Microarray analysis of a reversible model and an irreversible model of anti-Thy-1 nephritis.

A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin beta10. Increased expression of thymosin beta10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin beta10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin beta10 may play an important role in the progression of kidney disease.

Podocyte expression of the CDK-inhibitor p57 during development and disease.

BACKGROUND: The mature podocyte is a terminally differentiated cell with a limited proliferative capacity. The precise cell cycle proteins necessary for establishing podocyte quiescence during development or permitting podocyte cell cycle re-entry in disease states have not been fully defined. Accordingly, we studied the role of the cyclin dependent kinase (CDK)-inhibitor p57Kip2 (p57) in modulating these processes. METHODS: The expression of p57 protein in relation to markers of DNA synthesis was examined in developing mouse kidneys, and in the passive Heymann nephritis (PHN) and anti-glomerular antibody models of glomerular disease by immunohistochemistry. The role of p57 in glomerulogenesis was explored by examining renal tissue from embryonic p57-/- mice, and the expression of p21, p27 and p57 protein and mRNA was examined in podocytes in vitro. RESULTS: The de novo expression of p57 during glomerulogenesis coincides with the cessation of podocyte proliferation, and the establishment of a mature phenotype, and p57 is expressed exclusively in podocytes in mature glomeruli. However, p57 knockout mice have normal glomerular podocyte development. In addition, mRNA but not protein levels of p57 increased upon differentiation of podocytes in vitro. There was a marked decrease in p57 expression in both animal models of podocyte injury. This was diffuse in PHN, whereas in the murine model, loss of expression of p57 occurred predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA). CONCLUSION: Despite the de novo expression of p57 protein coinciding with the cessation of primitive podocyte proliferation during glomerulogenesis, embryonic p57-/- mice glomeruli were histologically normal. Cultured podocytes did not require changes in p57 protein levels to undergo differentiation. These data suggest that p57 alone is not required for podocyte differentiation, and that other cell cycle regulators may play a role. Furthermore, although injury to mature podocytes in experimental glomerular disease is associated with a decrease in p57, the levels of all three members of the Cip/Kip family of CDK inhibitors appear to determine the capability of podocytes to proliferate.

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression.

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.

A new member of the HCO3(-) transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney.

The kidneys play pivotal roles in acid-base homeostasis, and the acid-secreting (alpha-type) and bicarbonate-secreting (beta-type) intercalated cells in the collecting ducts are major sites for the final modulation of urinary acid secretion. Since the H(+)-ATPase and anion exchanger activities in these two types of intercalated cells exhibit opposite polarities, it has been suggested that the alpha- and beta-intercalated cells are interchangeable via a cell polarity change. Immunohistological studies, however, have failed to confirm that the apical anion exchanger of beta-intercalated cells is the band 3 protein localized to the basolateral membrane of alpha-intercalated cells. In the present study, we show the evidence that a novel member of the anion exchanger and sodium bicarbonate cotransporter superfamily is an apical anion exchanger of beta-intercalated cells. Cloned cDNA from the beta-intercalated cells shows about 30% homology with anion exchanger types 1-3, and functional expression of this protein in COS-7 cells and Xenopus oocytes showed sodium-independent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion exchanger activity. Furthermore, immunohistological studies revealed that this novel anion exchanger is present on the apical membrane of beta-intercalated cells, although some beta-intercalated cells were negative for AE4 staining. We conclude that our newly cloned transporter is an apical anion exchanger of the beta-intercalated cells, whereas our data do not exclude the possibility that there may be another form of anion exchanger in these cells.

Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages.

BACKGROUND: The 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) is almost exclusively expressed in the kidney. However, 1alpha-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1alpha-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1alpha-hydroxylase at a molecular level. METHODS: We used the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells by treatment with phorbol ester. The expression of 1alpha-hydroxylase in THP-1 cells was examined by Northern blotting and immunoblotting using an antibody raised against a synthetic peptide corresponding to the 14 C-terminal amino acids of 1alpha-hydroxylase. We investigated the regulation of 1alpha-hydroxylase mRNA expression by RNase protection assay. RESULTS: Northern blot and immunoblot analyses confirmed the expression of 1alpha-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1alpha-hydroxylase mRNA in THP-1 cells (198 +/- 9%). 1,25-(OH)2D3, known as a suppressor of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-gamma (2000 IU/mL) increased the expression of 1alpha-hydroxylase mRNA in differentiated THP-1 cells (922 +/- 25%). CONCLUSIONS: The present results suggest that 1alpha-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney. Interferon-gamma treatment increases macrophage 1alpha-hydroxylase levels via directly increasing gene expression of this enzyme.

