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A new member of water channels has been identified from rat testis. This gene, termed aquaporin 8 (AQP8), encoded a 263-amino-acid protein that contained the conserved NPA motifs of MIP family proteins. AQP8 has amino acid sequence identity with other aquaporins (approximately 35%) and highest with a plant water channel, AQP-gamma TIP (39%), suggesting that AQP8 is a unique member in mammalian aquaporins. The expression of AQP8 in Xenopus oocytes stimulated the osmotic water permeability (Pr) 8.5 folds. The increase of Pr was inhibited with 0.3 mM mercury chloride by 55%, which was reversed with mercaptoethanol. The Arrhenius activation energy for the stimulated water permeability was low (5.1 kcal/mol). AQP8 did not facilitate glycerol transport. Northern blot analysis revealed a 1.5-kb transcript of AQP8 abundantly in testis and slightly in liver. In situ hybridization of testis revealed the expression of AQP8 mRNA in all stages of spermatogenesis from primary spermatocytes to spermatids in seminiferous tubules. Together with previously cloned AQP7, AQP8 may also play an important role in spermatogenesis. The unexpected complexity of the presence of two aquaporins in testis may call for the further analysis of the role of aquaporins in the reproduction biology.

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability (Pf) of LLC-PK1 cells transfected with WT was 69.6 +/- 6.5 microm/s (24.8 +/- 2.2 microm/s for mock-transfected), and stimulation by 500 microM 8-(4-chlorophenylthio)-cAMP increased the Pf by 85 +/- 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the Pf was only 8 +/- 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 +/- 10%, significantly less than that for WT (88 +/- 5%). In addition, an in vivo [32P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.

Aquaporin-2 (AQP-2) water channel is a key molecule for urinary concentration whose expression is augmented by dehydration in vivo. To elucidate the regulatory mechanism of this phenomenon in vitro, mouse collecting duct cell lines were established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T antigen gene and then screened for the AQP-2 expression, using ribonuclease protection assay. In one cell line designated C4, the endogenous AQP-2 mRNA level measured by ribonuclease protection assay increased fourfold after treatment with chlorophenylthio-cAMP (cpt-cAMP) (400 microM). In contrast, phorbol 12-myristate 13-acetate did not affect the AQP-2 mRNA level. To identify the molecular mechanism(s) of cAMP-induced upregulation of AQP-2 mRNA in C4 cells, luciferase assay was performed using various 5'-flanking regions of the human AQP-2 gene. Luciferase activity in C4 cells transfected with constructs containing approximately 2.8-kbp or 224-bp 5'-flanking region showed a 3.5-fold increase by cpt-cAMP treatment, indicating that the 224-bp 5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms. This region contains cAMP-responsive element (CRE), and the deletion of the core sequence of CRE (GACGTCA) or introduction of mutation into CRE (GTGGTCA) completely abolished the responsiveness to cpt-cAMP, confirming the key role of CRE in the cAMP-induced transcriptional activation of the AQP-2 gene. Electrophoretic mobility shift assay revealed the existence of proteins binding to CRE in C4 cells and in rat kidney. The binding of CRE proteins to CRE was increased in the nuclear extract from cpt-cAMP-treated C4 cells and dehydrated rat kidney compared with those from controls. These results demonstrated that the CRE in the AQP-2 gene promoter is a key cis-element for cAMP-mediated transcriptional regulation of this gene and may be important for in vivo regulation of AQP-2 expression in a dehydrated state.

This study investigated the mRNA expression of the platelet-derived growth factor (PDGF) A-chain and B-chain and PDGF-beta receptor in glomeruli of 15 immunoglobulin A (IgA) nephropathy kidneys and those with minimal-change lesion (N = 7), membranous nephropathy (N = 3), and focal segmental glomerulonephritis (N = 5), by using competitive RT-PCR methods. The level of PDGF B-chain and beta receptor mRNA expression in IgA nephropathy was significantly higher than in the other forms of glomerulonephritis, but mRNA expressions of PDGF A-chain were not significantly different. Significant correlations were observed between the urinary protein level and the mRNA level of PDGF-beta receptor expression and PDGF B-chain expression, and between the serum creatinine level and the mRNA level of PDGF-beta receptor expression. The PDGF B-chain and beta-receptor may be upregulated and accelerate cell proliferation in a paracrine or autocrine manner and may play a role in the pathogenesis of IgA nephropathy.

