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Pendrin is expressed in the apical regions of type B and non-A, non-B intercalated cells, where it mediates [Cl.sup.-] absorption and HC[O.sup.-.sub.3] secretion through apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. Since pendrin is a robust [I.sup.-] transporter, we asked whether pendrin is upregulated with dietary [I.sup.-] restriction and whether it modulates [I.sup.-] balance. Thus [I.sup.-] balance was determined in pendrin null and in wild-type mice. Pendrin abundance was evaluated with immunoblots, immunohistochemistry, and immunogold cytochemistry with morphometric analysis. While pendrin abundance was unchanged when dietary [I.sup.-] intake was varied over the physiological range, [I.sup.-] balance differed in pendrin null and in wild-type mice. Serum [I.sup.-] was lower, while [I.sup.-] excretion was higher in pendrin null relative to wild-type mice, consistent with a role of pendrin in renal [I.sup.-] absorption. Increased [H.sub.2]O intake enhanced differences between wild-type and pendrin null mice in [I.sup.-] balance, suggesting that [H.sub.2]O intake modulates pendrin abundance. Raising water intake from ~4 to ~11 ml/day increased the ratio of B cell apical plasma membrane to cytoplasm pendrin label by 75%, although circulating renin, aldosterone, and serum osmolality were unchanged. Further studies asked whether [H.sub.2]O intake modulates pendrin through the action of AVP. We observed that [H.sub.2]O intake modulated pendrin abundance even when circulating vasopressin levels were clamped. We conclude that [H.sub.2]O intake modulates pendrin abundance, although not likely through a direct, type 2 vasopressin receptor-dependent mechanism. As water intake rises, pendrin becomes increasingly critical in the maintenance of [Cl.sup.-] and [I.sup.-] balance. chloride; apical anion exchange; vasopressin; intercalated cells; vasopressin escape doi: 10.1152/ajprenal.90581.2008.

Pendrin (encoded by Pds, Slc26a4) is a [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger expressed in the apical regions of type B and non-A, non-B intercalated cells of kidney and mediates renal [Cl.sup.-] absorption, particularly when upregulated. Aldosterone increases blood pressure by increasing absorption of both [Na.sup.+] and [Cl.sup.-] through increased protein abundance and function of [Na.sup.+] transporters, such as the epithelial [Na.sup.+] channel (ENaC) and the [Na.sup.+]-[Cl.sup.-] cotransporter (NCC), as well as [Cl.sup.-] transporters, such as pendrin. Because aldosterone analogs do not increase blood pressure in [Slc26a4.sup.-/-] mice, we asked whether [Na.sup.+] excretion and [Na.sup.+] transporter protein abundance are altered in kidneys from these mutant mice. Thus wild-type and Slc26a4-null mice were given a NaCl-replete, a NaCl-restricted, or NaCl-replete diet and aldosterone or aldosterone analogs. Abundance of the major renal [Na.sup.+] transporters was examined with immunoblots and immunohistochemistry. Slc26a4-null mice showed an impaired ability to conserve [Na.sup.+] during dietary NaCl restriction. Under treatment conditions in which circulating aldosterone is increased, [alpha]-, [beta]-, and 85-kDa [gamma]-ENaC subunit protein abundances were reduced 15-35%, whereas abundance of the 70-kDa fragment of [gamma]-ENaC was reduced ~70% in Slc26a4-null relative to wild-type mice. Moreover, ENaC-dependent changes in transepithelial voltage were much lower in cortical collecting ducts from Slc26a4-null than from wild-type mice. Thus, in kidney, ENaC protein abundance and function are modulated by pendrin or through a pendrin-dependent downstream event. The reduced ENaC protein abundance and function observed in Slc26a4-null mice contribute to their lower blood pressure and reduced ability to conserve [Na.sup.+] during NaCl restriction. intercalated cell; Slc26a4; apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger; epithelial sodium channel; aldosterone

