Salt-resistant Normotensive Rats Research Articles

Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats

AimsCardiovascular disease (CVD) is common in chronic kidney disease (CKD) patients. Indoxyl sulfate (IS) is a nephrovascular uremic toxin that induces oxidative stress in kidney and vascular system. The present study aimed to examine the effect of IS on fibrosis and oxidative stress in rat heart. Main methodsThe effects of IS on heart were examined by Masson's trichrome (MT) staining and immunohistochemistry using: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH+IS). Key findingsDH+IS rats showed significantly increased heart weight and left ventricle weight compared with DN. DH and DH+IS rats showed significantly increased diameter of cardiomyocytes, increased MT-positive fibrotic area, increased staining for transforming growth factor (TGF)-β1, α-smooth muscle actin (SMA), type 1 collagen, NADPH oxidase Nox 4, malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased staining for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the heart compared with DN. More notably, DH+IS rats showed significantly increased diameter of cardiomyocytes, increased fibrotic area, increased staining for TGF-β1, SMA, type 1 collagen, Nox4, 8-OHdG and MDA, and decreased staining for Nrf2 and HO-1 in the heart compared with DH. SignificanceIS aggravates cardiac fibrosis and cardiomyocyte hypertrophy with enhanced oxidative stress and reduced anti-oxidative defense in hypertensive rats.

Indoxyl sulfate, a uremic toxin, downregulates renal expression of Nrf2 through activation of NF-κB

BackgroundIndoxyl sulfate, a uremic toxin, is accumulated in the serum of chronic kidney disease (CKD) patients, accelerating the progression of CKD. In CKD rat kidney, the expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its related genes are downregulated. AST-120, an oral sorbent, reduces serum indoxyl sulfate and slows the progression of CKD. The present study aimed to determine whether indoxyl sulfate downregulates Nrf2 expression in human proximal tubular cells and rat kidneys and whether AST-120 upregulates Nrf2 expression in CKD rat kidneys.MethodsEffects of indoxyl sulfate on expression of Nrf2 were determined using HK-2 cells as human proximal tubular cells and the following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). Further, AST-120 was administered to subtotally nephrectomized CKD rats to determine its effect on the expression of Nrf2.ResultsIndoxyl sulfate downregulated Nrf2 expression in HK-2 cells. The indoxyl sulfate-induced downregulation of Nrf2 expression was alleviated by an inhibitor of nuclear factor-κB (NF-κB) (pyrrolidine dithiocarbamate) and small interfering RNA specific to NF-κB p65. DN+IS, DH, and DH+IS rats showed decreased renal expression of Nrf2 and its downstream target genes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and increased renal expression of 8-hydroxydeoxyguanosine (8-OHdG), a marker of reactive oxygen species (ROS), compared with DN. Thus, indoxyl sulfate, as well as hypertension, downregulated renal expression of Nrf2 in rats. AST-120 upregulated renal expression of Nrf2, HO-1 and NQO1 and suppressed renal expression of 8-OHdG compared with control CKD rats.ConclusionsIndoxyl sulfate downregulates renal expression of Nrf2 through activation of NF-κB, followed by downregulation of HO-1 and NQO1 and increased production of ROS. Further, AST-120 upregulates renal expression of Nrf2 in CKD rats by removing serum indoxyl sulfate, followed by upregulation of HO-1 and NQO1 and decreased production of ROS.