Members of the membrane intrinsic protein (MIP) family are expressed in various organisms including plants, insects, and vertebrates. E. coli is known to have a MIP member gene, glycerol facilitator (GlpF). Here we report the isolation of E. coli gene encoding BniP, bacterial nodulin-like intrinsic protein. BniP encodes a 231 amino acid, 24 kDa protein with 42 % amino acid identity to Nod26, 38 % amino acid identity to AQP1, and 29 % amino acid identity to GlpF. Analysis of deduced amino acid sequence predicted a hydrophobic protein with six membrane-spanning domains. Expression of BniP in Xenopus oocytes induced slight increase in osmotic water permeability, but not glycerol or ion permeability. Our results showed that BniP is a new member of the MIP channel-forming proteins of E. coli.

To elucidate the mechanisms of the cell cycle of mesangial cells, adenovirus vectors containing coding sequences of cyclin D1 (AxCAD1), p16INK4 (AxCAp16) and p21Cip1 (AxCAp21) were produced and investigated to determine whether transfer of these genes changes serum- and PDGF-induced proliferation of rat mesangial cells. Efficiency of the transfer of the genes was examined by Northern and Western blot analyses. The cell cycle of mesangial cells was evaluated by measurement of [3H]-thymidine incorporation, flow cytometry, and cyclin-dependent kinase 4 kinase assay. Expression of cyclin D1, p16INK4 and p21Cip1 was observed from 24 h after the infection, and the expression increased up to 48 h. AxCAp16 and AxCAp21 caused a significant inhibition in the [3H]-thymidine incorporation to 47% and 76%, respectively. AxCAp16 and AxCAp21 also inhibited the mitogen-induced increase in cyclin-dependent kinase 4 kinase activity and reduced the percentage of cells in S phase. Coinfection of AxCAp16 with AxCAp21 showed no additive inhibition. Overexpression of cyclin D1 reduced cell size and increased the percentage of the cells in S and G2 phase. 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 cell cycle disturbances. Adenovirus-mediated gene transfer of p16INK4 and p21Cip1 serves as a potential therapeutic approach to mesangial proliferative diseases.

Expression of aquaporin-2 (AQP2) is exclusively limited to kidney collecting duct cells, and this strictly limited expression could be mediated by transcription of the gene. We first examined AQP2 mRNA expression in many cultured epithelial cells derived from kidney. Northern blot using OK, LLC-PK1, Madin-Darby canine kidney, and outer medullary collecting duct (OMCD) cells and primary culture of inner medullary collecting duct (IMCD) cells did not reveal any significant signal. A more sensitive method, ribonuclease protection assay, could detect a faint signal in OMCD cells when they were bathed in a hypertonic medium. Reverse-transcribed polymerase chain reaction applied to primary culture of IMCD cells showed a rapid dissipation of AQP2 mRNA within 4 days after culture. A reporter gene assay performed in the 1st day of primary culture of IMCD cells showed that the 5' region up to -2.9 kb worked as a promoter. Deletion experiments showed that at least two regions, from -434 to -364 and from -153 to -84, contain negatively acting cis-elements. When connected to a heterologous promoter, these regions repressed the activity in an orientation-dependent manner. These results suggest that transcription of AQP2 gene is strictly regulated and its ability is rapidly depressed in culture condition. This cell differentiation-specific expression of the gene may be, at least in part, mediated by the repressors present in its 5'-flanking region.