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, [J.sub.Cl] in mouse CCD through a pendrin-dependent mechanism. [J.sub.Cl] and transepithelial voltage, [V.sub.T], were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaClreplete diet, [V.sub.T] and [J.sub.Cl] were not different from zero either in the presence or absence of angiotensin II ([10.sup.-8] M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative [V.sub.T] and absorbed [Cl.sup.-]. With angiotensin II in the bath, [Cl.sup.-] absorption doubled although [V.sub.T] did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, [Cl.sup.-] secretion and a [V.sub.T] of ~0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished [V.sub.T] although [J.sub.Cl] fell only ~50%. Thus substantial [Cl.sup.-] absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the [H.sup.+]-ATPase inhibitor bafilomycin abolished benzamil-insensitive [Cl.sup.-] absorption. In conclusion, angiotensin II increases transcellular [Cl.sup.-] absorption in the CCD through a pendrin- and [H.sup.+]-ATPase-dependent process. transepithelial voltage; bafilomycin; [H.sup.+]-ATPase; knockout mice; Slc26a4; intercalated cell

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 [Na.sup.+]-[K.sup.+]-2[C1.sup.-] cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst. aldosterone

Pendrin, encoded by Slc26a4, is a [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger expressed in the apical region of type B and non-A, non-B intercalated cells, which regulates renal NaCl excretion. Dietary C1 restriction upregulates total pendrin protein expression. Whether the subcellular expression of pendrin and whether the apparent vascular volume contraction observed in Slc26a4 null mice are [Cl.sup.-] dependent, but [Na.sup.+] independent, is unknown. Thus the subcellular distribution of pendrin and its role in acid-base and fluid balance were explored using immunogold cytochemistry and balance studies of mice ingesting a NaCl-replete or a [Na.sup.+]-replete, [Cl.sup.-]-restricted diet, achieved through substitution of NaCl with NaHCO3. Boundary length and apical plasma membrane pendrin label density each increased by ~60-70% in type B intercalated cells, but not in non-A, non-B cells, whereas cytoplasmic pendrin immunolabel increased ~60% in non-A, non-B intercalated cells, but not in type B cells. Following either NaC1 restriction or [Cl.sup.-] restriction alone, S1c26a4 null mice excreted more [Cl.sup.-] and had a higher arterial pH than pair-fed wild-type mice. In conclusion, 1) following dietary [Cl.sup.-] restriction, apical plasma membrane pendrin immunolabel increases in type B intercalated cells, but not in non-A, non-B intercalated cells; and 2) pendrin participates in the regulation of renal [C1.sup.-] excretion and arterial pH during dietary [C1.sup.-] restriction. acid-base and fluid balance; mammalian collecting duct

Lithium (Li) treatment for 4 wk has previously been shown to increase the fraction of intercalated ceils in parallel with a decrease in the fraction of principal cells in the kidney collecting duct (Christensen BM, Marples D, Kim YH, Wang W, Frokiaer J, and Nielsen S. Ant J Phvsiol Cell Physiol 286: C952-C964, 2004; Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frckiaer J, and Nielsen S. Am J Physiol Renal Physiol 285: F1244-F1257, 2003). To study how early this fractional change starts, the origin of the cells and the possible mechanism behind the changes, we did time course studies in rats subjected to different durations of Li treatment (i.e., for 4, 10, and 15 days). Increased urine output was already observed at day 4 of Li treatment with decreased AQP2 levels although not statistically significant. At days 10 and 15, both a significant polyuria and down-regulation in AQP2 expression were observed. At day 10, the density of [H.sup.+]-ATPase-positive cells was increased in the IMCD of Li-treated rats and this was further pronounced at day 15. Some of the [H.sup.+]-ATPase-positive cells did not costain with [Cl.sup.-]/HC[O.sup.-.sub.3]. exchanger AE1, indicating that they were not fully differentiated to type AIC. By double labeling for either [H.sup.4]-ATPase and proliferating-cell nuclear antigen (PCNA) or for AQP4 and PCNA, we found that proliferation mainly occulted in proximal IMCD cells at day 4 and it increased toward the middle part of the IMCD in response to prolonged Li treatment. Most cells expressing PCNA were stained with AQP4 but not with [H.sup.+]-ATPase. Triple-labeling for [H.sup.+]-ATPase, AQP4, and PCNA showed a subset of cells negative for all three proteins or only positive for PCNA. In contrast, a 4-wk recovery period after 4 wk of Li treatment reversed the enhanced proliferative rate to the control levels. In conclusion, the Li-induced increase in the density of intercalated cells is associated with a high proliferative rate of principal cells in the IM-1 and IM-2 rather than a selective proliferation of intercalated cells as expected. This is likely to contribute to the remodeling of the collecting duct after Li treatment. cell proliferation; collecting duct principal cell; collecting duct intercalated cells; lithium-induced nephrogenic diabetes insipidus

