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.     

Morphological heterogeneity of the rabbit collecting duct

The localization of carbonic anhydrase by histochemistry, of Na-K-ATPase by immunocytochemistry and of rod-shaped intramembranous particles by freeze-fracture electron microscopy, was determined in the collecting duct of rabbits. In the cortical collecting duct (CCD), rod-shaped particles, which are abundant in intercalated cells were observed in both the apical and basolateral membrane of all intercalated cells examined. In the outer stripe of the outer medullary collecting duct (OMCDo) a high density of rod-shaped particles was found only in the apical membrane of intercalated cells. All cells of the inner stripe of the outer medullary collecting duct (OMCDi) had rod-shaped particles in the apical membrane but not in the basolateral membrane. As the collecting duct entered the inner medulla the density of rod-shaped particles decreased until they were virtually absent in the terminal segment. Na-K-ATPase, localized to the basolateral membrane, was more abundant in principal cells than in intercalated cells in the CCD. In the OMCDo, staining was equal in principal and intercalated cells. All cells of the OMCDi and the inner medullary collecting duct (IMCD) stained for Na-K-ATPase. Carbonic anhydrase in the CCD was localized to the cell membranes and cytoplasm of intercalated cells. Principal cells did not stain for carbonic anhydrase. A similar pattern was seen in the OMCDo. In the outer region of the OMCDi most cells did not stain for carbonic anhydrase, whereas in the inner region the apical and lateral membranes of all cells stained for carbonic anhydrase. Weak cytoplasmic staining was occasionally seen. A similar pattern was seen in the initial half of the IMCD, while the terminal half of the IMCD did not stain. In this study, the localization of enzymes and rod-shaped intramembranous particles associated with Na+, K+, and H+ transport shows both segmental and cellular heterogeneity, and correlates with the known transport properties of tubule segments. The distribution of these enzymes and rod-shaped intramembranous particles is different in rabbits and rats, and may explain some of the functional differences between homologous segments in these species.     

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).     

Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct

Inward rectifier potassium channels (Kir) play an important role in the K(+) secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na(+), K(+)-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na(+), K(+)-ATPase and contribute to cell ion homeostasis and tubular K(+) secretion.     

Aquaporin-4 expression in adult and developing mouse and rat kidney

Aquaporin-4 (AQP4) is a member of the aquaporin water-channel family. AQP4 is expressed primarily in the brain, but it is also present in the collecting duct of the kidney, where it is located in the basolateral plasma membrane of principal cells and inner medullary collecting duct (IMCD) cells. Recent studies in the mouse also have reported the presence of AQP4 in the basolateral membrane of the proximal tubule. The purpose of this study was to establish the pattern of AQP4 expression during kidney development and in the adult kidney of both the mouse and the rat. Kidneys of adult and 3-, 7-, and 15-d-old mice and rats were preserved for immunohistochemistry and processed using a peroxidase pre-embedding technique. In both the mouse and the rat, strong basolateral immunostaining was observed in IMCD cells and principal cells in the medullary collecting duct at all ages examined. Labeling was weaker in the cortical collecting duct and the connecting tubule, and there was no labeling of connecting tubule cells in the mouse. In adult mouse kidney, strong AQP4 immunoreactivity was observed in the S3 segment of the proximal tubule. However, there was little or no labeling in the cortex or around the corticomedullary junction in 3- and 7-d-old mice. Between 7 and 15 d of age, distinct AQP4 immunoreactivity appeared in the S3 segment of the mouse proximal tubule concomitant with the differentiation of this segment of the nephron. Labeling of proximal tubules was never observed in the rat kidney. These results suggest that there are differences in transepithelial water transport between mouse and rat or that additional, not yet identified water channels exist in the rat proximal tubule.     

Renal concentrating and diluting function in deficiency of specific aquaporin genes

Aquaporins (AQP) are water-transporting proteins expressed in many fluid-transporting epithelia and endothelia. In kidney, AQP1 is expressed in plasma membranes of proximal tubule, thin descending limb of Henle and descending vasa recta, AQP2 in collecting duct luminal membrane, AQP3 and AQP4 in collecting duct basolateral membrane, AQP6 in intercalated cells, and AQP7 in the S3 segment of proximal tubule. Human mutations in AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus. Transgenic mice lacking the renal aquaporins have been useful in defining their role. Mice deficient in AQP1 are polyuric and unable to form a concentrated urine because of defective proximal tubule fluid absorption and countercurrent multiplication. Mice lacking AQP3 are markedly polyuric due to low water permeability across the cortical and outer medullary collecting duct. However, mice lacking AQP4, which is expressed mainly in inner medullary collecting duct, manifest only a mild defect in maximum urinary concentrating ability. The aquaporin null mice have normal urinary diluting ability. From many renal and extrarenal phenotype studies of aquaporin null mice, we conclude that aquaporins are important for rapid near-isosmolar transepithelial fluid absorption/secretion and for rapid vectorial water movement driven by osmotic gradients. The renal phenotype in aquaporin null mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents.     

