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Isolation of a novel zinc finger repressor that regulates the kidney-specific CLC-K1 promoter. CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, are transcriptionally regulated on a tissue-specific basis. We have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, a kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated as KKLF (kidney-enriched Kruppel-like factor) and the myc-associated zinc finger protein (MAZ) were cloned. KKLF was found to be abundantly expressed in the liver, kidney, heart, and skeletal muscle. In the kidney, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments where CLC-K1 and CLC-K2 were not expressed. Gel mobility shift assay revealed that recombinant KKLF and MAZ proteins exhibited sequence-specific binding to the CLC-K1GA element and that the consensus sequence for the KKLF binding site was GGGGNGGNG. In transient transfection, MAZ had a strong activating effect on the CLC-K1–luciferase reporter gene transcription. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue and nephron segment-specific expression of CLC-K1 and CLC-K2 channel genes through their GA cis element.

CLC-K1, a kidney-specific chloride channel, has been demonstrated to be involved in the urine concentration mechanism. Here, we investigated the developmental expression of CLC-K1 in the rat kidney. Using immunohistochemistry, we showed that CLC-K1 was not present in the thin ascending limb of Henle's loop during the early prenatal stages but was significantly expressed during the adult stage. CLC-K1 started to appear at day 5 and its expression increased during further development. In developing rats this increase coincided with the increase in the urine-concentrating capacity as the animals matured. We also investigated the expressions of other channels and transporters, including NKCC2, AQP-1, and AQP-2. NKCC2 was strongly expressed throughout the inner medulla in neonatal rat kidneys but was entirely undetectable at the adult stage. The decline in its expression took the form of a gradual recession from the inner medulla together with reciprocal increases in the expression of CLC-K1. AQP-1 was weakly expressed in the inner medulla during early development and showed a rapid increase in expression at a later stage. The collecting duct cells significantly expressed AQP-2 even at birth and maintained its expression throughout the development. These results suggest that CLC-K1 expression is one of the major determinants of the urine-concentrating capacity of the developing rat kidney.

A 58-year-old man developed proteinuria and renal dysfunction following pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin was administered, and prednisolone pulse therapy and plasmapheresis were performed. Subsequently, serum creatinine was decreased. Eight months later, creatinine and CRP were again elevated, and MRSA was detected. Vancomycin was again administered and plasmapheresis was performed. However, renal function was not improved and continuous hemodialysis was initiated. This case indicates that complete eradication of MRSA is necessary to treat MRSA-associated glomerulonephritis, and if this is not attained, a permanent loss of renal function occurs.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family of growth factors, is synthesized as a membrane-an- chored precursor (proHB-EGF) that is capable of stimulating adjacent cells in a juxtacrine manner. ProHB-EGF is cleaved in a protein kinase C-dependent process, to yield the soluble form. It was observed that HB-EGF acts as a morphogen for the collecting duct system in developing kidneys. HB-EGF protein was expressed in the ureteric bud of embryonic kid- neys. Cultured mouse ureteric bud cells (UBC) produced HB- EGF via protein kinase C activation. After stimulation with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) or re- combinant soluble HB-EGF, UBC cultured in three-dimen- sional collagen gels formed short tubules with varied abundant branches. When proHB-EGF-transfected UBC were stimulated with 12-O-tetradecanoylphorbol-13-acetate and cultured in collagen gels, they exhibited linear growth, forming long tu- bular structures with few branches at the time of appearance of proHB-EGF on the cell surface. The structures exhibited a strong resemblance to the early branching ureteric bud of embryonic kidneys. When UBC were cultured in the presence of transforming growth factor-b and soluble HB-EGF, they formed long tubules and few branches, similar to the structures observed in proHB-EGF-transfected UBC. These cells exhib- ited apical-basolateral polarization and expression of the water channel aquaporin-2. These findings indicate that soluble HB- EGF and proHB-EGF induce branching tubulogenesis in UBC in different ways. Juxtacrine activation by proHB-EGF or the synergic action of soluble HB-EGF with transforming growth factor-b is important for well balanced morphogenesis of the collecting duct system. The initial events in the process of mammalian kidney devel- opment are characterized by proliferation of ureteric bud cells (UBC) and tunneling of these cells into the surrounding undif- ferentiated mesenchyme (1-3). The ureteric bud forms branch- ing tubules, with subsequent differentiation into the collecting ducts. Simultaneously, cells of the metanephric mesenchyme differentiate into epithelial cells that eventually develop into nephrons. These processes can be affected by several locally produced growth factors, including epidermal growth factor (EGF), transforming growth factor-a (TGF-a), hepatocyte growth factor (HGF), glial cell line-derived neutrophic growth

CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.

Liddle's syndrome is a rare form of hereditary hypertension caused by mutations of the epithelial sodium (Na(+)) channel (ENaC). Analysis of the diseased pedigrees indicates an autosomal dominant inheritance, and the identified mutations are heterozygotes of gain-of-function mutations. However, sporadic cases of Liddle's syndrome have been reported in the literature, including one recently reported case caused by a de novo mutation of ENaC. We identified two patients with Liddle's syndrome who did not have family histories of hypertension. Sequence analysis showed a mutation in each case (P616L in betaENaC and W576X in gammaENaC), both confirmed to be de novo mutations. These data indicate that Liddle's syndrome should be considered even in patients without a family history of hypertension.