Mammalian urea transporters are facilitated membrane transport proteins belonging to two families, UT-A and UT-B. They are best known for their role of maintaining the renal inner medullary urinary concentrating gradient. Urea transporters have also been identified in tissues not typically associated with urea metabolism. The purpose of this study was to survey the major organs in rat to determine the distribution of UT-A and UT-B mRNA transcripts and protein forms and determine their cellular localization. Five kidney subregions and 17 extrarenal tissues were screened by Northern blot analysis using two UT-A and three UT-B probes and by Western blot analysis using polyclonal COOH-terminal UT-A and UT-B antibodies. Immunohistochemistry was performed on 16 extrarenal tissues using the same antibodies. In kidney, we detected mRNA transcripts and protein bands consistent with previously-identified UT-A and UT-B isoforms, as well as novel forms. We found that UT-A mRNA and protein are widely expressed in extrarenal tissues in various forms that are different from the known isoforms. We determined the cellular localization of UT-A and UT-B in these tissues. We found that both UT-A and UT-B are ubiquitously expressed as numerous tissue-specific mRNA transcripts and protein forms that are localized to cell membranes, cytoplasm, or nuclei. urea transporter; Northern blot analysis; Western blot analysis; immunohistochemistry

Chronic metabolic acidosis indramatic increases in net acid excretion that are predomidue to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HC1 ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion. connecting segment; collecting duct

NKCC1 null mice are hypotensive, in part, from the absence of NKCC1-mediated vasoconstriction. Whether these mice have renal defects in NaCl and water handling which r contribute to the hypotension is unexplored. Therefore, we asked 1) whether NKCC1 (-/-) mice have a defect in the regulation of NaCl and water balance, which might contribute to the observed hypotension and 2) whether the hypotension observed in these mice is accompanied by endocrine abnormalities and/or downregulation of renal [Na.sup.+] transporter expression. Thus we performed balance studies, semi-quantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (-/-) mice which consumed either a high (2.8% NACl)- or a low-NaCl (0.01% NaCl) diet for 7 days. Blood pressure was lower in NKCC1 (-/-) than NKCC1 (+/+) mice following either high or low dietary NaCl intake. Relative to wild-type mice, NKCC1 null mice had a lower plasma ANP concentration, a higher plasma renin and a higher serum [K.sup.+] concentration with inappropriately low urinary [K.sup.+] excretion, although serum aldosterone was either the same or only slightly increased in the mutant mice. Expression of NHE3, the a-subunit of the Na-K-ATPase, NCC, and NKCC2 were higher in NKCC1 null than in wild-type mice, although differences were generally greater during NaCl restriction. NKCC1 null mice had a reduced capacity to excrete free water than wild-type mice, which resulted in hypochloremia following the NaCl-deficient diet. Hypochloremia did not occur from increased aquaporin-1 (AQP1) or 2 protein expression or from redistribution of AQP2 to the apical regions of principal cells. Instead, NKCC1 null mice had a blunted increase in urinary osmolality following vasopressin administration, which should increase free water excretion and attenuate the hypochloremia. In conclusion, aldosterone release is inappropriately low in NKCC1 null mice. Moreover, the action of aldosterone and vasopressin is altered within kidneys of NKCC1 null mice, which likely contributes to their hypotension. Increased [Na.sup.+] transporter expression, increased plasma renin, and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thus minimizing hypotension. [Na.sup.+] transporters; chloride; water; renin; aldosterone

Slc26a4 (Pds) encodes pendrin, a C1 /HCO3 exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH equivalents. Thus genetic disruption of S1c26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in S1c26a4 (-/-) mice under basal conditions? 2) In the absence of pendrin-mediated OH secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell [H.sup.+]/O[H.sup.-] transporter expression? To answer these questions, net acid excretion and [H.sup.+]/O[H.sup.-] transporter expression were examined in Slc26a4 (-/-) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and P[CO.sub.2] were much lower in Sic26a4 null relative to wild-type mice due to reduced urinary buffering of secreted [H.sup.+] by HC[O.sup.-.sub.3]. Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of S1c26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced [H.sup.+]-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell [H.sup.+]/O[H.sup.-] transporter expression is observed in S1c26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4. NBC3; AEl; acid-base balance