Role of aquaporin water channels in kidney function studied using transgenic mice

Several aquaporin (AQP) water transporting proteins are expressed in mammalian kidney: AQP1 in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in collecting duct luminal membrane; AQP3 and AQP4 in collecting duct basolateral membrane; AQP6 in intercalated cells; and AQP7 in the S3 segment of proximal tubule. To define the role of aquaporins in renal physiology, we have generated and characterized transgenic null mice deficient in AQP1, AQP3, and AQP4, individually and in combinations, as well as AQP2 mutant mice, in which the T126M mutation causing human nephrogenic diabetes insipidus was introduced. AQP1-deficient mice are polyuric and unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion greatly reduces osmotic water permeability in proximal tubule, thin descending limb of Henle, and descending vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3 have low basolateral membrane water permeability in cortical collecting duct and excrete large quantities of dilute urine. Mice lacking AQP4 have low water permeability in inner medullary collecting duct, but manifest only a mild defect in maximum urinary concentrating ability. These data, taken together with phenotype analyses of brain, lung, and gastrointestinal organs, support the paradigm that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. The renal phenotype data in aquaporin knockout mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents. Received: March 19, 2001 / Accepted: March 22, 2001     

Distribution of postsynaptic density proteins in rat kidney: relationship to neuronal nitric oxide synthase

BACKGROUND: Neuronal nitric oxide synthase (nNOS) is expressed in skeletal muscle beneath the sarcolemma associated with dystrophin complex. In brain, nNOS is anchored to synaptic membranes by specific postsynaptic density proteins (PSD)-95 and PSD-93. We have investigated the cellular and subcellular localization of these PSD proteins in the kidney and their relationship to nNOS and the cell membrane. METHODS: Kidneys from male Sprague-Dawley rats were processed for light and electron microscopic immunohistochemistry with polyclonal antibodies against PSD and nNOS proteins. RESULTS: Western blot analysis of rat kidney revealed a specific band for PSD-93 at the molecular weight of 103 kDa. Immunostaining for PSD-93 was located in the thick ascending limb of the loop of Henle, macula densa cells, distal convoluted tubules, cortical collecting ducts, outer and inner medullary collecting duct, glomerular epithelium, and Bowman's capsule. A pre-embedding electron microscopic immunoperoxidase procedure localized PSD-93 to the basolateral membrane of these tubular cells. Using different sized immunogold particles, a portion of nNOS in the macula densa colocalized with PSD-93 adjacent to cytoplasmic vesicles and the basolateral membrane. In contrast, PSD-95 protein was detected only weakly in the cortex by Western blot. Immunostaining for PSD-95 was located only faintly in the apical membrane of the thick ascending limb, macula densa, distal convoluted tubule and cortical collecting duct cells. CONCLUSION: PSD-93 is the predominant PSD expressed in the rat kidney. It is located primarily in the basolateral membranes of distal nephron and colocalizes with a pool of nNOS in cytoplasmic vesicles and basolateral membranes of macula densa cells.     

Carbonic anhydrase in the human kidney: a histochemical and immunocytochemical study

The intracellular distribution of carbonic anhydrase was studied in nine human donor kidneys by the histochemical method of Hansson and by an immunofluorescence technique using antisera specific against the cytoplasmic isoenzymes HCA B and HCA C. Only HCA C was found in the renal tubules. Convoluted proximal tubules showed enzyme staining at the brush border and the basolateral membranes, and in the cytoplasm. No staining was observed in straight proximal tubules. The initial part of the thin limb of Henle's loop displayed cytoplasmic staining, whereas the distal part was unstained in most nephrons. In the medullary ascending thick limb enzyme was present at apical and basolateral cell membranes as well as in the cytoplasm, whereas in the apical cell region of the cortical ascending thick limb there was no distinct staining. The cells of the macula densa showed staining only at the cell membranes. The distal convoluted tubule exhibited heavy staining in the cytoplasm and at the cell membranes. In the initial collecting tubule and the collecting duct very intensely stained intercalated cells were found among less strongly stained chief cells. In the inner medullary segment of the collecting duct, the staining for carbonic anhydrase gradually disappeared. Many peritubular capillaries showed enzyme staining, while glomerular capillaries and larger vessels were negative. Specific fluorescence for HCA B was observed in the capillary walls.     

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.     