It has been reported that deposition of β2-microglobulin (BMG) is associated with the occurrence of dialysis-related amyloidosis (DRA) in long-term hemodialysis (HD) patients. Though reduction of the BMG burden is essential in preventing DRA, simple BMG kinetic models applicable to clinical practice have not been established. We have reported a modified variable-volume one-compartment model (1CM) for analyzing urea nitrogen (UN) kinetics, in which no specific parameters other than a modification factor are necessary. If there is a constant relation between the BMG concentration of the dialyzer arterial line and the mean BMG concentration in the body during HD, the modified 1CM may also be applied to BMG. As such, in order to verify its validity, we analyzed UN and BMG kinetics by the modified 1CM in 28 HD patients in whom polysulfone dialyzers were used, and compared the calculated and measured solute rebound. In 3 of the patients, the spent dialysate was collected in a tank, and the BMG removal mass, calculated by the modified 1CM, was compared with that recovered in the tank. The BMG rebound ratio (%), calculated by the modified 1CM, was not different from the measured one (36.3 ± 9.9 vs. 36.5 ± 11.5, p = 0.954), as in the case of UN (15.8 ± 4.5 vs. 16.3 ± 4.6, p = 0.541). The solute dilution in the blood circuit by solute disequilibrium and blood recirculations for BMG was estimated to be 74% stronger than that for UN. The normalized solute generation rate (mg/min/l) and the time-averaged solute concentration over a 1-week period (mg/l) were 0.142 ± 0.023 and 448 ± 68 for UN and 0.00578 ± 0.00125 and 22.4 ± 4.6 for BMG, respectively. The differences between these two solutes resulted in a Dilution index for BMG that was 23% lower than that for UN (2.62 ± 0.44 vs. 3.21 ± 0.39, p < 0.0001). The modified 1CM overestimated the BMG removal mass (mg) by about 20% compared with that recovered in the tank (195 ± 22 vs. 162 ± 17, p < 0.0001). One of the causes of this discrepancy was speculated to be adsorption of BMG by polysulfone dialyzers. It was concluded that, despite several problems in quantitative analysis, the modified 1CM would be a useful model for estimating the dialysis efficiency for BMG removal in clinical practice.

The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1−/− mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1−/− mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/− and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1−/− mice. These results indicate that the polyuria observed in Clcnk1−/− mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1−/− mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1−/− mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1−/− mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1−/− mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1−/− mice. We concluded that NDI in Clcnk1−/− mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems.

We treated a female patient suffering from immunoglobulin A (IgA) nephropathy and congenital deficiency of the ninth component of the complement system (C9). She was admitted with hematuria and proteinuria, and the C9 deficiency was diagnosed based on the low hemolytic activity of 50 % of the hemolytic unit of the complements (CH50) and the normal C3 level in the plasma. Renal biopsy revealed mild mesangial proliferation, and immunofluorescence examination revealed mild mesangial deposits of IgA and C3 with the same distribution. We discuss the pathogenesis of IgA nephropathy and the role of the complements in its progression.

Impaired mobility at the onset of dialysis is considered one of the most important risk factors for short-term mortality after initiation of dialysis in elderly patients. However, whether a decline in mobility after starting dialysis also affects mortality is unclear.A total of 202 patients (age,75 years; mean, 80.4 ± 4.3) were enrolled in this retrospective cohort study in Yokosuka, Japan. They were divided into three subgroups by mobility: independent mobility at onset of dialysis and preservation of mobility after starting dialysis (group 1, n = 104); independent mobility at onset of dialysis and decline in mobility after starting dialysis (group 2, n = 48); and impaired mobility at onset of dialysis (group 3, n = 50). They were followed for 6 months after starting dialysis. A Cox proportional hazards model was used to evaluate the association between mobility and mortality.A total of 24.8% of patients had impaired mobility at the start of dialysis, and 68.9% declined in mobility after starting dialysis. In multivariate Cox proportional hazards analysis, the adjusted hazard ratios of groups 2 and 3 compared with group 1 were 3.80 (95% confidence interval, 1.02-14.10) and 4.94 (95% confidence interval, 1.42-17.10), respectively.Not only impaired mobility at the start of dialysis but also a decline in mobility after starting dialysis is associated with short-term mortality after initiation of dialysis.

A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.

CLC-K1 and CLC-K2 are two kidney-specific members of the CLC chloride channel family (1, 35). Both are present in the plasma membranes of tubular cells in the kidney (36, 37), and it has been speculated that both serve as routes for transepithelial chloride transport. Mutations of CLCNKB (the human homologue of rat CLC-K2) were recently found in patients with Bartter's syndrome (30), and the CLC-K1 gene knockout in mice results in nephrogenic diabetes insipidus (21), confirming the important role of these channels in chloride transport in the kidneys. Although the two clones are highly homologous (80% amino acid identity in the rat sequence and 90% in the human sequence), their intrarenal localizations are completely different (39). Accordingly, the analysis of transcriptional regulation of these two genes is expected to elucidate mechanisms of kidney-specific and nephron segement-specific gene expression. To this end, we previously isolated the promoters of the rat CLC-K1 (34) and CLC-K2 (26) genes. Surprisingly, proximal 5′-flanking regions that include the transcriptional start sites are highly homologous and characterized by a GA element, GGGGAGGGGAGGGGGAGGG (26). Reporter gene assays and gel retardation assays (26, 34) revealed that this GA element is indispensable for the basal promoter activities of both genes, suggesting that one or more proteins binding to this element may be involved in the kidney-specific expression of the CLC-K1 and CLC-K2 genes. In the present study, we isolated two cDNAs that bind to the GA element, i.e., MAZ, the previously isolated myc-associated zinc finger protein, and KKLF, a novel kidney-enriched Kruppel-like factor. MAZ and KKLF have opposite effects on the CLC-K1 promoter activity, suggesting that the kidney-specific expression of CLC-K genes may be regulated by a series of zinc finger proteins through the GA element. The spatial pattern of KKLF expression overlapped with negative expressions of CLC-K1 and CLC-K2 in the kidneys, supporting the idea that KKLF may contribute to the strict nephron segment-specific expression of the CLC-K genes in vivo. Furthermore, we also found that KKLF repressed the promoter activity of the α2(I) collagen gene. Given the localization of KKLF in interstitial fibroblasts in cardiac and skeletal muscle and in potentially fibrogenic cells such as the mesangial cells in the kidneys or stellate cells in the liver, it is reasonable to assume that KKLF may be involved in the fibrogenesis in these organs. In a unilateral ureteral obstruction (UUO) model of mouse kidney, a well-characterized model of progressive tubulointerstitial fibrosis, a rapid decrease of KKLF and subsequent increase of α2(I) collagen expression were observed, suggesting that KKLF is involved in type I collagen synthesis and tissue fibrosis.