Cell-specific expression of the IP(3) receptor gene family in the kidney.

BACKGROUND/AIM: The localization of inositol trisphosphate (IP(3)) receptor isoforms, types 1-3, in the kidney and their role in the regulation of the intracellular calcium concentration - [Ca(2+)](i) - are discussed. METHODS: Immunohistological studies with isoform-specific antibodies were performed to reveal the localization of IP(3) receptor isoforms. To examine the role of IP(3) receptor type 1 in the glomeruli, the responses of [Ca(2+)](i) to hormonal stimuli were examined in IP(3) receptor type 1 knockout mice. RESULTS: In the immunohistological study, type 1 receptor was present in arteries, afferent arterioles, and mesangial cells. Double staining with antibodies against aquaporin 2 and IP(3) type 2 receptor revealed that type 2 receptor was localized mainly in the intercalated cells. The type 3 receptor showed characteristic intracellular localization in the collecting duct cells of the cortex to the outer medulla. Immunostaining of type 3 receptor was most intense in the cytoplasm on the basolateral membrane side and was not seen on the apical side. The responses of [Ca(2+)](i) to angiotensin II and endothelin in the glomeruli were markedly attenuated in IP(3) receptor type 1 knockout mice. CONCLUSIONS: The three isoforms of the IP(3) receptor showed distinctive localization in the kidney, and the type 1 receptor plays a major role in the regulation of [Ca(2+)](i) in the glomeruli. The physiological significance of the cell-specific localization, however, remains to be determined.

Calcitonin induces 25-hydroxyvitamin D3 1alpha-hydroxylase mRNA expression via protein kinase C pathway in LLC-PK1 cells.

The biosynthesis of 1alpha, 25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) in renal proximal tubules. It was recently demonstrated that LLC-PK1 cells express CYP27B1 mRNA, which is regulated by intracellular cAMP but not vitamin D3. To clarify the effect of calcitonin on vitamin D3 metabolism in vitro, LLC-PK1 cells were incubated with hormonal factors, and expression of CYP27B1 mRNA was measured by quantitative reverse transcription-PCR. Calcitonin at 100 nmol/L significantly increased CYP27B1 mRNA expression by 24 h (271 +/- 21% of control). Incubation with calcitonin over a range of 1 micromol/L to 1 pmol/L resulted in a concentration-dependent increase in CYP27B1 mRNA levels. It is known that the calcitonin receptor has dual intracellular signaling pathways, via protein kinases A and C. Both 500 micromol/L 8-bromo-cAMP, a protein kinase A activator, and 100 nmol/L phorbol 12-myristate 13-acetate, a protein kinase C activator, increased CYP27B1 mRNA levels at 24 h (207 +/- 54 and 246 +/- 58% of control, respectively). However, calcitonin-induced CYP27B1 mRNA expression was only inhibited by the protein kinase C inhibitors staurosporine and calphostin C. The protein kinase A inhibitors Rp-cAMPS at 10 and 100 micromol/L and H-89 at 10 micromol/L had no effect on the action of calcitonin, in spite of cAMP-activation by calcitonin. The present data suggest that calcitonin upregulates CYP27B1 mRNA expression via the protein kinase C pathway in LLC-PK1 cells.

Intracellular calcium concentration in the inositol trisphosphate receptor type 1 knockout mouse.

Recently, mice with a disrupted inositol trisphosphate (IP3) receptor type 1 allele were produced by gene targeting. To examine the role of IP3 receptor type I in the regulation of intracellular calcium concentration ([Ca2+]i) of glomerular cells, [Ca2+]i was measured with fura 2-acetoxymethyl-ester in the superfused glomeruli from homozygous and wild-type mice. [Ca2+]i was determined in calcium-free medium before and after the addition of 10(-7) M endothelin-1 (ET-1) and 10(-6) M angiotensin II (AngII). The expression of mRNA of IP3 receptor isoforms and hormone receptors in the glomeruli from these animals also was measured by quantitative reverse transcription-PCR with specific primers for IP3 receptor isoforms (types 1, 2, and 3), AngII receptor type 1, and ET receptors (types A and B). In homozygous mutants, the shorter mRNA of IP3 receptor type 1, which lacks the first exon, is transcribed. Basal [Ca2+]i and the responses to ET-1 and AngII in homozygous mutants (ET-1, 55 +/- 7 nM to 73 +/- 7 nM; AngII, 66 +/- 6 to 91 +/- 8 nM) were significantly lower than those in the wild-type mice (ET-1, 93 +/- 13 nM to 162 +/- 13 nM; AngII, 87 +/- 7 to 147 +/- 9 nM; P < 0.05 for both hormones) without significant changes in mRNA expression of hormone receptors. The results with quantitative reverse transcription-PCR also revealed that mRNA expression of the IP3 receptor gene family was not significantly different between the two groups. The present study clearly shows that IP3 receptor type 1 plays a major role in the regulation of [Ca2+]i in the glomeruli and that lack of an isoform of IP3 receptor in the glomeruli does not induce expression of the other isoforms of the IP3 receptor.