Indoxyl Sulfate Enhances p53-TGF-β1-Smad3 Pathway in Proximal Tubular Cells

Background/Aim: Indoxyl sulfate-induced activation of nuclear factor (NF)-ĸB promotes transforming growth factor (TGF)-β₁ in human proximal tubular cells (HK-2 cells). The present study aimed to elucidate the cross talk among indoxyl sulfate, p53 and TGF-β₁-Smad3 signaling in proximal tubular cells. Methods: The effects of indoxyl sulfate on the expression of TGF-β₁, Smad3, and α-smooth muscle actin (α-SMA) were determined using HK-2 cells. As for in vivo experiments the following animals were used: Dahl salt-resistant normotensive rats (DN) and indoxyl sulfate-administered Dahl salt-resistant normotensive rats (DN+IS). Results: Both indoxyl sulfate and nutlin-3, a specific p53 inducer, stimulated TGF-β₁ expression, which was suppressed by pifithrin-α, p-nitro, a p53 inhibitor. Further, indoxyl sulfate stimulated TGF-β₁-induced expression of α-SMA by enhancing Smad3 expression and TGF-β₁-induced Smad3 phosphorylation. Indoxyl sulfate induced phosphorylation of extracellular signal-regulated kinase (ERK). U0126, an inhibitor of ERK pathway, prevented indoxyl sulfate-induced upregulation of Smad3 expression. Immunohistochemistry demonstrated that TGF-β₁ and Smad3 were localized in renal tubular cells, and that indoxyl sulfate increased the TGF-β₁ and Smad3-positive area in the kidney. Conclusion: Indoxyl sulfate stimulates p53-induced TGF-β₁ expression and TGF-β₁-induced α-SMA expression in proximal tubular cells. Indoxyl sulfate-induced Smad3 accelerates TGF-β₁-induced α-SMA expression through ERK activation. Thus, indoxyl sulfate enhances p53-TGF-β₁-Smad3 pathway in proximal tubular cells.

Indoxyl sulfate upregulates renal expression of ICAM-1 via production of ROS and activation of NF-κB and p53 in proximal tubular cells

AimsIntercellular adhesion molecule 1 (ICAM-1) plays an important role in adhesion of monocytes/macrophages to injured tubulointerstitial tissue. The present study aimed to determine if indoxyl sulfate, a uremic toxin, regulates renal expression of ICAM-1. Main methodsThe effect of indoxyl sulfate on expression of ICAM-1 was determined using human proximal tubular cells (HK-2 cells) and the following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). Key findingsDN+IS, DH, and DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DN rats. DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DH rats. Immunohistochemistry revealed that ICAM-1 was localized in the cytoplasm of renal tubular cells, and was most prominently expressed in DH+IS rats. Indoxyl sulfate upregulated mRNA and protein expression of ICAM-1 in HK-2 cells. Inhibitors of NADPH oxidase (diphenylene iodonium chloride), NF-κB (isohelenin) and p53 (pifithrin-α,p-nitro) suppressed indoxyl sulfate-induced expression of ICAM-1 mRNA and protein in HK-2 cells. SignificanceIndoxyl sulfate upregulated renal expression of ICAM-1 through production of reactive oxygen species (ROS) such as superoxide, and activation of NF-κB and p53 in proximal tubular cells. Further, administration of indoxyl sulfate promoted ICAM-1 expression in rat kidneys. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of ICAM-1 in the kidney.

Indoxyl sulfate upregulates renal expression of icam-1 via production of ros and activation of nf-κb and p53 in proximal tubular cells

Intercellular adhesion molecule 1 (ICAM-1) plays an important role in adhesion of monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of ICAM-1. The effect of indoxyl sulfate on expression of ICAM-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). DN+IS, DH, and DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DN rats. DH+IS rats showed significantly increased mRNA expression of ICAM-1 in the kidneys compared with DH rats. Indoxyl sulfate upregulated mRNA expression of ICAM-1 in HK-2 cells. Inhibitors of NADPH oxidase, NF-κB and p53 suppressed indoxyl sulfate-induced mRNA expression of ICAM-1 in HK-2 cells. In conclusion, indoxyl sulfate upregulates renal expression of ICAM-1 through production of ROS and activation of NF-κB and p53 in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of ICAM-1 in the kidneys.

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells

AimsMonocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1. Main methodsThe effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). Key findingsDN+IS, DH, and DH+IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH+IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells. SignificanceIndoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.