A new outwardly rectifying Cl channels (ORCC) that belongs to ClC Cl channel family has been identified from rat kidney and designated as ClC-5. ClC-5 cDNA encodes a polypeptide of 746 amino acids, which is indicated by hydrophobicity analysis to have structural features that are common of the ClC family. However, the amino acid sequence was weakly homologous to those of other ClC Cl channels except for ClC-3, which we recently identified as a Ca-sensitive ORCC. Northern blot analysis of rat tissues showed that ClC-5 mRNA was predominantly expressed in the kidney and colon. To characterize the functional properties of ClC-5 by whole cell patch-clamp technique, we established the stably transfected CHO-K1 cell line using intranuclear microinjection technique. The transfected cells induced outwardly rectifying and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive Cl currents on whole cell configuration. Following the identification of two highly homologous ORCCs, ClC-3 and ClC-5, a new subfamily encoding ORCC has emerged in the ClC family. Furthermore, ClC-5 was almost identical to a partial sequence of human cDNA that is related to Dent's disease. The molecular structure and functional properties of ClC-5 will provide an important insight into ORCCs and the pathogenesis of Dent's disease.

Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct. AQP-2 is selectively permeable to water molecule and is translocated between the apical membrane and subapical endosomes in response to vasopressin. To investigate the localization and structure of the aqueous pathway of the AQP-2 water channel, a series of site-directed mutants was constructed and functionally analyzed. Insertion of N-glycosylation reporter sequence into each hydrophilic loop (HL) indicated that AQP-2 has a six-membrane spanning topology and that insertional mutations in HL-2 or HL-5 do not alter water channel function. Mercury-sensitive site of AQP-2 is located near the second asparagine-proline-alanine (NPA) domain at cysteine 181, but not near the first NPA domain. Replacement of HL-3 or HL-4 with the corresponding part of Escherichia coli glycerol facilitator abolished water channel function without changing plasma membrane expression of the channel protein. Introduction of cysteine residues in His-122, Asn-123, Gly-154, Asp-155, or Asn-156 induced partial mercury sensitivity, and point mutations in asparagine 123 significantly altered water permeability. Our results implicate that the structure of AQP-2 is different from models previously proposed for AQP-1 and that HL-3 and HL-4 are closely located to the aqueous pathway.

Renal nephron segments are heterogeneous, and receptors for endothelin (ET)-1, ET-3, Angiotensin II (AII), epidermal growth factor (EGF), and insulin-like growth factor I distribute differently along the nephron segments. Recently, growth factors and vasoactive substances are reported to stimulate mitogen-activated protein kinase (MAP-K). In this study, we showed that mRNA and proteins of MEK-K, Raf-1-K, MAPK-K, MAP-K (p42 and p44), and S6-K are expressed ubiquitously in intact nephron segment. We demonstrated that four tiers of a cascade composed of the Raf-1-K, MAP-K, MAP-K, and S6-K are stimulated by ET-1 and ET-3 in rat intact glomeruli (Glm) via primarily B-type ET receptors and PKC. The stimulatory effect of EGF and IGF-I to MAP-K activity is inhibited by a tyrosine kinase inhibitor in Glm. IGF-I significantly stimulates MAP-K activity and EGF and All moderately stimulate MAP-K activity in the proximal convoluted tubule (PCT). EGF significantly increased MAP-K cascades and ET-1 and ET-3 slightly increased MAP-K cascades in the medullary thick ascending limb (MTAL). EGF significantly stimulated MAP-K cascades, and ET-1 and ET-3 moderately stimulate MAP-K cascades in the outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD). MAPK-K and S6-K are similarly stimulated by these agonists in each segment. This study shows that MAP-K cascades are expressed in every nephron segment. ET-1, ET-3, All, EGF, and IGF-I stimulate MAP-K cascades heterogeneously along the nephron segment. It was concluded that MAP-K cascades play an important role in the regulation of renal function.

