+, K+-ATPase, is thought to account for most of the kaliuretic effect of furosemide. Because piretanide is reported to be less kaliuretic than equidiuretic doses of furosemide, the authors compared the effects of 1-wk treatment with the two loop diuretics on urinary potassium excretion and on Na+, K+-ATPase activity in the collecting duct. At equidiuretic and equinatriuretic doses, furosemide increased urinary potassium excretion as well as collecting duct Na+, K+-ATPase activity, whereas piretanide had no effect on either parameter. These effects of furosemide were curtailed by concomitant administration of the angiotensin-converting enzyme inhibitor enalapril, but they were not altered either by clamping changes in plasma aldosterone or by blocking type I angiotensin receptors. Treatment with the antagonist of bradykinin B2 receptors Hoe140 mimicked the two effects of furosemide. In addition, the effects of Hoe140 and furosemide were not additive. Finally, piretanide increased urinary bradykinin excretion, whereas furosemide did not. These results suggest that induction of collecting duct Na+, K+-ATPase (a) accounts for the kaliuretic effect of furosemide, (b) is independent of the renin/angiotensin/aldosterone system, (c) results from increased Na+ delivery to the collecting duct and enhanced intracellular Na+ concentration, and (d) is prevented in piretanide treated rats by increased bradykinin production that may limit apical Na+ entry in collecting duct principal cells.

RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.

Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.

Increased synthesis and avp unresponsiveness of Na,K-ATPase in collecting duct from nephrotic rats.

Renal sodium retention is responsible for ascites and edema in nephrotic syndrome. In puromycin aminonucleoside (PAN)-induced nephrosis, sodium retention originates in part from the collecting duct, and it is associated with increased Na,K-ATPase activity in the cortical collecting duct (CCD). The aims of this study were to evaluate whether the outer medullary collecting duct (OMCD) also participates to sodium retention and to determine the mechanisms responsible for stimulation of Na,K-ATPase in CCD. PAN nephrosis increased Na,K-ATPase activity in the CCD but not in OMCD. The two-fold increase of Na,K-ATPase activity in CCD was associated with two-fold increases in the number of alpha and beta Na,K-ATPase subunits mRNA determined by quantitative RT-PCR and of the total amount of Na,K-ATPase alpha subunits estimated by Western blotting. PAN nephrosis also increased two-fold the amount of Na,K-ATPase alpha subunit at the basolateral membrane of CCD principal cells, as determined by Western blotting after biotinylation and streptavidin precipitation and by immunofluorescence. The intracellular pool of latent Na,K-ATPase units also increased in size and was no longer recruitable by vasopressin and cAMP. This unresponsiveness of the intracellular pool of Na,K-ATPase to vasopressin was not the result of any alteration of the molecular and functional expression of the vasopressin V(2) receptor/adenylyl cyclase (AC) complex. It is concluded that PAN nephrosis (1) does not alter sodium reabsorption in OMCD, (2) is associated with increased synthesis and membrane expression of Na,K-ATPase in the CCD, and (3) alters the normal trafficking of intracellular Na,K-ATPase units to the basolateral membrane.