We determined alterations in renal aquaporin-2 (AQP2) gene expression in association with impaired water excretion in glucocorticoid-deficient rats. After adrenalectomy, Sprague-Dawley rats were administered aldosterone alone by osmotic pumps (glucocorticoid-deficient rats). As a control, both aldosterone and dexamethasone were administered. These animals were subjected to the studies on days 7–14. The expressions of AQP2 mRNA and protein in kidney of the glucocorticoid-deficient rats were increased by 1.6- and 1.4-fold compared with the control rats, respectively. An acute oral water load test verified the marked impairment in water excretion in the glucocorticoid-deficient rats. One hour after the water load, the expressions of AQP2 mRNA and protein were significantly reduced in the control rats, but they remained unchanged in the glucocorticoid-deficient rats. However, there was no alteration in [3H]arginine vasopressin (AVP) receptor binding and AVP V2receptor mRNA expression in the glucocorticoid-deficient rats. A V2-receptor antagonist abolished the increased expressions of AQP2 mRNA and protein in the glucocorticoid-deficient rats. These results indicate that augmented expression of AQP2 participates in impaired water excretion, dependent on AVP, in glucocorticoid deficiency.

Genes of the IL-1 family encode three different peptides, IL-1alpha, IL-1beta, and IL-1Ra, respectively. IL-1 operates through IL-1RI, and is involved in airway inflammation in asthmatic subjects, whereas IL-1Ra appears to be a specific competitive inhibitor of IL-1. All genes are on chromosome 2q12-21 where genomewide searches have identified linkage for asthma. To test whether variants of IL-1 relate to asthma, we conducted a genetic association study in a Japanese population. We show that the A2 allele of IL1RN (encoding IL-1Ra) associates with nonatopic asthma [OR = 5.71, 95% CI: 1.63-19. 8, Pc = 0.007]. Both atopic and nonatopic asthmatics with the A2 allele had significantly lower serum IL-1Ra levels in both types of asthmatics. Peripheral blood cells from asthmatics with A2 alleles, however, produced as much IL-1 as did those with A1 homozygotes. Since Th1 and Th2 cytokines differentially regulate the ratio between IL-1beta and IL-1Ra, these findings suggest that dysregulation of IL-1beta/IL-1Ra, probably due to interaction between epithelium and immuno-competent cells in the airway, is important in asthma inflammation.

The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression was demonstrated at the tail of late spermatids [Ishibashi et al: J Biol Chem 1997;272:20782–20786]. AQP7 is also expressed in the kidney. The localization of AQP7 in the kidney is unknown. We examined the cellular localization of AQP7 in the kidney with Northern blot, reverse transcribed PCR, Western blot and immunohistochemistry in the rat kidney. In Northern blot, AQP7 was expressed in the cortex and the outer medulla but absent in the inner medulla of the kidney. Reverse transcribed PCR of rat nephron segments revealed the selective expression of AQP7 at the proximal straight tubules (PST). Western blot of the membrane fraction of outer medulla revealed a single band of ∼33 kDa. Immunohistochemistry of the rat kidney showed the selective expression of AQP7 at the brush border membranes of PST (S3 segment). AQP7 is now shown to be localized selectively at the brush border membranes of PST in the rat kidney. The result suggests that AQP7 may function as a pathway for transcellular water transport in PST in concert with more widely expressed AQP1 in proximal tubules. Alternatively, as AQP7 transports urea as well as water, AQP7 may function as a passive urea secretory pathway in this segment and may play a role in the formation and/or maintenance of the medullary urea concentration gradient.