Cloning of porcine 25-hydroxyvitamin D3 1alpha-hydroxylase and its regulation by cAMP in LLC-PK1 cells.

The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.

Two novel 1alpha-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type I1.

BACKGROUND: Vitamin D dependency rickets type I (VDDR-I) is an autosomal recessive disorder in which 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase) activity in renal proximal tubules is deficient. VDDR-I is recognized throughout the world, but occurs more frequently in a subset of the French-Canadian population. We and others have recently cloned the human 1alpha-hydroxylase cDNA and gene, making it possible to screen for mutations. The first VDDR-I mutations were reported in one American and four Japanese patients. In this study, we screened for 1alpha-hydroxylase mutations in French-Canadian patients with VDDR-I. METHODS: The nine exons of the 1alpha-hydroxylase gene were amplified by polymerase chain reaction (PCR) from genomic DNA of four unrelated French-Canadian patients with VDDR-I and their parents, and sequenced. RESULTS: Three of the patients were homozygous for a single base-pair deletion (G) at position 262 in the cDNA that lies in exon 2, and causes a premature termination codon upstream from the putative ferredoxin- and heme-binding domains. The fourth patient was homozygous for a 7-bp insertion (CCCCCCA) at position 1323 of the cDNA that lies in exon 8, and causes a premature termination upstream from the putative heme-binding domain. In each family, obligate carriers have one copy of the mutant allele. These mutations, which could be detected by PCR-restriction fragment length polymorphism and polyacrylamide gel electrophoresis of the PCR products, were not found in 25 normal French-Canadians. CONCLUSION: We describe two novel 1alpha-hydroxylase mutations that are consistent with loss of function in four French-Canadian patients with VDDR-I and suggest that the 1alpha-hydroxylase mutations arise from more than one founder in this population.

Serum leptin concentrations in patients with thyroid disorders.

OBJECTIVE: Leptin, the obese gene product, is secreted exclusively by adipocytes and is thought to act as a lipostatic signal that regulates body weight homeostasis. We previously reported that thyroid hormone is one of the up-regulating factors of leptin in vitro. T3, at physiological concentrations, stimulates leptin mRNA expression and leptin secretion by 3T3-L1 adipocytes. The aim of this study was to explore the role of thyroid hormone in the regulation of leptin in humans. DESIGN AND PATIENTS: A total of 59 non-obese women aged 38.4 +/- 1.8 years (mean +/- SEM) were studied: 19 patients with hyperthyroidism, 17 patients with hypothyroidism, and 23 normal control subjects. The correlation between serum leptin concentrations and body mass index (BMI) was analyzed, and serum leptin levels were compared among the three groups. MEASUREMENTS: Serum leptin concentrations were measured by radioimmunoassay. RESULTS: Serum leptin concentrations after logarithmic transformation were correlated significantly (P < 0.05) with BMI in the hyperthyroid (r = 0.46), the hypothyroid (r = 0.84), and normal (r = 0.63) groups. Even though age, body weight, and BMI were similar in all groups, serum leptin levels in the hypothyroid patients (5.30 +/- 1.12 micrograms/l) were significantly (P < 0.05) lower than in the hyperthyroid and normal groups (6.87 +/- 0.66 and 6.58 +/- 0.68 micrograms/l, respectively). CONCLUSIONS: These results indicate that thyroid hormone may play an important role in the appropriate secretion of leptin in humans.

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

Molecular cloning of cDNA and genomic DNA for human 25-hydroxyvitamin D3 1 alpha-hydroxylase.

The 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase) is a cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3. This enzyme plays an important role in calcium homeostasis. Here we report the molecular cloning of cDNA and gene for human 1 alpha-hydroxylase. The cDNA clone was obtained from a human kidney cDNA library by cross-hybridization with a previously cloned rat cDNA probe. The cDNA consists of 2469 bp and encodes a protein of 508 amino acids that shows 82.5% sequence identity with the rat enzyme. A computer-aided homology search revealed that 1 alpha-hydroxylase shares a relatively high homology with vitamin D3 25-hydroxylase (about 40% amino acid identity). Northern blot analysis showed that the 2.5-kb mRNA is most abundant in kidney. The gene for human 1 alpha-hydroxylase spans approximately 6 kb, is composed of nine exons, and is present as a single copy. This molecular cloning makes it possible to investigate the genetic mechanism of diseases related to calcium metabolism, including vitamin D-dependency rickets type I.