The ability to concentrate the urine was an essential step in the adaptation of animals to life in a terrestrial environment. Only mammals and birds produce a urine which is hypertonic to plasma. A concentrated urine is formed in the kidney collecting duct in response to vasopressin by the massive reabsorption of water from luminal fluid. The most prominent feature of water transport in the collecting duct is the rapid and large change in water permeability induced by vasopressin. In isolated perfused rabbit collecting duct segments, for example, the lag time between the addition of vasopressin and the appearance of an increase of water permeability is less than 30 seconds [1], and more than 10-fold increases in water permeability have been reported [2, 3]. The mechanisms responsible for this drastic change in water permeability have been studied extensively over the past three decades. Current models, as reviewed by Jo and Harris in this issue, hold that the vasopressin-induced changes in water permeability are due to the insertion and retrieval of “water channel” proteins to and from the apical membrane of collecting duct cells. There is now abundant functional and morphological evidence supporting the presence of water channels in the apical membrane of collecting duct. Following the discovery of AQP-CHIP as the first identified water channel protein [4, 5], which is present in many water transporting tissues including the kidney proximal tubules and thin descending limb of Henle [6–8], we succeeded in cloning another water channel, AQP–CD, which is expressed in collecting ducts [9]. Accumulating evidence indicates that AQP–CD is the vasopressin-regulated water channel of collecting duct. Very recently we have also cloned an additional water channel protein, named AQP3, which is expressed at the basolateral membrane of collecting ducts of the kidney [10]. This review will focus mainly on these collecting duct specific water channels.

A cDNA encoding rat AQP3, a water channel and a member of the MIP family, that is expressed predominantly in kidney medulla and colon was cloned recently. To determine the structure, tissue distribution, and chromosomal localization of the human AQP3 gene, we screened a human kidney cDNA library with rat AQP3 probe and isolated a cDNA coding for human AQP3 protein. The deduced amino acid sequence of human AQP3 was 91% identical to rat AQP3. Human AQP3 mRNA was expressed in colon, kidney, liver, pancreas, lung, peripheral leukocytes, spleen, and prostate. The human AQP3 gene was mapped to 7q36.2-q36.3 by chromosome fluorescence in situ hybridization.

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg- Arg-Gln- Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.

To investigate the physiological role of a kidney-specific chloride channel (ClC-K1), we sought to determine its exact localization by immunohistochemistry and its functional regulation using Xenopus oocyte expression system. The antiserum specifically recognized a 70-kD protein in SDS-PAGE of membrane protein from rat inner medulla and an in vitro translated ClC-K1 protein. Immunohistochemistry revealed that ClC-K1 was exclusively localized to the thin limb of Henle's loop in rat inner medulla. In comparison with the immunostaining with anti-aquaporin-CHIP antibody that only stains the descending thin limb of Henle's loop (tDL), ClC-K1 was found to be localized only in the ascending limb (tAL) which has the highest chloride permeability among nephron segments. Immunoelectron microscopy confirmed that the staining of ClC-K1 in tAL was observed in the region of both apical and basolateral plasma membranes. Expressed chloride current in Xenopus oocytes by ClC-K1 cRNA was regulated by extracellular pH and extracellular calcium. Furosemide inhibited the expressed current (Ki = 100 microM), whereas N-ethyl-maleimide stimulated the current. These functional characteristics were consistent with the in vitro perfusion studies of chloride transport in tAL. The localization and the functional characteristics described here indicate that ClC-K1 is responsible for the transepithelial chloride transport in tAL.

Background Elderly patients with chronic kidney disease (CKD) are usually at a high risk of fractures due to both osteoporosis and CKD-mineral bone disease (MBD). A new marker is needed to prevent fractures and control CKD-MBD from the early to advanced stages of CKD. In the early stage of CKD, fibroblast growth factor 23 (FGF23) level increases before parathyroid hormone (PTH) and phosphate levels increase, and steadily increases with the progression of kidney disease. It has been reported that FGF23 is related to the overall fracture risk. We investigated the usefulness of FGF23 as a marker for evaluating the risk of vertebral fracture and CKD-MBD in elderly CKD patients. Methods One hundred and five elderly predialysis CKD patients who had never been treated for osteoporosis and had never used calcium supplements, vitamin D supplements, or phosphate binders were enrolled in this cross-sectional study in Tokyo, Japan. We investigated the prevalence of vertebral fracture and measured serum calcium, phosphate, 1,25(OH)2 vitamin D [1,25(OH)2D], intact PTH, FGF23, alkaline phosphatase, and urinary N-terminal telopeptide levels. Then, we examined the relationship between the level of FGF23 and those of bone-metabolism-related markers and identified markers associated with vertebral fractures in elderly CKD patients. Results The background features of the patients were as follows: female, 32.4%; diabetes mellitus, 39.0%; average age (standard deviation), 73.2 (7.7) years; and estimated glomerular filtration rate (eGFR), 45.7 (24.1) ml/min/1.73 m2. Adjusted multivariate regression analysis showed that the natural logarithm value of FGF23 level [ln(FGF23)] was positively associated with body mass index (p = 0.002), serum phosphate level (p = 0.0001), and negatively with eGFR (p = 0.0006). Multivariate logistic regression analysis showed that vertebral fracture was independently associated with ln(FGF23) (adjusted odds ratio, 4.44; 95% confidence interval, 1.13-17.46). A receiver-operating-characteristic curve of ln(FGF23) showed that the optimal cutoff level of FGF23 indicative of vertebral fracture was 56.8 pg/ml (sensitivity, 0.82; specificity, 0.63). Conclusions FGF23 level was independently associated with the levels of bone-metabolism-related markers and vertebral fracture. FGF23 is a new candidate marker for detecting abnormalities of bone metabolism and vertebral fracture in elderly CKD patients.