Activated STAT1 suppresses proliferation of cultured rat mesangial cells. Background JAK-STAT signaling has been shown to promote development and proliferation in lymphopoietic and hematopoietic lineages. We investigated the effect of activated STAT1 on mesangial cell proliferation. Methods Rat mesangial cells of primary culture (rMCs) were used in the following experiments: ( 1 ) Whole cell lysates were immunoblotted against JAK1 and JAK2. ( 2 ) Whole cell lysates and nuclear proteins were extracted from rMCs with or without treatment with interferon-γ, and immunoblotting was performed against both STAT1 and tyrosine (701)-phosphorylated STAT1. ( 3 ) rMCs and rMCs electroporated with either wild-type STAT1, mutated STAT1, or antibody against STAT1 were incubated with interferon-γ for 20 hours, followed by a further incubation with [ 3 H]-thymidine for four hours. Results JAK1, JAK2, and STAT1 were detected in whole cell lysates, suggesting that JAK-STAT signaling could be activated by interferon-γ (INF-γ). Using an antibody specific for tyrosine-phosphorylated STAT1, we detected signal in the INF-γ–treated nuclear extracts, which showed translocation of phosphorylated STAT1 to the nucleus. [ 3 H]-thymidine incorporation in the presence of INF-γ was significantly lower than that of control in a dose-dependent manner. The introduction of wild-type STAT1 enhanced the effect of interferon-γ and decreased [ 3 H]-thymidine incorporation, whereas tyrosine-mutated (Y701F) STAT1 and SH2 domain (R602T)-mutated STAT1 reversed INF-γ–induced suppression of [ 3 H]-thymidine incorporation. Electroinjected antibody against STAT1 increased [ 3 H]-thymidine incorporation upon stimulation with INF-γ. Conclusion STAT1 activated by interferon-γ suppresses mesangial cell proliferation.

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.

We observed severe overhydration in an 18-month-old Japanese girl with primary polydipsia. The secretion of antidiuretic hormone (ADH) was decreased, and urinary excretion of aquaporin-2, a vasopressin-sensitive water channel protein, was suppressed under basal conditions, but the response of aquaporin-2 to ADH was essentially preserved. These findings suggest that the water channel itself was intact and that overhydration resulting from polydipsia was responsible for the decreased ADH secretion and suppression of the water channel protein.

In order to demonstrate the localization of an ethacrynic acid-sensitive Cl− pump in the rat kidney, immunohistochemical analysis was performed using an anti-Cl− pump antibody raised against rat brain Cl− pump protein with confocal laser scanning microscopy. The antibodies against Na+,K+-ATPase, aquaporin 2 and a type B intercalated cell marker, 43-kDa protein, were also used for comparison. Anti-Cl− pump antibody recognized a 51-kDa renal protein of the same size as that in the brain on Western blots. Cl−-pump-like immunoreactivity was observed on the basolateral membranes of 42 ±3% of cortical collecting duct (CCD) cells and of 38 ± 1% of outer medullar collecting duct (OMCD) cells. Such immunoreactivity in CCD was sometimes co-localized with Na+,K+-ATPase, but in OMCD, the Cl− pump-like immunoreactivity co-existed with neither Na+,K+-ATPase, aquaporin 2 nor the type B intercalated cell marker 43-kDa protein. Thus, the Cl− pump was demonstrated to be localized on the basolateral membranes of type A intercalated cells of cortical and medullary collecting ducts in the rat kidney.

Roles of E2F1 in mesangial cell proliferation in vitro. Background The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. Methods To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. Results E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. Conclusions In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins.

ClC-4 gene was isolated as a putative Cl−channel. Due to a lack of functional expression of ClC-4, its physiological role remains unknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletal muscles and stably transfected it to Chinese hamster ovary cells. Whole cell patch-clamp studies showed that the hClC-4sk channel was activated by external acidic pH and inhibited by DIDS. It passed a strong outward Cl−current with a permeability sequence of I−> Cl−> F−. The hClC-4sk has consensus sites for phosphorylation by protein kinase A (PKA); however, stimulation of PKA had no effect on the currents. hClC-4sk mRNA was expressed in excitable tissues, such as heart, brain, and skeletal muscle. These functional characteristics of hClC-4sk provide a clue to its physiological role in excitable cells.

Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated protein kinase cascade. Mitogen-activated protein kinases (MAPKs) have been shown to play an important role in transducing extracellular signals into cellular responses. The classic MAPK pathway is commonly activated by growth factors and has been shown to play a crucial role in cell proliferation. Transforming growth factor-β (TGF-β)–activating kinase-1 (TAK1) is a novel MAPK kinase kinase that is reported to stimulate the MKK6-p38K pathway. To elucidate the functional roles of the TAK1 pathway, we transfected its constitutive active form (TAKdN) and negative form (TAKK63W) to LLC-PK1 cells. TAKdN stimulated MKK6 phosphorylation and p38K activity and inhibited the percentages of the S and G2/M phases. TAKK63W, the constitutive negative form, reduced TGF-β–stimulated MKK6 phosphorylation and p38K activity and increased the percentages of the S and G2/M phases. The cyclin D1 protein level is reduced by the TAK1 pathway. We also examined the effects of the TAK1 pathway on cyclin D1 promoter-luciferase assay. The overexpression of TAKdN or p38K inhibited cyclin D1 promoter activity. In contrast, overexpression of the active form of MKK1, the classic MAPK-activator, MKK1 increased cyclin D1 promoter activity and protein level, as well as the percentages of S and G2/M phases.