Regulation of expression of leptin mRNA and secretion of leptin by thyroid hormone in 3T3-L1 adipocytes.

Leptin, the obese gene product, is secreted exclusively by adipocytes and regulates energy balance. We examined the effects of thyroid hormones on the regulation of leptin in 3T3-L1 adipocytes. Fully differentiated adipocytes were incubated with thyroid hormones, and the expression of leptin mRNA was measured by quantitative reverse transcription-polymerase chain reaction. After a 24-h incubation, triiodothyronine (T3) at 10-1000 nmol/l significantly increased the expression of leptin mRNA (237-337%). These stimulatory effects were not observed in preadipocytes. By contrast, thyroxine (T4) at 1-1000 nmol/l did not affect leptin mRNA expression in adipocytes. We also measured the levels of secreted leptin in conditioned media using radioimmunoassay. T3 at 10 and 1000 nmol/l significantly increased the levels of secreted leptin (132% and 126%, respectively) after a 24-h incubation. Our present data suggest that thyroid hormone is a novel regulator of leptin mRNA expression and protein secretion in 3T3-L1 adipocytes.

Expression and localization of the water channels in human autosomal dominant polycystic kidney disease.

To characterize the cyst-lining cells in human autosomal dominant polycystic kidney disease (ADPKD), we performed immunohistological studies with specific antibodies against human aquaporin-2 (AQP-2, the vasopressin-regulated water channel) and aquaporin-3 (AQP-3), which are expressed only in collecting duct cells in the normal kidney. The polycystic kidney samples were obtained from 2 hemodialysis patient at uninephrectomy. Immunohistochemical studies revealed two types of staining of cyst-lining cells. Approximately 30% of all the cysts were simultaneously immunostained by both antibodies. Among these AQP-positive cysts, more than 90% of the cysts were intensely stained, with well-polarized localization of AQP-2 and AQP-3. In fewer than 10% of AQP-positive cysts, by contrast, immunostaining for AQP-2 and AQP-3 was faint and no clearly polarized localization of the channels was observed. We examined the immunostaining in further detail by electron microscopy. Staining specific for AQP-2 was mainly observed in the apical membrane of cyst-lining cells. Moreover, staining specific for AQP-3 was observed in all of the AQP-2-positive cysts. It appeared unlikely that the variations in immunostaining observed under the light microscope had been induced by total disruption of water-channel polarity. The present study suggests that about 30% of the cysts in our cases of ADPKD were derived from the collecting duct cells and that the cyst-lining cells were well differentiated in terms of AQP expression.

Na+/H+-exchanger 3 activity and its gene in the spontaneously hypertensive rat kidney.

OBJECTIVE: To examine the mechanisms affecting proximal tubule sodium reabsorption, we studied the gene expression and functional changes of the Na+/H+-exchanger 3 (NHE3) and its gene expression in spontaneously hypertensive rats (SHR). METHODS: The S1 segment of the superficial proximal convoluted tubules (PCT) was microdissected from collagenase-treated kidneys of SHR and Wistar-Kyoto (WKY) rats. The tubules were then superfused in a HEPES-buffered solution. The intracellular pH was measured by monitoring changes in fluorescent dye emission. By measuring the amiloride-sensitive intracellular pH recovery rate after intracellular acid loading with NH4Cl, we estimated the NHE3 activity. Northern blotting was performed to quantitate the NHE3 messenger RNA (mRNA) expression, and the NHE3 complementary DNA sequence was determined both in SHR and in WKY rat strains. RESULTS: NHE3 activity was significantly higher in SHR than it was in WKY rats both at age 4 weeks and at age 7 weeks. Northern blot analysis showed that the mRNA expression did not differ significantly in the two strains at either age. Sequence analysis of complementary DNA of SHR NHE3 revealed five allelic variations compared with the known wild-type sequence in Sprague-Dawley rats. The same variations were also identified in the WKY rats. CONCLUSION: NHE3 activity is increased in SHR compared with WKY rats of the same age. Changes in the NHE3 mRNA expression level and protein structure are not responsible for this difference between SHR and WKY rat strains.

Regulation of obese mRNA expression by hormonal factors in primary cultures of rat adipocytes.