We recently cloned a cDNA of the collecting duct apical membrane water channel of rat kidney, which is important for the formation of concentrated urine (Fushima, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. 1993. Nature [Lond.]. 361:549-552). Since urine concentrating ability varies among mammalian species, we examined whether an homologous protein is present in human kidney. By screening a human kidney cDNA library, we isolated a cDNA clone, designated human aquaporin of collecting duct (hAQP-CD), that encodes a 271-amino acid protein with 91% identity to rat AQP-CD. mRNA expression of hAQP-CD was predominant in the kidney medulla compared with the cortex, immunohistochemical staining of hAQP-CD was observed only in the collecting duct cells, and the staining was dominant in the apical domain. Functional expression study in Xenopus oocytes confirmed that hAQP-CD worked as a water channel. Western blot analysis of human kidney medulla indicated that the molecular mass of hAQP-CD is 29 kD, which is the same mass expected from the amino acid sequence. Chromosomal mapping of the hAQP-CD gene assigned its location to chromosome 12q13. These results could be important for future studies of the pathophysiology of human urinary concentration mechanisms in normal and abnormal states.

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.

A new chloride channel has recently been identified by expression cloning (Paulmiehl, M., et al. Nature 356: 238). To date there is no information available on the distribution of this channel in mammalian tissues. We cloned rat homologue of this Cl channel and found 92% identity in deduced amino-acid sequence. We studied the tissue distribution of its mRNA and regulation by protein kinases. Its mRNAwas expressed mall l7 bovine tissues we studied, most abundantly in the brain. In the kidney, it was expressed more in the medulla than cortex. Dehydration up to 5 days did not change its mRNA level in the rat kidney. Both phorbol myristate acetate (PMA) and forskolin down regulated its expression in MDCK cells, suggesting that both PKC and PKA modulated its expression. The physiological role of this chloride channel remains to be clarified.

Concentrating urine is mandatory for most mammals to prevent water loss from the body. Concentrated urine is produced in response to vasopressin by the transepithelial recovery of water from the lumen of the kidney collecting tubule through highly water-permeable membranes. In this nephron segment, vasopressin regulates water permeability by endo- and exocytosis of water channels from or to the apical membrane. CHIP28 is a water channel in red blood cells and the kidney proximal tubule, but it is not expressed in the collecting tubule. Here we report the cloning of the complementary DNA for WCH-CD, a water channel of the apical membrane of the kidney collecting tubule. WCH-CD is 42% identical in amino-acid sequence to CHIP28. WCH-CD transcripts are detected only in the collecting tubule of the kidney. Immunohistochemically, WCH-CD is localized to the apical region of the kidney collecting tubule cells. Expression of WCH-CD in Xenopus oocytes markedly increases osmotic water permeability. The functional expression and the limited localization of WCH-CD to the apical region of the kidney collecting tubule suggest that WCH-CD is the vasopressin-regulated water channel.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.