Cell cycle inhibitors (p27 Kip1 and p21 CIP1 ) cause hypertrophy in LLC-PK 1 cells. Background Angiotensin II has been reported to induce renal tubular hypertrophy, but the mechanisms of this hypertrophy are not well known. We evaluated the roles of cyclin-dependent kinase (CDK) inhibitors in renal tubular hypertrophy. Methods To elucidate whether CDK inhibitors cause renal tubular hypertrophy, we produced adenovirus vectors containing coding sequences of the CDK inhibitors p27 Kip1 (AxCAp27), p21 CIP1 (AxCAp21), and p16 INK4 (AxCAp16), and we investigated the effect of these gene transfers on epidermal growth factor (EGF)-induced proliferation in LLC-PK 1 cells. We evaluated the cell cycle and hypertrophy by measurements of the [ 3 H]-leucine and [ 3 H]-thymidine incorporation, the protein:DNA ratio, flow cytometry, and CDK4 and CDK2 kinase assays. Results AxCAp27 and AxCAp21 caused significant increases in [ 3 H]-leucine incorporation and the protein:DNA ratio but did not change the [ 3 H]-thymidine incorporation. Conversely, AxCAp16 inhibited EGF-stimulated [ 3 H]-thymidine incorporation but did not change the [ 3 H]-leucine incorporation. AxCAp27, AxCAp21, and AxCAp16 all inhibited EGF-stimulated CDK4 kinase activity (to 15.6, 14.1, and 21.9% of control, respectively). Forward light-scatter analysis demonstrated that AxCAp27 and AxCAp21 increased the cell size but that AxCAp16 effected no change in cell size. Conclusion These findings suggest that p27 Kip1 and p21 CIP1 may play an important role in hypertrophy of renal tubule cells by reducing pRb phosphorylation. On the other hand, p16 INK4 was not found to cause hypertrophic changes in EGF-treated LLC-PK 1 cells.

A new aquaporin was isolated from rat liver based on homology to known aquaporins. A 1408 bp cDNA was sequenced (designated rAQP9L) with a 885 bp open reading frame encoding a 295 amino acid hydrophobic protein. rAQP9L has the greatest amino-acid sequence identity with human AQP9 (75%) and a less homology with AQP3 (49%) and AQP7 (47%). Northern blot analysis indicated a 1.4-kb transcript expressed strongly in liver > testis > brain = lung. Expression of rAQP9L cRNA in Xenopus oocytes increased osmotic water permeability by 6-folds which was inhibited by 0.3 mM mercury chloride by 42%. rAQP9L also facilitated glycerol and urea transport by 2- and 5-folds, respectively. The large discrepancy of tissue distribution between hAQP9 and rAQP9L suggest that rAQP9L is a new aquaporin, which is involved in transport of urea as well as water in liver.

最近10年間にチャネル蛋白の遺伝子異常に基づく疾患が続々と同定され,チャネル病(channelopathy)と呼ばれるようになっている.チャネルが神経・筋の刺激伝導の主役であることを反映して,チャネル病には神経・筋疾患が多いが,心臓のQT延長症候群や腎の水・電解質疾患も含まれる.分子生物学により臨床と基礎の垣根が一挙に消失することがあるが,チャネル病はその典型例である.チャネル病解析から,障害されている遺伝子に基づく疾患分類の大切さが浮かび上がってきた.また遺伝子変異の種類から,コードされているチャネル蛋白の溝造・機能共関についての示唆を得ることもできた.治療に対する試みは始ったばかりであるが,今後の進展が期待できる.

CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) in the inner medulla 1, 2 . Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system 3, 4, 5 , but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo , we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1 –/– mice were physically normal appearance, but produced approximately five times more urine than Clcnk1 +/– and Clcnk1 +/+ mice. After 24 hours of water deprivation, Clcnk1 –/– mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1 +/– and Clcnk1 +/+ mice, whereas only a minimal increase was seen in Clcnk1 –/– mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1 –/– mice in contrast to Clcnk1 +/+ and Clcnk1 +/– mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.

The cDNA and the gene of human aquaporin8 (AQP8) were cloned from human testis cDNA and a genomic library, respectively. The AQP8 cDNA encodes 261 amino acids. The identity of the amino acid sequence to other aquaporins is highest with a plant water channel, gamma-TIP (40.4%), while AQP2 and AQP3 are 28.9 and 29.5% identical to human AQP8, respectively. The human AQP8 is only 74.9% identical to rat AQP8 and 76.0% identical to mouse AQP8. In Northern blot analysis, approximately 1.35-kb human AQP8 mRNA was expressed in pancreas and colon, but not in other tissues. Absence of human AQP8 in testis is noteworthy as rat AQP8 was abundantly expressed in testis. The expression of human AQP8 cRNA in Xenopus oocytes increased osmotic water permeability by 10-fold. AQP8 was not permeable to urea nor to glycerol. The AQP8 gene has five introns, and the locations of exon-intron boundaries were different from those of the other mammalian aquaporins, suggesting its separate phylogenetic origin.

▪ Abstract Since the molecular identification of the first aquaporin in 1992, the number of proteins known to belong to this family has been rapidly increasing. These members may be separated into two subgroups based on gene structure, sequence homology, and function. Regulation of the water permeability of the collecting ducts of the kidney is essential for urinary concentration. Aquaporin-2 and -3, which are representative of these subgroups, are colocalized in the collecting ducts. Understanding these subgroups will elucidate the differences between aquaporin-2 and -3. Aquaporin-2 is a vasopressin-regulated water channel located in the apical membrane, and aquaporin-3 is a constitutive water channel located in the basolateral membrane. In contrast to aquaporin-3, which appears to be less well regulated, many studies have now identified multiple regulational mechanisms at the gene, protein, and cell levels for aquaporin-2, thus reflecting its physiological importance. Evidence of the participation of aquaporin-2 in the pathophysiology of water-balance disorders is accumulating.

