Contribution of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) to transepithelial transport of H(+), NH(4)(+), K(+), and Na(+) in rat outer medullary collecting duct

In rat kidney, the "secretory" isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the alpha intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl(-) uptake across the basolateral membrane in series with Cl(-) secretion across the apical membrane in rat OMCD. NKCC1 transports NH(4)(+), K(+), and Na(+) as well as Cl(-); therefore, a role for the cotransporter in the process of HCl, NH(4)Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)-treated rats were perfused in vitro. Hydration of CO(2), rather than NH(4)(+), provides the principle source of H(+) for net acid secretion. In HCO(3)(-)/CO(2)-buffered solutions, no effect of bumetanide on net K(+) flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H(+) equivalents. Transepithelial Na(+) flux, J(Na), was -1.5 +/- 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, J(Na) was +5.2 +/- 1.3 pmol/mm per min (P < 0.05), which indicates net Na(+) absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na(+) and Cl(-). The role of NKCC1 in the secretion of net H(+) equivalents is small.     

氨转运蛋白Rh b型糖蛋白在低蛋白饮食大鼠肾脏的表达

目的 研究低蛋白饮食对氨转运蛋白Rh b型糖蛋白（Rhbg ）在大鼠肾脏表达的影响。 方法 利用Western印迹分别检测Rhbg在低蛋白饮食组和正常对照组大鼠肾脏皮质、内髓、外髓的表达。利用单标记免疫组化法、双标记免疫组化法和定量免疫组化法检测Rhbg在两组中肾小管主细胞和暗细胞的表达。 结果 与正常对照组比较,在大鼠肾脏皮质,低蛋白饮食组Rhbg 的蛋白水平显著增高（954778±509288比275701±262374,P < 0.05）;在大鼠肾脏内髓和外髓,低蛋白饮食组Rhbg 的蛋白水平无明显变化;在大鼠肾脏皮质集合管主细胞,低蛋白饮食组免疫组化Rhbg表达像素值（1310±357）显著高于对照组（896±154,P < 0.05）;在大鼠肾脏皮质集合管暗细胞,低蛋白饮食组免疫组化Rhbg表达像素值（1550±497）显著高于对照组（926±251,P < 0.05）;在大鼠肾脏内髓集合管和外髓集合管的主细胞或暗细胞中,两组间免疫组化Rhbg表达无显著差异。 结论 饮食中蛋白的限制可能会增加Rhbg在大鼠肾脏皮质集合管的主细胞和暗细胞的表达。     

Developmental changes in carbonic anhydrase II in the rat kidney

We examined the distribution and maturational changes of carbonic anhydrase I (CAI) and carbonic anhydrase II (CAII) in microdissected nephron segments of Sprague-Dawley rats. CAI and CAII proteins were measured by enzyme-linked immunosorbent assay. CAI was not detected in any nephron segment in 7-week-old rats. CAII was present in the collecting ducts, proximal tubules, and thick ascending limbs of loop of Henle in 7-week-old rats. CAII contents were significantly higher in the early proximal tubules (S1) than in second (S2) and late (S3) portions of the proximal tubules, while the contents in S1 were less than in cortical collecting ducts (CCD), outer stripe and inner stripes of the outer medullary collecting ducts (OMCDo and OMCDi). CAII content in each of S1, CCD, and OMCD of 1-week-old rats was only 14% or less of that of adults, but increased steeply during the 2nd and 3rd weeks of life, reaching almost 40% at 3 weeks of age and 97% at 7 weeks. Our results indicate that CAII is present throughout the entire nephron of the rat, and that CAII content in S1, CCD, and OMCD increases exponentially during the first 7 weeks of life. Our data suggest that the immature low levels of CAII may explain, at least in part, the limited capacity of urinary acidification during neonatal life. Further studies are necessary to establish the role of such changes in CAII content in acid-base homeostasis during neonatal life. Received December 6, 1996; received in revised form September 8, 1997; accepted September 19, 1997     

Postnatal disappearance of type A intercalated cells in carbonic anhydrase II-deficient mice