The obese (ob) gene has been cloned recently and its protein product is called "leptin". Leptin is an adipocyte-derived satiety factor that regulates body weight homeostasis. Several hormonal factors have been reported to regulate ob mRNA expression. To determine which factors are most important for regulation of ob mRNA expression, we examined the effects of insulin, dexamethasone, a beta3-adrenergic agonist (CGP12177A), 8-bromo-cAMP, 8-bromo-cGMP and 1-methyl-3-isobutylxanthine (MIX) on primary cultured adipocytes. Rat adipocytes obtained from epididymal fat were cultured using the ceiling method. Total RNA was extracted and the expression of ob mRNA was measured by quantitative reverse transcription-polymerase chain reaction. After 24 h of incubation, 100 nmol/l insulin significantly increased the expression of ob mRNA (21.4-fold compared to control). Moreover, insulin increased ob mRNA expression in a dose-dependent manner over a range of 1-100 nmol/l. The effect of 100 nmol/l insulin was similar to that seen with 20% newborn calf serum. Dexamethasone (25-1000 nmol/l) also increased ob mRNA expression (2.5-2.9-fold). The effect of dexamethasone occurred more rapidly than insulin. CGP12177A (1-10 micromol/l) and 0.5 mmol/l 8-bromo-cAMP had no effects, whereas 0.5 mmol/l 8-bromo-cGMP and 0.5 mmol/l MIX had stimulatory effects (2.8- and 2.4-fold increase in ob mRNA, respectively). The combination of 250 nmol/l dexamethasone and 0.5 mmol/l MIX did not have an additive effect on ob mRNA levels. Our present data suggest that, of these agents, insulin is the most important factor regulating ob mRNA expression.

Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.

To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Role of vasopressin V2 receptor in acute regulation of aquaporin-2.

Aquaporin-2 (AQP-2) has been shown to be a vasopressin-sensitive water channel in collecting duct (CD) cells of the kidney. To prove the role of the vasopressin V2 receptor (V2R) in the regulation of intracellular AQP-2 shuttling, we examined the acute effects of vasopressin and V2R antagonist on the distribution of AQP-2 in the cells. Normal Wistar rats were given continuous infusions of vasopressin, vasopressin V2R antagonist (OPC31260), or both. The kidneys were then processed for immunofluorescent studies with an affinity-purified specific antibody to AQP-2. One hour after the infusion of the V2R antagonist, AQP-2 staining was diffusely distributed in the CD cells from the cortex to the inner medulla. This tendency was not changed by the concomitant infusion with vasopressin. Vasopressin infusion without antagonist, however, induced intensified AQP-2 staining of the apical membrane in the CD cells. The ratio of the fluorescence intensity of the apical to subapical region was determined by confocal laser microscopy. In the inner medulla, this ratio was significantly increased in the vasopressin treatment group (2.26 +/- 0.76) as compared to the V2R antagonist group (1.03 +/- 0.34) and the combined treatment group (0.84 +/- 0.43). The increase in the ratio was also demonstrated in the cortex and the outer medulla in the vasopressin-treated group. In addition, Northern blotting studies clearly revealed that mRNA of AQP-2 in the vasopressin-treated group was increased when compared to the combined treatment animals. Our present results reveal that localization and gene expressions of AQP-2 are acutely regulated via vasopressin V2R.

Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits.

The inositol 1,4,5-trisphosphate receptor (IP3R) exists as a tetrameric complex to form a functional inositol 1,4,5-trisphosphate-gated Ca2+ channel. Molecular cloning studies have shown that there are at least three types of IP3R subunits, designated type 1, type 2, and type 3. The levels of expression of IP3R subunits in various cell lines were investigated by Western blot analysis using type-specific antibodies against 15 C-terminal amino acids of each IP3R subunit. We found that all the three types of IP3R subunits were expressed in each cell line examined, but their levels of expression varied. To determine whether IP3Rs form heterotetramers, we employed immunoprecipitation experiments using Chinese hamster ovary cells (CHO-K1 cells), in which all three types are abundantly expressed. Each type-specific antibody immunoprecipitated not only the respective cognate type but also the other two types. This result suggests that distinct types of IP3R subunits assemble to form heterotetramers in CHO-K1 cells. We also detected heterotetramers in rat liver, in which IP3R type 1 and type 2 are expressed abundantly. Previous studies have shown some functional differences among IP3R types, suggesting the possibility that various compositions of subunits show distinct channel properties. The diversity of IP3R channels may be further increased by the co-assembly of different IP3R subunits to form homo- or heterotetramers.