The expressions of mRNAs of hepatocyte growth factor (HGF) and its receptor (c-met) and its effects were examined in cultured renal epithelial cell lines (OK, LLCPK1, and MDCK cells) and rat mesangial cells in primary culture. Northern blot analysis revealed the presence of HGF mRNA in mesangial cells, but not in epithelial cells. c-met mRNA was detected in epithelial cells, but not in mesangial cells. HGF stimulated [3H]-thymidine incorporation (DNA synthesis) dose-dependently in OK and LLCPK1 cells, but not in MDCK and mesangial cells. Ouabaine sensitive rubidium uptake (Na,K-ATPase activity) was stimulated by 63% with HGF (10 ng/ml) treatment for 16hr in MDCK cells. The results suggest that HGF is produced in the kidney, at least in mesangial cells and works on epithelial cells to stimulate the proliferation and/or to modify cell functions in a paracrine manner.

The parathyroid hormone (PTH) effects on luminal Na-H exchange and basolateral Na-HCO3 cotransport were examined in isolated perfused rabbit proximal convoluted tubules. Lumen pH (pHi) and cell pH (pHi) were measured by a fluorescent technique using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The time course of the PTH effect on proximal acidification was first determined by continuous monitoring of pHi in a stop-flow condition. PTH 10(-8) M added to the bath promptly inhibited the acid pHl generation. Adenosine 3',5'-cyclic monophosphate (cAMP) (10(-4) M) added to the bath mimicked the PTH effect. Luminal Na-H exchange was assayed by monitoring changes in pHi (dpHi/dt) in response to luminal Na+ removal in HCO3- free condition. The addition of PTH or cAMP for 5 min decreased dpHi/dt by 30 and 37%, respectively. Basolateral Na-HCO3 cotransport was assayed by measuring dpHi/dt caused by bath Na+ removal. The addition of PTH or cAMP for 5 min did not change the dpHi/dt. PTH also did not alter the dpHi/dt induced by reducing the bath HCO3- from 25 to 5 mM. The addition of PTH or cAMP to the bath slightly reduced pHi by 0.05. These results suggest that 1) PTH inhibits proximal acidification very rapidly and the effect reached a maximum within 10 min, 2) PTH rapidly inhibits luminal Na-H exchange but not basolateral Na-HCO3 cotransport in a short period, and 3) this effect is at least partly mediated by cAMP-dependent mechanisms.

During the last ten years, the mechanisms and regulation of HCO 3 − /H+ transport across the cell membranes of proximal tubule cells, have been extensively studied. The generally accepted view is that tubular acidification is performed by H+ secretion into the lumen. This secretion is mostly due to a luminal Na/H exchange; a small portion is mediated by an ATP-dependent H+ pump. Most basolateral HCO 3 − exit is through a Na-(HCO3)3 cotransport; a small fraction (15%) is mediated by Na+ coupled Cl−/HCO 3 − exchange. The amount of passive HCO 3 − leak from the peritubular to the tubular lumen is relatively small compared to the active H+ secretion. Based on this concept, I will discuss possible mechanisms of proximal RTA; (1) decreased luminal H+ secretion, (2) decreased basolateral HCO 3 − exit, (3) inhibition of Na-K-ATPase pump, (4) decreased generation of ATP, and (5) increased passive permeability of HCO 3 − .

We measured the single cell pH of principal cells (PC) and intercalated cells (IC) in the outer stripe of the outer medullary collecting duct (OMCDo) of the rabbit using fluorescence ratio imaging.