Despite chronic acidosis, collecting ducts in adult carbonic anhydrase II-deficient (CAD mice) are depleted of intercalated cells, including those of type A, which are acid-secreting cells. We hypothesized that this depletion could occur during postnatal development. Principal cells were identified by immunofluorescence using an antibody to rat aquaporin-2 (AQP-2), and type A intercalated cells using an antibody specific for anion exchanger (AE1). In CAD mice the proportion of AQP2-positive cells, normal at 11 days, increased progressively in the cortical (CCD) and outer medullary collecting duct (OMCD), to reach almost 100% in the OMCD in adults. The percentage of AE1-positive cells in the OMCD of CAD mice decreased by half by 6 weeks of age and further by adulthood. In controls, however, the proportion of AQP2-positive cells and that of AE1-positive cells in the OMCD remained stable after 10 days of age. AE1-positive cells accounted for the majority of intercalated cells in the OMCD. The mechanisms leading to selective postnatal cell depletion in the collecting duct in CAD mice remain to be determined. Received: 9 August 2000 / Revised: 12 December 2000 / Accepted: 14 December 2000     

The hyperpolarization-activated cyclic nucleotide-gated HCN2 channel transports ammonium in the distal nephron

Recent studies have identified Rhesus proteins as important molecules for ammonia transport in acid-secreting intercalated cells in the distal nephron. Here, we provide evidence for an additional molecule that can mediate NH3/NH4 excretion, the subtype 2 of the hyperpolarization-activated cyclic nucleotide-gated channel family (HCN2), in collecting ducts in rat renal cortex and medulla. Chronic metabolic acidosis in rats did not alter HCN2 protein expression but downregulated the relative abundance of HCN2 mRNA. Its cDNA was identical to the homolog from the brain and the protein was post-translationally modified by N-type glycosylation. Electrophysiological recordings in Xenopus oocytes injected with HCN2 cRNA found that potassium was transported better than ammonium, each of which was transported significantly better than sodium, criteria that are compatible with a role for HCN2 in ammonium transport. In microperfused rat outer medullary collecting duct segments, the initial rate of acidification, upon exposure to a basolateral ammonium chloride pulse, was higher in intercalated than in principal cells. A specific inhibitor of HCN2 (ZD7288) decreased acidification only in intercalated cells from control rats. In rats with chronic metabolic acidosis, the rate of acidification doubled in both intercalated and principal cells; however, ZD7288 had no significant inhibitory effect. Thus, HCN2 is a basolateral ammonium transport pathway of intercalated cells and may contribute to the renal regulation of body pH under basal conditions.     

Low tonicity mediates a downregulation of cyclooxygenase-1 expression by furosemide in the rat renal papilla

It is well known that loop diuretics enhance the renal excretion of prostanoids; therefore, this study aimed to characterize the influence of loop diuretics on the intrarenal expression of cyclooxygenases, which are the key enzymes for prostanoid formation. Male Sprague-Dawley rats were infused with furosemide (12 mg/kg per d) for 6 d, and the expression of cyclooxygenase-1 and -2 (Cox-1 and Cox-2) was analyzed in the different kidney zones. Furosemide increased Cox-2 mRNA expression approximately twofold in the cortex, but it left Cox-1 mRNA expression unaltered there. In the outer medulla, furosemide changed neither Cox-1 nor Cox-2 mRNA expression. In the inner medulla, however, furosemide decreased Cox-1 and Cox-2 mRNA levels to approximately 30% and 60% of their control levels, respectively. The downregulation of mRNA was paralleled by a decrease of Cox protein in the collecting ducts and interstitial cells. Moreover, tissue prostaglandin E(2) (PGE(2)) concentrations in the papilla were markedly decreased by furosemide to about 30% of the control level. Furosemide lowered urine osmolality from 1550 mosmol/kg to 480 mosmol/kg; therefore, further consideration was given to the influence of tonicity as a possible mediator of the effects of furosemide on the Cox expression. Water loading was therefore used to reduce the medullary tonicity by a second maneuver. Water loading led to a similar reduction in papillary Cox mRNA expression and PGE(2) content like furosemide. To investigate the influence of the osmolarity on the expression of Cox and the production of PGE(2) under defined in vitro conditions, inner medullary collecting duct cells were incubated with culture medium containing graded amounts of NaCl ranging from 200 mmol/L to 600 mmol/L, and Cox-1 and Cox-2 mRNA abundance were determined after 24 h an 48 h. Cox-1 and Cox-2 mRNA abundance changed in parallel with the osmolarity. The data suggest that loop diuretics decrease the expression of cyclooxygenases and consequently tissue PGE(2) concentrations in the kidney inner medulla. This effect could be related to the breakdown of the papillary osmotic gradient induced by loop diuretics.     