Recent studies have shown that mitogen-activated protein kinase (MAPK) consists of at least 3 subfamilies, including the classical MAPK, stress-activated protein kinase/c-Jun N-terminal kinase, and p38 kinase. Transforming growth factor (TGF)-β-activating kinase-1 (TAK1) is a novel MAP kinase kinase (MAPKK) that is reported to stimulate the p38 kinase and/or c-Jun N-terminal kinase pathway. TAKdN, the active form of TAK1, inhibits [3H]-thymidine uptake, and reduces the percentages of cells in the S and G2/M phases of the cell cycle. TAKK63W, the negative, or inactive form of TAK1, ameliorates the inhibition of3H-thymidine uptake and the percentages of cells in S and G2/M phases induced by TGF-β. Overexpression of TAKdN inhibits cyclin D1 gene promoter activity and protein expression. In contrast, constitutive active MKK1, the classical p42/44 MAPK activator, increases cyclin D1 promoter activity and protein level. Overexpression of the active form of MKK1 increases [3H]-thymidine uptake, while the inactive form decreases the uptake. To elucidate the mechanisms of the cell cycle of mesangial cells, we produced adenovirus vectors containing the coding sequences of cyclin D1 (AxCAD1), p16INK4 (AxCAp16), and p21CIP1 (AxCAp21), and investigated whether transfer of these genes changes platelet-derived growth factor-and endothelin-1-induced proliferation of rat mesangial cells. AxCAp16 and AxCAp21 inhibits [3H]-thymidine incorporation and the mitogen-induced increase in cyclin-dependent kinase-4 activity, and reduces the percentage of cells in the S phase as well. Overexpression of cyclin D1 increases the percentage of the cells in the S and G2 phases, and reduces cell size. These findings suggest that p16INK4 and p21CIP1 function as inhibitors of the proliferation of mesangial cells, induced by growth-promoting factors, and that deregulated expression of cyclin D1 causes disturbances in the cell cycle.

Stanniocalcin (STC) is a Ca- and phosphate-regulating hormone produced by the corpuscles of Stannius in bony fishes. The mammalian homologue of STC has recently been reported (STC1), which stimulates the phosphate uptake of kidney. Here we report the cloning of a second mammalian stanniocalcin (STC2) from the human osteosarcoma cDNA library. STC2 has 302 amino acid residues with 34% identity with STC1 and eel STC. STC2 has a conserved N-glycosylation site and is rich in cysteines as is the case with other stanniocalcins. STC2 has the same exon–intron boundaries as STC1. The culture medium of STC2-transfected CHO cells inhibited the promoter activity of Na-phosphate cotransporter (NaPi-3) and also inhibited the phosphate uptake of a kidney cell line (OK cells). Therefore, the function of STC2 seems to be opposite to that of STC1 on Na-phosphate cotransporter. Northern blot analysis revealed multiple transcripts in number of human tissues with high levels being present in skeletal muscle and heart. STC2 was also expressed in mice widely and its expression was lower in hypophosphatemic mice (Hypmice) in many organs. We have cloned a widely expressed new human stanniocalcin homologue which suppressed the expression of renal Na-phosphate cotransporter.

Chronic renal failure due to granulomatous interstitial nephritis in sarcoidosis is a rare phenomenon, and its response to corticosteroid therapy is not well known. We report a patient with sarcoidosis who presented with chronic renal failure and hypercalcemia, but who did not exhibit nephrocalcinosis. Renal histology findings showed the presence of noncaseating granuloma and heavy interstitial nephritis. Although hypercalcemia was remarkably improved by corticosteroid therapy, chronic renal failure, due to interstitial fibrosis and scarring, remained unchanged. This case reinforces evidence supporting the effectiveness of corticosteroid therapy in granulomatous interstitial nephritis of sarcoidosis, and suggests the need for early initiation of the therapy to avoid permanent renal dysfunction.

Novel mutations of the aquaporin-2 (AQP2) gene have been detected in Japanese female siblings with autosomal-recessive nephrogenic diabetes insipidus. The patients were compound heterozygote for point mutations at nucleotide position 374 (C374T) and at position 523 (G523A) in exon 2 of the AQP2 gene, resulting in substitution of methionine for threonine at codon 125 (T125M) and arginine for glycine at codon 175 (G175R). The water permeability (Pf) of oocytes injected with wild-type complementary RNA increased 9.0-fold compared with the Pf of water-injected oocytes, whereas the increases in the Pf of oocytes injected with T125M and G175R complementary RNA were only 1.7-fold and 1.5-fold, respectively. Immunoblot and immunocytochemistry indicated that the plasma membrane expressions of T125M and G175R AQP2 proteins were comparable to that of the wild-type, suggesting that although neither the T125M nor G175R mutation had a significant effect on plasma membrane expression, they both distorted the structure and function of the aqueous pore of AQP2. These results provide evidence that the nephrogenic diabetes insipidus in patients with T125M and G175R mutations is attributable not to the misrouting of AQP2, but to the disrupted water channel function.

Our understanding of the cell-cycle mechanisms has progressively advanced in the past few years. Cyclins and cyclin-dependent kinases play major roles as positive cell-cycle regulatory proteins and CDK inhibitors; while the Kip family and INK4 family are negative regulatory proteins in mesangial cells and renal tubular cells. An understanding of the cell cycle is essential for the rational design of novel pharmacotherapeutic approaches to suppress the excessive proliferation of mesangial cells in glomerular disease and hypertrophic tubular disease.