Background Congenital nephrogenic diabetes insipidus (NDI) is characterised by an inability to concentrate urine despite normal or elevated plasma levels of the antidiuretic hormone arginine vasopressin. We report a Japanese extended family with NDI caused by an 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the Rho GTPase-activating protein 4 (ARHGAP4) locus. ARHGAP4 belongs to the RhoGAP family, Rho GTPases are critical regulators of many cellular activities, such as motility and proliferation which enhances intrinsic GTPase activity. ARHGAP4 is expressed at high levels in hematopoietic cells, and it has been reported that an NDI patient lacking AVPR2 and all of ARHGAP4 showed immunodeficiency characterised by a marked reduction in the number of circulating CD3+ cells and almost complete absence of CD8+ cells. Methods PCR and sequencing were performed to identify the deleted region in the Japanese NDI patients. Immunological profiles of the NDI patients were analysed by flow cytometry. We also investigated the gene expression profiles of peripheral blood mononuclear cells (PBMC) from NDI patients and healthy controls in microarray technique. Results We evaluated subjects (one child and two adults) with 11.2-kb deletion that includes the entire AVPR2 locus and approximately half of the ARHGAP4. Hematologic tests showed a reduction of CD4+ cells in one adult patient, a reduction in CD8+ cells in the paediatric patient, and a slight reduction in the serum IgG levels in the adult patients, but none of them showed susceptibility to infection. Gene expression profiling of PBMC lacking ARHGAP4 revealed that expression of RhoGAP family genes was not influenced greatly by the lack of ARHGAP4. Conclusion These results suggest that loss of ARHGAP4 expression is not compensated for by other family members. ARHGAP4 may play some role in lymphocyte differentiation but partial loss of ARHGAP4 does not result in clinical immunodeficiency.

We investigated whether ET-1 is synthesized by and released from mesangial cells. ET-1-like immunoreactivity (LI) released into medium increased time-dependently under a serum-free condition. The amounts of ET-1-LI released into the medium was augmented in the presence of fetal calf serum. Reverse-phase HPLC profile of the conditioned media revealed a major component coeluting with standard ET-1. Northern blot analysis of poly(A) + RNA extracted from mesangial cells showed a single major band corresponding to the size of preproET-1 mRNA (2.3 kb). These findings demonstrate that ET-1 is synthesized by and released from rat mesangial cells and suggest a possibility that it acts on their own cells as an autocrine factor.

Unilateral renal agenesis with impaired renal function was found in all 3 siblings of a single family, in association with various metabolic disorders such as diabetes mellitus, hyperuricemia and hyperbilirubinemia with gallstones. The present report suggests that unilateral renal agenesis could occur as a manifestation of a genetic disorder.

Direct effect of azosemide on the mouse thick ascending limb of Henle's loop was examined by the isolated tubules perfusion technique, and the effect was compared with that of furosemide. Azosemide added to the tubular lumen reversibly suppressed the lumen positive potential (PDt) and decreased the net chloride reabsorption. Dose-response studies of these two drugs suggested that azosemide was about 5 times as potent as furosemide. The more potent effect of azosemide compared with furosemide was confirmed by a paired study in the same tubules. At a concentration of 10(-5) mol/l azosemide suppressed PDt by 91.4%, furosemide by 65.6%.

Ischemic acute renal failure (ARF) is the most common form of ARF in the adult population. The molecular mechanisms of tubular regeneration after ischemic renal injury remain largely unknown. An understanding of the mechanisms that lead to renal cell proliferation and regeneration will be necessary for the exploration of novel therapeutic strategies for the treatment of ARF. It has been suggested that regeneration processes may recapitulate developmental processes in order to restore organ or tissue function. The adult tubular epithelial cells have a potent ability of regenerate after cellular damage. We examined functional role of two developmental genes, Wnt-4 and Ets-1, in renal tubular regeneration in ARF. The Wnt-β-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in the embryonic development. To clarify the significance of the Wnt-4-β-catenin pathway in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. After clamping left rat renal artery for 1 hour, we examined whole kidney homogenate and total RNA extracted at 3, 6, 12, 24, 48, and 72 hours after reperfusion by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR). Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and coexpressed with aquaporin 1 and proliferating cell nuclear antigen (PCNA). Cyclin D1 and cyclin A were expressed at 12 to 48 hours after reperfusion. Furthermore, overexpression of Wnt-4 and β-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 and cyclin A in LLC-PK1 cells. These data suggest that the Wnt-4-β-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. To clarify the significance of Ets-1 in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. Ets-1 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after the ischemia, respectively. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with PCNA. Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 hours and 6 hours in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell cycle progression of renal tubules in ARF. Our data suggest that Wnt-4-β-catenin and Ets-1 pathways regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. These developmental genes may play key roles in dedifferentiation and regeneration of the renal tubular cells after ARF.