Regulation of acid-base transporters by vasopressin in the kidney collecting duct of Brattleboro rat

AIM: The objective of these studies was to examine the effects of long-term vasopressin treatment on acid-base transporters in the collecting duct of rat kidney. METHODS: Brattleboro rats were placed in metabolic cages and treated with daily injections of 1-desamino-8-D-arginine vasopressin (dDAVP), a selective V2-receptor agonist, or its vehicle (control) for up to 8 days. RESULTS: dDAVP treatment resulted in a significant reduction in serum bicarbonate concentration, and caused the upregulation of key ammoniagenesis enzymes, along with increased urinary NH4+ excretion. Northern hybridization and immunofluorescence labeling indicated a significant increase (+80%) in mRNA expression of the apical Cl-/HCO3- exchanger pendrin (PDS), along with a sharp increase in its protein abundance in B-type intercalated cells in the cortical collecting duct in dDAVP-treated rats. In the inner medullary collecting duct, the abundance of basolateral Cl-/HCO3- exchanger (AE1) and apical H+-ATPase was significantly reduced in dDAVP-treated rats. Kidney renin mRNA increased significantly and correlated with an increase in serum aldosterone levels in dDAVP-injected rats. Serum corticosterone levels were, however, reduced and correlated with increased mRNA levels of renal 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD2) and decreased mRNA expression of 11beta-hydroxylase in the adrenal gland of dDAVP-injected rats. CONCLUSION: Chronic administration of dDAVP to Brattleboro rats is associated with the upregulation of PDS and downregulation of H+-ATPase and AE1 in the collecting duct, along with increased ammoniagenesis. Stimulation of the renin-angiotensin-aldosterone system and/or decreased glucocorticoid levels likely plays a role in the transduction of these effects.     

Reverse pharmacological effect of loop diuretics and altered rBSC1 expression in rats with lithium nephropathy

BACKGROUND: Renal urinary concentration is associated with enhanced expression of rBSC1, a rat sodium cotransporter, in the thick ascending limb of Henle. Increased expression of rBSC1 was reported recently in nephrogenic diabetes insipidus induced by lithium chloride (Li nephropathy). However, the pathophysiological implication of altered rBSC1 expression has not yet been investigated. METHODS: Li nephropathy was induced in rats by an oral administration of 40 mmol lithium/kg dry food. In rats with reduced urinary osmolality to less than 300 mOsm/kg H2O, we examined the expression of rBSC1 mRNA and protein, plasma arginine vasopressin (AVP) and RNA expression of kidney-specific water channel, aquaporin-2 (AQP2), of collecting ducts. Rats with Li nephropathy were treated with furosemide (3 mg/kg body weight), which blocks the activity of rBSC1, and changes in urine concentration, plasma AVP, medullary accumulation of Li ions, and apical AQP2 expression were determined. RESULTS: Rats with Li nephropathy showed increased rBSC1 RNA and protein expression and reduced AQP2 RNA. In these rats, furosemide, which induces dilution of urine and polyuria in normal rats, resulted in a progressive and significant rise in urine osmolality from 167 +/- 11 (mean +/- SD) at baseline to 450 +/- 45 mOsm/kg H2O at three hours after administration, and significant oliguria. In the same rats, plasma AVP decreased significantly from 5.7 to 3.0 pg/mL. In addition, recovery of apical AQP2 expression was noted in a proportion of epithelial cells of the collecting ducts. Although Li+ in the renal medulla was slightly lower in rats with Li nephropathy treated with furosemide, statistical significance was not achieved. CONCLUSIONS: Our results suggest that dehydration or high plasma AVP results in an enhanced rBSC1 expression in Li nephropathy, and that rBSC1 expression is closely associated with the adverse effects of Li ions on collecting duct function.     