Lithium treatment is associated with development of nephrogenic diabetes insipidus, caused in part by downregulation of collecting duct aquaporin-2 (AQP2) and AQP3 expression. In the present study, we carried out cDNA microarray screening of gene expression in the inner medulla (IM) of lithium-treated and control rats, and selected genes were then investigated at the protein level by immunoblotting and/or immunohistochemistry. The following genes exhibited significantly altered transcription and mRNA expression levels, and these were compatible with the changes in protein expression. 11[beta]-Hydroxysteroid dehydrogenase type 2 protein expression in the IM was markedly increased (198 [+ or -] 25% of controls, n = 6), and immunocytochemistry demonstrated an increased labeling of IM collecting duct (IMCD) principal cells. This indicated altered renal mineralocorticoid/glucocorticoid responses in lithium-treated rats. The inhibitor of cyclin-dependent kinases p27 (KIP) protein expression was significantly decreased or undetectable in the IMCD cells, pointing to increased cellular proliferation and remodeling. Heat shock protein 27 protein expression was decreased in the IM (64 [+ or -] 6% of controls, n = 6), likely to be associated with the decreased medullary osmolality in lithium-treated rats. Consistent with this, lens aldose reductase protein expression was markedly decreased in the IM (16 [+ or -] 2% of controls, n = 6), and immunocytochemistry revealed decreased expression in the thin limb cells in the middle and terminal parts of the IM. Ezrin protein expression was upregulated in the IM (158 [+ or -] 16% of controls, n = 6), where it was predominantly expressed in the apical and cytoplasmic domain of the IMCD cells. Increased ezrin expression indicated remodeling of the actin cytoskeleton and/or altered regulation of IMCD transporters. In conclusion, the present study demonstrates changes in gene expression not only in the collecting duct but also in the thin limb of the loop of Henle in the IM, and several of these genes are linked to altered sodium and water reabsorption, cell cycling, and changes in interstitial osmolality. nephrogenic diabetes insipidus; 11[beta]-hydroxysteroid dehydrogenase type 2; aldose reductase; inhibitor of cyclin-dependent kinases p27; heat shock protein 27; urine concentration

AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic N[H.sub.4]Cl loading. Am J Physiol Renal Physiol 286:F1163--F1170, 2004. First published January 28, 2004; 10.1152/ajprenal.00409.2003.--Three splice variants of anion exchanger (AE)2 (AE2a, b, and c) have been described in the rat, but their relative distribution in rat kidney is not known. The purpose of this study was to describe the segmental and cellular distribution of the AE2 isoforms in the rat kidney and to evaluate whether the expression levels of these AE2 isoforms are regulated independently in response to chronic N[H.sub.4]Cl loading. Two polyclonal antibodies were generated, respectively, recognizing a N[H.sub.2]-terminal peptide unique to AE2a and an amino acid sequence common to AE2a and AE2b. Antibody specificities were tested using cells transfected separately with the AE2a, AE2b, and AE2c isoforms, Immunohistochemistry on sections of paraffin-embedded rat kidneys showed a distribution of AE2a/AE2b labeling in the kidney similar to the distribution of AE2 in the rat kidney reported previously. AE2 is highly expressed in the medullary thick ascending limb, cortical thick ascending limb (cTAL), and macula densa. The pattern of AE2a-specific labeling differed from the pattern of AE2a/AE2b labeling in that relatively more of the total immunolabel was observed in the terminal inner medullary collecting duct. N[H.sub.4]Cl loading (0.033 mrnol N[H.sub.4]Cl/g body wt for 7 days) did not change the labeling of AE2 isoforms in the medulla, whereas the labeling in the cortex was intensified and included more distal parts of the cTAL. Immunoblotting confirmed upregulation of AE2a/b expression in the cortex. These results indicate that AE2a and AE2b are differentially expressed and regulated in the rat kidney. The regulation following N[H.sub.4]Cl loading of AE2b in the cTAL suggests a role for AE2 in transepithelial bicarbonate reabsorption in this segment. immunohistochemistry; collecting duct; nephron segments

Primary cultures of rat inner medullary collecting duct (IMCD) cells [Na.sup.+] dependently import HC[O.sup.-.sub.3] across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral [Na.sup.+]-HC[O.sup.-.sub.3] cotransport. The mRNA encoding the electroneutral [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter NBCnl was detected as the only [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter in the rat inner medulla (IM) among the five characterized [Na.sup.+]-dependent HC[O.sup.-.sub.3] transporters. The mRNA of a yet uncharacterized transporter-like protein, BTRI, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an ~180kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, [Na.sup.+]- and HC[O.sup.-.sub.3]-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemideinduced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHC[O.sup.3]-loaded animals, and the NBCn1 abundance increased only in the ISOM after N[H.sub.4]C1 loading. In conclusion, NBCn1 is a basolateral [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb. acid-base balance; hydrogen-ATPase; bicarbonate transport; bicarbonate metabolism; immunohistochemistry; intracellular pH; 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein

Christensen, Birgitte Monster, David Marples, Young-Hee Kim, Weidong Wang, Jorgen Frokiaer, and Soren Nielsen. Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI. Am J Physiol Cell Physiol 286: C952-C964, 2004. First published November 12, 2003: 10.1152/ ajpcell.00266.2003.--Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H.sup.+]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H.sup.+]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 [+ or -] 3.4 vs. 62 [+ or -] 1.8% of controls (P < 0.05; n = 4) and in inner medullary collecting ducts, 58 [+ or -] 1.6 vs. 81 [+ or -] 1.3% of controls (P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H.sup.+]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus. nephrogenic diabetes insipidus; aquaporin; exchanger

Prolonged lithium treatment of humans and rodents often results in hyperchloremic metabolic acidosis. This is thought to be caused by diminished net [H.sup.+] secretion and/or excessive back-diffusion of acid equivalents. To explore whether lithium treatment is associated with changes in the expression of key renal acid-base transporters, semiquantitative immunoblotting and immunocytochemistry were performed using kidneys from lithium-treated (n = 6) and control (n = 6) rats. Rats treated with lithium for 28 days showed decreased urine pH, whereas no significant differences in blood pH and plasma HC[O.sup.-.sub.3] levels were observed. Immunoblot analysis revealed that lithium treatment induced a significant increase in the expression of the [H.sup.+]-ATPase ([B.sub.1]-subunit) in cortex (190 [+ or -] 18%) and inner stripe of the outer medulla (190 [+ or -] 9%), and a dramatic increase in inner medulla (900 [+ or -] 104%) in parallel to an increase in the expression of type 1 anion exchanger (400 [+ or -] 40%). This was confirmed by immunocytochemistry and immunoelectron microscopy, which also revealed increased density of intercalated cells. Moreover, immunoblotting and immunocytochemistry revealed a significant increase in the expression of the type 1 electrogenic [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) in cortex (200 [+ or -] 23%) and of the electroneutral NBCn1 in inner stripe of the outer medulla (250 [+ or -] 54%). In contrast, there were no changes in the expression of [Na.sup.+]/[H.sup.+] exchanger-3 or of the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger pendrin. These results demonstrate that the expression of specific renal acid-base transporters is markedly altered in response to long-term lithium treatment. This is likely to represent direct or compensatory effects to increase the capacity for HC[O.sup.-.sub.3] reabsorption, N[H.sup.+.sub.4] reabsorption, and proton secretion to prevent the development of systemic metabolic acidosis. acid-base balance; distal tubular acidosis; hydrogen ion-adenosinetriphosphatase; electroneutral sodium-HC[O.sup.-.sub.3] cotransporter; pendrin

Lithium-induced nephrogenic diabetes insipidus is associated with increased renal sodium excretion in addition to severe urinary concentrating defects. However, the molecular basis for this altered renal sodium excretion remains undefined. The amiloride-sensitive sodium channel (ENaC) is expressed in the renal connecting tubule and collecting duct and is essential in renal regulation of body sodium balance and blood pressure. We hypothesized that dysregulation of ENaC subunits may be responsible for the increased sodium excretion associated with lithium treatment. Lithium treatment for 28 days resulted in severe polyuria, increased fractional excretion of sodium, and increased plasma aldosterone concentration. Immunoblotting revealed that lithium treatment induced a marked decrease in the protein abundance of [beta]-ENaC and [gamma]-ENaC in the cortex and outer medulla. Moreover, immunohistochemistry and laser confocal microscopy demonstrated an almost complete absence of [beta]-ENaC and [gamma]-ENaC labeling in cortical and outer medullary collecting duct, which was not affected by dietary sodium intake. In contrast, immunohistochemistry showed increased apical labeling of all ENaC subunits in the connecting tubule and inner medullary collecting duct in rats on a fixed sodium intake but not in rats with free access to sodium. Except for a modest downregulation of the thiazide-sensitive Na-Cl cotransporter, the key renal sodium transporters upstream from the connecting tubule (including the [[alpha].sub.1]-subunit of Na-K-ATPase, type 3 Na/H exchanger, and Na-K-2Cl cotransporter) were unchanged. These results identify a marked and highly segment-specific downregulation of [beta]-ENaC and [gamma]-ENaC in the cortical and outer medullary collecting duct, chief sites for collecting duct sodium reabsorption, in rats with a lithium-induced increase in fractional excretion of sodium. aldosterone; collecting duct; epithelial sodium channel; hypertension; nephrogenic diabetes insipidus; sodium reabsorption; sodium wasting

Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urinary pH from 6.15 [+ or -] (SE) 0.1 to 5.63 [+ or -] 0.07. To explore the mechanism of the decrease in urinary pH, we examined the expression of [H.sup.+]-ATPase in kidneys of AQP1 null mice. There was strong labeling for [H.sup.+]-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild-type mice. Strong [H.sup.+]-ATPase immunostaining was also present in the apical plasma membrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice, whereas no [H.sup.+]-ATPase labeling was observed in IMCD cells in wild-type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, which normally occurs during postnatal kidney development, was impaired. Western blot analysis of [H.sup.+]-ATPase expression in the different regions of the kidney demonstrated a significant increase in [H.sup.+]-ATPase protein in the inner medulla of AQP1 null mice compared with wild-type mice. There were no changes in [H.sup.+]-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical [H.sup.+]-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urinary pH observed in AQP1 null mice is due to upregulation of [H.sup.+]-ATPase in the IMCD. The induction of [H.sup.+]-ATPase expression in IMCD cells of AQP1 null mice may be related to the chronically low interstitial osmolality in these animals. The challenge will be to identify the molecular signal(s) responsible for the de novo [H.sup.+]-ATPase expression. urine acidification; intercalated cells; immunohistochemistry

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) [Na.sup.+] transporters. Rats were on a fixed intake of [Na.sup.+] and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng * [min.sup.-1] * [kg.sup.-1] ANG II (sc). Semiquantitative immunoblotting revealed that [Na.sup.+]/[H.sup.+] exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 [+ or -] 28 (ANG II-50, n = 5), 166 [+ or -] 23 (ANG II-100, n = 7), and 167 [+ or -] 19% (ANG II-200, n = 4) of control levels (n = 6, P 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive [Na.sup.+] -[K.sup.+] -2[Cl.sup.-] cotransporter (BSC-1) in the ISOM was also increased to 187 [+ or -] 28 (ANG II-50), 162 [+ or -] 23 (ANG II-100), and 166 [+ or 1] 19% (ANG II-200) of control levels (P < 0.05), but there were no changes in the abundance of [Na.sup.+] -[K.sup.+]-ATPase and the electroneutral [Na.sup.+]-HC[O.sub.3] cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng * [min.sup.-1] * [kg.sup.-1] for 5 days, n = 6) had a higher urinary pH compared with controls. N[H.sub.4]Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal [Na.sup.+] and HC[O.sub.3] reabsorption in response to ANG II. bumetanide-sensitive sodium-potassium-2chloride cotransporter; Na-K-ATPase; type 3 sodium-hydrogen exchanger; bicarbonate transport; sodium transport

Ammonia is both produced and transported by renal epithelial cells, and it regulates renal ion transport. Recent studies have identified a family of putative ammonium transporters; mRNA for two members of this family, Rh B-glycoprotein (RhBG) and Rh C-glycoprotein (RhCG), is expressed in the kidney. The purpose of this study was to determine the cellular location of RhBG and RhCG protein in the mouse kidney. We generated RhBG- and RhCG-specific anti-peptide antibodies. Immunoblot analysis confirmed that both proteins were expressed in the mouse kidney. RhBG localization with immunohistochemistry revealed discrete basolateral labeling in the connecting segment (CNT) and in the majority of initial collecting tubule (ICT) and cortical collecting duct (CCD) cells. In the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD) only a subpopulation of cells exhibited basolateral immunoreactivity. Colocalization of RhBG with carbonic anhydrase II, the thiazide-sensitive transporter, and the anion exchangers AE1 and pendrin demonstrated RhBG immunoreactivity in all CNT cells and all CCD and ICT principal cells. In the ICT and CCD, basolateral RhBG immunoreactivity is also present in A-type intercalated cells but not in pendrin-positive CCD intercalated cells. In the OMCD and IMCD, only intercalated cells exhibit RhBG immunoreactivity. Immunoreactivity for a second putative ammonium transporter, RhCG, was present in the apical region of cells with almost the same distribution as RhBG. However, RhCG immunoreactivity was present in all CCD cells, and it was present in outer stripe OMCD principal cells, in addition to OMCD and IMCD intercalated cells. Thus the majority of RhBG and RhCG protein expression is present in the same epithelial cell types in the CNT and collecting duct but with opposite polarity. These findings suggest that RhBG and RhCG may play important and cell-specific roles in ammonium transport and signaling in these regions of the kidney. Rh B-glycoprotein; Rh C-glycoprotein; collecting duct; intercalated cell; principal cell; immunohistochemistry; connecting segment