The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression demonstrated at the tail of late spermatids (Ishibashi et al., J. Biol. Chem. 272 (1997) 20,782-20,786). Here we report the isolation of the mouse and the human AQP7 cDNA and the human AQP7 gene. The human AQP7 gene is identical with human adipose AQP (AQPap or AQP7L). The deduced amino acid sequences of human and mouse AQP7 were 68% and 79% identical to those of rat AQP7, respectively. The mouse AQP7 is 67% identical to the human AQP7. Such a lower conservation of AQP7 among species is unusual in the aquaporin family. The human AQP7 gene is composed of six exons distributing over 6.5 kb. The exon-intron boundaries are identical to those of the human AQP3 gene. The intron sizes are also similar. Moreover, chromosomal localization of AQP7 was assigned to 9p13 by fluorescent in situ hybridization, where AQP3 is also localized, suggesting that 9p13 may be another site of an aquaporin cluster.

MIP family proteins can be divided into two groups according to their primary sequences. The CHIP group is predominant in the plant and animal kingdoms and functions primarily as water channels. The GLP group is a minor group with limited prevalence and functions primarily as glycerol transporters. Both prototypes are present in bacteria and may have evolved separately.

Mutations in the CLCN5 gene have been demonstrated in three disorders of hypercalciuric nephrolithiasis, i.e., Dent's disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphatemic rickets. Recently, a number of Japanese children with low molecular weight proteinuria (LMWP) showing symptoms similar to those shown by patients with Dent's disease in British families have also been reported to have mutations in the CLCN5 gene. The present study examines five unrelated Japanese families with LMWP, two of which lacked any signs other than LMWP, and three of which had several signs other than LMWP, i.e., hypercalciuria, aminoaciduria, hypophosphatemia, and rickets. One nonsense (E118X) and one missense (W22G) mutation were found in three patients in the two families having only LMWP. One genomic deletion including exons 5 to 8 in the CLCN5 gene was found in a patient with hypophosphatemic rickets, and a nonsense mutation (R347X) was found in one patient with LMWP and slight hypercalciuria. No mutations of the exons and exon-intron boundaries in the CLCN5 gene were found in one patient with LMWP, aminoaciduria, and hypokalemia. In addition to the predicted loss of chloride channel function in these nonsense and deletion mutations, the loss of function in the missense mutation W22G was confirmed in the Xenopus oocyte expression system. These results clarified four novel mutations in the CLCN5 genes, and additionally suggested that the loss-of-function mutation of the CLCN5 does not necessarily lead to hypercalciuria and nephrocalcinosis in the early stage of the disease, and that LMWP is an early and essential manifestation of disorders of the CLC-5 chloride channel.

Aquaporin (AQP) is the water channel protein associated with water-permeability, in which AQP1, AQP3, AQP4, and AQP5, are expressed in respiratory organs. Because water filling the fetal lung has to be absorbed after birth, aquaporins are postulated to contribute to this water reabsorption. To examine the potential role of AQP3 around birth, we studied mRNA expression and localization of AQP3 in fetal and postnatal rat lung. AQP3 mRNA was most prominently expressed in rat lung on the day of birth, began to decrease on the 2nd day after birth, and was not observed on the 4th day and later. In situ hybridization demonstrated that AQP3 mRNA was expressed in alveolar epithelium. The expression of AQP3 mRNA may influence water reabsorption of the perinatal rat lung.

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle's loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5'-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the approximately 2.5-kb pair 5'-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7-to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the approximately 2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5'-flanking region but was lost in the -29 construct, clearly demonstrating that the 22 bp from -51 to -30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGG-GAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.

Using 196 serum samples obtained from both children patients who showed allergic symptoms and normal donors, the usefulness of LUMIWARD immunoassay system was investigated for diagnosis of allergic diseases throughout comparison with skin test and CAP RAST.On fundamental test results, excellent performances were obtained on both reproducibility tests and dilution tests. The interference substances had no effect on the measured values in the assay system. The minimum detectable concentrations were 0.04IU/ml on specific IgE antibody and 0.11IU/ml on total IgE. On 858 tests from 145 patients, the correlation of 8 allergens between LUMIWARD and CAP RAST showed high performance, that concordance rate, positive concordance rate and negative one were 91.8, 90.3, 92.8%, and the correlation coefficient was 0.916.On the clinical evaluations, LUMIWARD showed a little sensitivity and more excellent specificity than the skin test. In regard to the appearance frequency of qualitatively positive sera by age group, only egg white- and/or milk-specific IgE in food allergens were detected on the positive zone at age 0. All allergen-specific IgE had peak frequency at age 2 and decreased gradually at age 6. The IgE against cat epithelium and house dust mite increased in order of age, especially mite-specific IgE reached 70% in frequency at age 6. In the retrospective study of 6-aged patients who showed positive to egg white, seven of nine had been positive to egg white at age 0. One of residual 2 samples showed low concentration IgE (0.04-0.35IU/ml) against egg white, though it deemed to be negative. At age 0, one sample was positive and two sample had low concentration on milk-specific IgE. On mite-specific IgE, all of nine samples showed negative but four samples had low concentration of IgE.These observations suggested that LUMIWARD had the clinical usefulness in detecting IgE antibodies in allergic subjects and earlier diagnosis on childfood.