Potential physiologic roles for epidermal growth factor in the kidney

Epidermal growth factor (EGF) is a 53-amino acid polypeptide that is known to produce a number of biologic effects both in vitro and in vivo. High concentrations of EGF are found in urine, and high concentrations of prepro-EGF mRNA have been detected in kidney, localized to thick ascending limb of Henle (TALH) and distal convoluted tubule. Specific high-affinity EGF receptors have been demonstrated in mesangial cells, proximal tubule, and cortical and inner medullary collecting duct, as well as in medullary interstitial cells. In the proximal tubule, EGF binding and EGF receptor-associated tyrosine kinase activity are localized to basolateral membrane, and functional responses in collecting duct are observed only with basolateral administration of EGF. A number of renal responses to administration of EGF have recently been described, including modulation of glomerular hemodynamics, renal metabolism, tubular transport functions, and eicosanoid synthesis. In addition, EGF has been shown to be a potent mitogen in vitro for a variety of cell types in the kidney and may be an important mediator of renal repair following injury.     

Basolateral carbonic anhydrase IV in the proximal tubule is a glycosylphosphatidylinositol-anchored protein

Carbonic anhydrase (CA) IV facilitates HCO(3) reabsorption in the renal proximal tubule by catalyzing the reversible hydration of CO(2). CAIV is tethered to cell membranes via a glycosylphosphatidylinositol (GPI) lipid anchor. As there is basolateral as well as apical CAIV staining in proximal tubule, the molecular identity of basolateral CAIV was examined. Biotinylation of confluent monolayers of rat inner medullary collecting duct cells stably transfected with rabbit CAIV showed apical and basolateral CAIV, and in the cell transfectants expressing high levels of CAIV, a transmembrane form was targeted to the basolateral membrane. Basolateral expression of CAIV ( approximately 46 kDa) was confirmed in normal kidney tissue by Western blotting of vesicle fractions enriched for basolateral membranes by Percoll density fractionation. We examined the mode of membrane linkage of basolaterally expressed CAIV in the kidney cortex. CAIV detected in basolateral or apical membrane vesicles exhibited similar molecular size by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis following deglycosylation, and was equally sensitive to phosphatidylinositol-specific phospholipase C digestion, indicating that CAIV is expressed on the basolateral membrane as a GPI-anchored protein. Half of the hydratase activity of basolateral vesicles was resistant to SDS denaturation, compatible with being CAIV. Thus, GPI-anchored CAIV resides in the basolateral membrane of proximal tubule epithelia where it may facilitate HCO(3) reabsorption via association with kNBC1.     

Expression of the ammonia transporter, rh C glycoprotein, in normal and neoplastic human kidney

Recent studies have identified the presence of a novel Mep/Amt/Rh glycoprotein family of proteins that may play an important role in transmembrane ammonia transport. One of the mammalian members of this family, Rh C glycoprotein (RhCG), transports ammonia, is expressed in distal nephron sites that are critically important for ammonia secretion, exhibits increased expression in response to chronic metabolic acidosis, and originally was cloned as a tumor-related protein. The purpose of our studies was to determine the localization of RhCG in the normal and neoplastic human kidney. Immunoblot analysis of human renal cortical protein lysates demonstrated RhCG protein expression with a molecular weight of approximately 52 kD. Immunohistochemistry revealed both apical and basolateral Rhcg expression in the distal convoluted tubule, connecting segment, and initial collecting tubule and throughout the collecting duct. Co-localization with calbindin-D28k, H(+)-ATPase, aquaporin-2, and pendrin showed that distal convoluted tubule and connecting segment cells, A-type intercalated cells, and non-A, non-B cells express RhCG and that B-type intercalated cells, principal cells, and inner medullary collecting duct cells do not. In renal neoplasms, RhCG was expressed by chromophobe renal cell carcinoma and renal oncocytoma but not by clear cell renal cell carcinoma or by papillary renal cell carcinomas. These studies suggest that RhCG contributes to both apical and basolateral membrane ammonia transport in the human kidney. Furthermore, renal chromophobe renal cell carcinoma and renal oncocytoma seem to originate from the A-type intercalated cell.     

Expression of the alpha-subunit of Na/K-ATPase in renal collecting duct epithelium during development

Aldosterone enhances synthesis and enzyme activity of Na/K-ATPase and has been found to stimulate sodium reabsorption by the renal cortical collecting duct. Moreover, chronic exposure to aldosterone is associated with a remarkable morphological-functional adaptation. This is seen as a magnification in basolateral membrane area of principal cells. In the present paper we investigated the acquistion of Na/K-ATPase alpha-subunit in renal tissue and examined whether aldosterone initiates functional and adaptive changes in cultured collecting duct cells similar to those observed in vivo. Using a monoclonal antibody, immunofluorescence microscopy demonstrated the acquisition of the alpha-subunit of Na/K-ATPase in the developing cortical collecting ducts of the kidney of neonatal rabbits. The mature collecting ducts in the medulla and papilla of the developing kidney were strongly labelled at the basolateral side, while in the cortical portion of the fetal collecting duct adjacent to the embryonic ampullae the immunolabel was found at the apical and the basolateral aspect of the epithelium. However, the embryonic collecting duct ampullae in the outer cortex did not show any reaction with the antibody. In the collecting duct cells cultured for 24 hours the alpha-subunit of Na/K-ATPase was found to be distributed at both the apical and basolateral aspect of the epithelium. After two to 16 days, the immunolabel was strictly found distributed at the basolateral side. Culturing collecting duct cells in the presence of aldosterone (10(-6) M), the hormone modulated the cellular shape of epithelia after five days by infolding the lateral plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of carbonic anhydrase in the rat kidney