Recent studies have demonstrated that a novel anion exchanger, pendrin, is expressed in the apical domain of type B intercalated cells in the mammalian collecting duct. The purpose of this study was 1) to determine the expression and distribution of pendrin along the collecting duct and connecting tubule of mouse and rat kidney and establish whether pendrin is expressed in the nonA-non-B intercalated cells and 2) to determine the intracellular localization of pendrin in the different populations of intercalated cells by immunoelectron microscopy. A peptide-derived affinity-purified antibody was generated that specifically recognized pendrin in immunoblots of rat and mouse kidney. Immunohistochemistry and confocal laser scanning microscopy demonstrated the presence of pendrin in apical domains of all type B intercalated cells in mouse and rat connecting tubule and collecting duct. In addition, strong pendrin immunostaining was observed in non-A-non-B intercalated cells. There was no labeling of type A intercalated cells. Immunoelectron microscopy demonstrated that pendrin was located in the apical plasma membrane and intracellular vesicles of both type B intercalated cells and non-A-non-B cells; the latter was identifled by the presence of [H.sup.+]-ATPase in the apical plasma membrane. The results of this study demonstrate that both pendrin and [H.sup.+] -ATPase are expressed in the apical plasma membrane of non-A-non-B intercalated cells, suggesting that these cells are capable of both H[CO.sup.-.sub.3] and proton secretion. Furthermore, the presence of pendrin in both the apical plasma membrane and the apical intracellular vesicles of type B and non-A-non-B intercalated cells suggests that H[CO.sup.-.sub.3] secretion may be regulated by trafficking of pendrin between the two membrane compartments. acid-base metabolism; connecting tubule; collecting duct; bicarbonate secretion

Newborn rats are not capable of producing concentrated urine. With development of the concentrating system and a hypertonic medullary interstitium, intracellular osmolytes, such as sorbitol, accumulate in the renal medulla. Sorbitol is produced from glucose in a reaction catalyzed by aldose reductase (AR). The purpose of this study was to establish the time of expression and distribution of AR in the developing rat kidney. Kidneys from 16-, 18-, and 20-day-old fetuses and 1-, 3-, 4-, 5-, 7-, 14-, and 21-day-old pups were processed for immunohistochemistry and immunoblot analysis. In adult animals, AR was expressed only in the inner medulla, in which it was localized in ascending thin limbs (ATLs), inner medullary collecting ducts (IMCDs), and interstitial cells. AR immunoreactivity was not detected in fetal kidneys but was observed in the terminal part of the descending thin limb and IMCD in the renal papilla of 1-day-old pups. At birth, all of the loops of Henle are configured as short loops and there are no ATLs. After birth, papillary thick ascending limbs are gradually transformed into ATLs by a process that involves apoptotic deletion of cells from the thick ascending limb. During this time, AR immunoreactivity appeared in the cells undergoing transformation in the ascending limb, beginning at the papillary tip and ascending to the border between the outer medulla and the inner medulla. However, there was no labeling of apoptotic cells. The expression of AR in both the ATL and the IMCD gradually increased during kidney development. We conclude that AR expression in the inner medulla coincides with the increase in medullary tonicity that is known to occur during the first 3 wk after birth. On the basis of the observation that only AR-negative cells were deleted by apoptosis in the differentiating ATL, we propose that AR may protect ATL cells against apoptosis. ascending thin limb; development; apoptosis

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4- and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14- and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine. UT-A; UT-B; immunohistochemistry; urine concentration

The metal ion sensing properties of the prepared, 8-hydroxyquinoline derivatives are talked about. The results show a selective chromogenic behavior toward Hg ions.