Aldosterone decreases UT-A1 urea transporter expression via the mineralocorticoid receptor

Adrenalectomy in rats is associated with urinary concentrating and diluting defects. This study tested the effect of adrenal steroids on the UT-A1 urea transporter because it is involved in the urine-concentrating mechanism. Rats were adrenalectomized and given normal saline for 14 d, after which they received (1) vehicle, (2) aldosterone, or (3) spironolactone plus aldosterone. Adrenalectomy alone significantly increased UT-A1 protein in the inner medullary tip after 7 d, whereas aldosterone repletion reversed the effect. Spironolactone blocked the aldosterone-induced decrease in UT-A1, indicating that aldosterone was working via the mineralocorticoid receptor. For verifying that glucocorticoids downregulate UT-A1 protein through a different receptor, three groups of adrenalectomized rats were prepared: (1) vehicle, (2) adrenalectomy plus dexamethasone, and (3) adrenalectomy plus dexamethasone and spironolactone. Dexamethasone significantly reversed UT-A1 protein abundance increase in the inner medullary tip of adrenalectomized rats. When spironolactone was given with dexamethasone, it did not affect the dexamethasone-induced decrease in UT-A1. There was no significant change in serum vasopressin level, aquaporin 2, or Na(+)-K(+)-2Cl(-) co-transporter NKCC2/BSC1 protein abundances or UT-A1 mRNA abundance in any of the groups. In conclusion, either mineralocorticoids or glucocorticoids can downregulate UT-A1 protein. The decrease in UT-A1 does not require both steroid hormones, and each works through a different receptor.     

Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.

This study tested whether glucocorticoids regulate tubular urea transport. Urea permeability was measured in perfused inner medullary collecting duct (IMCD) subsegments from rats that underwent adrenalectomy, adrenalectomy plus replacement with a physiologic dose of glucocorticoid (dexamethasone), or sham operation. Compared with sham rats, basal urea permeability in terminal IMCD was significantly increased in adrenalectomized rats and reduced in dexamethasone-treated rats. Vasopressin significantly increased urea permeability in all three groups. In contrast, there was no difference in basal or vasopressin-stimulated urea permeability in initial IMCD between the three groups. Next, membrane and vesicle fraction proteins were isolated from inner medullary tip or base and Western analysis was performed by use of an antibody to the rat vasopressin-regulated urea transporter. Vasopressin-regulated urea transporter protein was significantly increased in both membrane and vesicle fractions from the inner medullary tip of adrenalectomized rats. There was no change in vasopressin-regulated urea transporter protein in the inner medullary base, and Northern analysis showed no change in urea transporter mRNA abundance in either inner medullary region. It was concluded that glucocorticoids can downregulate function and expression of the vasopressin-regulated urea transporter in rat terminal IMCD.     

Acute and chronic effects of hyperosmolality on mRNA and protein expression and the activity of Na-K-ATPase in the IMCD.

We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase alpha and beta isoforms and Na-K-ATPase activity in the rat inner medullary collecting duct (IMCD). Incubation of IMCD in hypertonic medium for 30 min reduced the Na-K-ATPase activity by 50%. The Na-K-ATPase activity of dehydrated rats measured in isotonic medium was decreased, and incubation in hypertonic medium did not further decrease the activity. Incubation of IMCD in hypertonic medium for 6 h did not change alpha(1) mRNA. In contrast, dehydration decreased alpha(1) subunit mRNA and protein and beta(1) protein expressions without changing beta(1) mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing alpha(1) and beta(1) mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing alpha(1) and beta(1) proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is different between acute and chronic exposure to hyperosmolality.     

Differential regulation of basolateral Cl-/HCO3- exchangers SLC26A7 and AE1 in kidney outer medullary collecting duct