The rat kidney was studied by light and electron microscope after it was histochemically stained for carbonic anhydrase activity. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed intense activity at the brush border and the basolateral membranes. Cytoplasmic activity also was found. Straight proximal tubules had considerable enzyme activity at basolateral membranes but only low activity at the brush border and in the cytoplasm. In nephrons with long loops, the descending thin limb contained cytoplasmic enzyme activity, whereas the ascending thin limb was inactive. Thin limbs of short loops showed a varying enzyme pattern. In the thick limb of Henle's loop, most enzyme activity was found at the luminal cell border. Distal convoluted tubules showed enzyme activity only at basal infolded membranes. In the late distal tubule, intercalated cells appear among the "ordinary" distal cells, and they contained abundant cytoplasmic enzyme. Many highly active intercalated cells were found also in the cortical and outer medullary segments of the collecting duct. The chief cells in these segments also showed some cytoplasmic enzyme activity. In the inner medullary segment of the collecting duct, enzyme activity disappeared gradually, and the tip of the papilla lacked activity. Acetazolamide (10 microM) completely abolished visible staining, whereas Cl 13850 (10 microM), an inactive acetazolamide analogue, did not interfere with the staining.     

Physiology and pathophysiology of renal aquaporins

The discovery of aquaporin membrane water channels by Agre and coworkers answered a long-standing biophysical question of how water specifically crosses biologic membranes, and provided insight, at the molecular level, into the fundamental physiology of water balance and the pathophysiology of water balance disorders. Of nine aquaporin isoforms, at least six are known to be present in the kidney at distinct sites along the nephron and collecting duct. Aquaporin-1 (AQP1) is extremely abundant in the proximal tubule and descending thin limb, where it appears to provide the chief route for proximal nephron water reabsorption. AQP2 is abundant in the collecting duct principal cells and is the chief target for vasopressin to regulate collecting duct water reabsorption. Acute regulation involves vasopressin-regulated trafficking of AQP2 between an intracellular reservoir and the apical plasma membrane. In addition, AQP2 is involved in chronic/adaptational regulation of body water balance achieved through regulation of AQP2 expression. Importantly, multiple studies have now identified a critical role of AQP2 in several inherited and acquired water balance disorders. This concerns inherited forms of nephrogenic diabetes insipidus and several, much more common acquired types of nephrogenic diabetes insipidus where AQP2 expression and/or targeting are affected. Conversely, AQP2 expression and targeting appear to be increased in some conditions with water retention such as pregnancy and congestive heart failure. AQP3 and AQP4 are basolateral water channels located in the kidney collecting duct, and AQP6 and AQP7 appear to be expressed at lower abundance at several sites including the proximal tubule. This review focuses mainly on the role of AQP2 in water balance regulation and in the pathophysiology of water balance disorders.     

Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval

The Na/K-ATPase plays a fundamental role in the physiology of various mammalian cells. In the kidney, previous immunocytochemical studies have localized this protein to the basolateral membrane in different tubule segments. However, intercalated cells (IC) of the collecting duct (CD) in rat and mouse were unlabeled with anti-Na/K-ATPase antibodies. An antigen retrieval technique has been recently described in which tissue sections are pretreated with sodium dodecyl sulfate before immunostaining. This procedure was used to reexamine the presence of Na/K-ATPase in IC along the rat nephron using monoclonal antibodies against the Na/K-ATPase alpha-subunit. Subtypes of IC along the nephron were identified by their distinctive staining with polyclonal and monoclonal antibodies to the 31-kD vacuolar H+ -ATPase subunit, whereas principal cells (PC) were labeled with a polyclonal antibody to the water channel aquaporin-4 (AQP-4). In PC, the Na/K-ATPase and AQP-4 staining colocalized basolaterally. In contrast to previous reports, we found that IC of all types showed basolateral labeling with the anti-Na/K-ATPase antibody. The staining was quantified by fluorescence image analysis. It was weak to moderate in IC of cortical and outer medullary collecting ducts and most intense in IC of the initial inner medullary collecting duct. IC in the initial inner medulla showed a staining intensity that was equivalent or stronger to that in adjacent principal cells. Models of ion transport at the cellular and epithelial level in rat kidney, therefore, must take into account the potential role of a basolateral Na/K-ATPase in intercalated cell function.