Cyclin D1, p16, and retinoblastoma gene regulate mitogenic signaling of endothelin in rat mesangial cells. To elucidate the mechanisms by which endothelin (ET)-1 induces proliferation of mesangial cells, we investigated the involvement of the first gap phase of the cell cycle (G1) cyclin, cyclin-dependent kinase 4 (CDK4) activity, and the retinoblastoma gene product (pRb) in ET-1-stimulated cell cycle progression. In the present study, ET-1 stimulated CDK4 activity and cell cycle progression via ET A-type receptors and induced cyclin D1 mRNA and protein expression in rat mesangial cells. We also found that ET-1 stimulation of mesangial cell proliferation was inhibited by antisense oligonucleotides directed against cyclin D1 and by overexpression of a nonphosphorylatable form of pRb. To investigate the functional roles of p16INK4 and p21cip1 in ET-1-stimulated mesangial cell proliferation, we used adenovirus-mediated gene transfer. Endothelin-1-stimulated [3H]-thymidine incorporation, CDK4 kinase activity, and the percent of cells in S phase were found to be significantly inhibited by overexpression of p16INK4 and slightly inhibited by overexpression of p21cip1. Thus, ET-1 induced cyclin D1 expression and stimulated CDK4 activity and cell cycle progression via the A-type receptor in rat mesangial cells. These effects were regulated by the expression of cyclin D1, p16INK4, p21cip1, and phosphorylatable form of pRb. The results of the present study provide the basis for further investigation of basic and therapeutic approaches towards mesangial proliferative diseases.

The promoters of rat and mouse aquaporin-2 (AQP-2) genes were cloned and compared with that of human genes. Nucleotide identity up to -593 bp was 62%, and consensus sequences such as TATA box and adenosine 3',5'-cyclic monophosphate responsive element were conserved. Deoxyribonuclease I footprint assay revealed a footprinted region at -210 to -184 bp in rat AQP-2 gene promoter produced by nuclear extract from nonexpressing (liver) tissue. The sequence of this region included a GATA motif but otherwise showed no homology with any other previously known cis-elements. Electromobility shift assay and ultraviolet cross-linking analysis confirmed that specific binding proteins to this element were present in kidney, spleen, and liver and that these proteins were distinct from GATA factors. Both deletion and mutation of this cis-element abolished the protein DNA binding and increased promoter activity in in vitro reporter gene assay using rat cultured hepatocyte Ac2F cells, suggesting the negative regulatory role of this cis-element. These results indicate that tissue-specific expression of AQP-2 gene may in part be regulated by this novel negative acting cis-element.

A new member of the aquaporin (AQP) family has been identified from rat testis. This gene, referred as aquaporin 7 (AQP7), encodes a 269-amino acid protein that contained the conserved NPA motifs of MIP family proteins. AQP7 has the amino acid sequence homology with other aquaporins ( approximately 30%), and it is highest with AQP3 (48%), suggesting that both AQP3 and AQP7 belong to a subfamily in the MIP family. Injection of AQP7-cRNA into Xenopus oocytes expressed a 26-kDa protein detected by immunoblotting. The expression of AQP7 in oocytes stimulated the osmotic water permeability by 10-fold which was not inhibited by 0.3 mM mercury chloride. The Arrhenius activation energy for the stimulated water permeability was low (2.1 kcal/mol). AQP7 also facilitated glycerol and urea transport by 5- and 9-fold, respectively. The activation energy for glycerol was also low (5.3 kcal/mol after the correction of the endogenous glycerol permeability of oocytes). Northern blot analysis revealed a 1.5-kilobase pair transcript expressed abundantly in testis. In situ hybridization of testis revealed the expression of AQP7 at late spermatids in seminiferous tubules. The immunohistochemistry of testis localized the AQP7 expression at late spermatids and at maturing sperms. AQP7 may play an important role in sperm function.

We determined whether alteration in urinary excretion of aquaporin-2 (UAQP-2) is of value to diagnose central diabetes insipidus (CDI). First, UAQP-2 was determined in 16 normal subjects under ad libitum water drinking (n = 6) and after an overnight dehydration (n = 10). UAQP-2 has a positive correlation with plasma arginine vasopressin (AVP) levels (r = 0.61, P < 0.05) but not with urinary osmolality (Uosm). Second, a hypertonic saline (5% NaCl)-infusion test was studied in 5 normal subjects (21 to 25 yr old) and 10 patients with CDI (22–68 yr). After drinking water ad libitum, they were given 20 mL/kg water orally and then given 5% NaCl (0.05 mL/kg·min) iv for 120 min. Finally, 0.1 U of AVP was administered iv. During the period, 30-min urine collections were made. In the normal subjects, after the infusion of 5% NaCl, plasma AVP levels and Uosm markedly increased in parallel with an increase in plasma osmolality (Posm, 294–320 mOsm/kg H2O; Uosm, 102–737 mOsm/kg H2O; AVP, 0.4–2.6 pg/mL, P < 0.001). In t...

To evaluate the functional role of GATA motifs in the 5′-flanking region of a kidney-specific AQP-2 water channel gene, we sought to isolate a GATA factor(s) expressed in collecting ducts and determined the role on the AQP-2 promoter. Two cDNAs encoding GATA factors were isolated from rat kidney, whose sequences were highly homologous with human GATA-2 and -3. Reverse-transcription PCR using dissected nephron segments revealed that rat GATA-3 but not GATA-2 was expressed in collecting ducts, thus indicating that GATA-3 could interact with GATA motifs in the AQP-2 promoter. Transactivation experiments utilizing the rat GATA-3 expression vector indicated that rat GATA-3 increased the AQP-2 promoter activity about fourfold. These results indicated that GATA motifs in the 5′-flanking region of the hAQP-2 gene were functional cis-elements and that GATA-3 in collecting ducts may be one of the important regulators of AQP-2 expressioninvivo.