SLC26A7 is a recently identified Cl(-)/HCO(3)(-) exchanger that co-localizes with AE1 on the basolateral membrane of Alpha intercalated cells (A-IC) in outer medullary collecting duct (OMCD). The purpose of these studies was to determine whether AE1 and SLC26A7 are differentially regulated in OMCD in pathophysiologic states. Toward this end, the expression and regulation of AE1 and SLC26A7 was examined in water deprivation, a condition known to increase the osmolality of the medulla. Rats were subjected to 3 d of water deprivation while having free access to food. Northern hybridizations demonstrated that in the outer medulla, the mRNA expression of SLC26A7 increased by approximately 300% (P < 0.01 versus control; n = 3), whereas the expression of AE1 decreased by approximately 50% (P < 0.05 versus control, n = 3) in water-deprived rats. Immunoblot analysis studies demonstrated that in the outer medulla, SLC26A7 abundance increased by approximately 3.5-fold (P < 0.02 versus control; n = 3), whereas the AE1 abundance decreased by approximately 55% (P < 0.05 versus control) in water deprivation. The expression of SLC26A7 remained unchanged in the kidney cortex and stomach in water deprivation, indicating the specificity of SLC26A7 upregulation in outer medulla. In situ hybridization indicated the exclusive expression of SLC26A7 in the outer medulla and double immunofluorescence labeling confirmed the co-localization of AE1 and SLC26A7 on the basolateral membrane of A-IC cells in OMCD. It is concluded that AE1 and SLC26A7 are differentially regulated in OMCD in water deprivation. On the basis of these results and previous functional studies indicating the activation of SLC26A7 activity by high osmolality, it is proposed that SLC26A7 may play an important role in bicarbonate reabsorption and or cell volume regulation in OMCD (specifically under hypertonic conditions).     

Regulation of the basolateral chloride/base exchangers AE1 and SLC26A7 in the kidney collecting duct in potassium depletion.

In the present study, the effect of potassium depletion on the expression of acid-base transporters in the collecting duct was examined. Toward this end rats were fed a potassium-free diet for 3 weeks. Thereafter, the expression of the basolateral chloride/bicarbonate exchangers AE1 and SLC26A7 and the apical H(+)-ATPase was examined by northern hybridization, immunoblot analysis and immunofluorescence labelling. The mRNA expression of AE1 increased by a robust approximately 500% in the cortex and approximately 70% in the outer medulla, which translated into a huge increase in AE1 protein abundance in the cortex and a moderate increase in the outer medulla in K-depletion. The mRNA expression of SLC26A7 did not change significantly but its protein abundance showed a robust increase in the outer medulla. The expression of SLC26A7 remained undetected in the cortex in K-depleted rats. The post translational increase in SLC26A7 membrane abundance in potassium depletion was recapitulated in vitro using epitope-tagged SLC26A7. H(+)-ATPase displayed enhanced apical plasma membrane immunoreactivity in the OMCD in K-depletion. We suggest that the up-regulation of SLC26A7 and AE1 on the basolateral membrane of A-intercalated cells in the OMCD and CCD, respectively, along with H(+)-ATPase on the apical membrane, contributes to enhanced bicarbonate absorption in the collecting duct in K-depletion.     

Posttranscriptional compensation for heterozygous disruption of the kidney-specific NaK2Cl cotransporter gene

Mice homozygous for a loss of function mutation of the kidney-specific NaK2Cl cotransporter, BSC1/NKCC2, do not survive. Here the effects of loss of one copy of the gene are studied. NKCC2 mRNA of NKCC2 +/- kidney was 55 +/- 6% of +/+, yet no differences were found between NKCC2 +/+ and +/- mice in BP, blood gas, electrolytes, creatinine, plasma renin concentration, urine volume and osmolality, ability to concentrate and dilute urine, and response to furosemide. When mice were challenged with 180 mM NH(4)Cl, plasma ammonia and urinary ammonia excretion were increased twofold and fivefold, respectively, but there was still no difference between the two genotypes. NKCC2 +/- mice had a near-normal level of NKCC2 protein and no clear change in the distribution of NKCC2 in the thick ascending limb (TAL) cells. In vitro microperfusion of isolated TAL showed no significant difference between the two genotypes in the basal and vasopressin-stimulated capacity to reabsorb NaCl. There was no difference in the mRNA expressions of thiazide-sensitive NaCl cotransporter, epithelial Na channel (ENaC), aquaporin-2, ROMK, and NaKATPase. Halving the mRNA expression of NKCC2 does not affect BP or fluid balance because of compensatory factors that restore the protein level to near normal. One possible factor is a regulated increase in the movement of cytoplasmic protein to the luminal membrane leading to a restoration of functional transporter to an essentially wild type level.