Indoxyl Sulfate Induces Epithelial-to-Mesenchymal Transition in Rat Kidneys and Human Proximal Tubular Cells

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic kidney disease (CKD). This study aimed to determine if IS induces epithelial-to-mesenchymal transition (EMT) in the kidneys of hypertensive rats and human proximal tubular cells (HK-2). Methods: EMT was evaluated by immunohistochemistry, reverse transcription-polymerase chain reaction and immunoblotting of the epithelial markers E-cadherin and zonula occludens-1 (ZO-1), and the mesenchymal marker α-smooth muscle actin (α-SMA). Rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH+IS). HK-2 cells were incubated with or without IS. Results: In kidneys, DH rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN rats. DN+IS and DH+IS rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN and DH rats, respectively. DH+IS and DH rats showed increased Masson’s trichrome-positive fibrosis areas compared with DH and DN, respectively. IS-treated HK-2 cells showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA. Conclusion: IS induces EMT in the kidneys of hypertensive rats and in human proximal tubular cells.

Indoxyl Sulfate Downregulates Renal Expression of Klotho through Production of ROS and Activation of Nuclear Factor-ĸB

Background/Aim: Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease (CKD). The present study aimed to examine whether renal expression of Klotho is regulated by indoxyl sulfate, a uremic toxin, using rat kidneys and human proximal tubular cells (HK-2). Methods: The effect of indoxyl sulfate on renal expression of Klotho was examined using (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). The effects of indoxyl sulfate, inhibitors of nuclear factor-ĸB (NF-ĸB) and an antioxidant on the expression of Klotho in HK-2 cells were examined. Results: DH+IS and DN+IS rats showed decreased expression of Klotho mRNA in the kidneys as compared with DH and DN rats, respectively. Indoxyl sulfate suppressed the expression of Klotho mRNA and protein in HK-2 cells, whereas an antioxidant, N-acetylcysteine, and NF-ĸB inhibitors, pyrrolidine dithiocarbamate and isohelenin, alleviated these effects. Conclusions: Indoxyl sulfate downregulates Klotho expression in kidneys through production of reactive oxygen species and activation of NF-ĸB in proximal tubular cells. Indoxyl sulfate may be involved in reduced renal expression of Klotho in CKD.

Indoxyl sulfate, a uremic toxin, promotes cell senescence in aorta of hypertensive rats

We demonstrated that administration of indoxyl sulfate, a uremic toxin, promotes aortic calcification in hypertensive rats. This study aimed to clarify if indoxyl sulfate could contribute to cell senescence in the aorta of hypertensive rats. The rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS). After 32 weeks, their arcuate aortas were excised for histological and immunohistochemical analysis. Cell senescence was evaluated by immunohistochemistry of senescence-associated β-galactosidase (SA-β-gal), and senescence-related proteins such as p16 INK4a, p21 WAF1/CIP1, p53 and retinoblastoma protein (Rb). Both DH and DH + IS rats showed significantly higher systolic blood pressure than DN and DN + IS rats, respectively. Serum indoxyl sulfate levels were significantly higher in DN + IS and DH + IS rats than in DN and DH rats, respectively. In aorta, DH rats showed significantly increased aortic calcification and wall thickness, and increased expression of SA-β-gal, p16 INK4a, p21 WAF1/CIP1, p53 and Rb in the calcification area of arcuate aorta as compared with DN rats. More notably, DH + IS rats showed significantly increased aortic calcification and wall thickness, and significantly increased expression of SA-β-gal, p16 INK4a, p21 WAF1/CIP1, p53 and Rb in the cells embedded in the calcification area as compared with DH rats. In conclusion, indoxyl sulfate promotes cell senescence with aortic calcification and expression of senescence-related proteins in hypertensive rats.

Indoxyl sulphate promotes aortic calcification with expression of osteoblast-specific proteins in hypertensive rats

Stage 5 chronic kidney disease (CKD) is associated with enhanced aortic calcification. The aim of this study was to determine if the administration of indoxyl sulphate (IS), a uraemic toxin, stimulates the progression of aortic calcification. The rat groups consisted of (i) Dahl salt-resistant normotensive rats (DR) with intake of 0.3% salt, (ii) Dahl salt-sensitive hypertensive rats (DS) with intake of 2.0% salt and (iii) Dahl salt-sensitive hypertensive IS-administered rats (DS-IS) with intake of 2.0% salt and 200 mg/kg of IS in water. After 30 weeks, their aortic and kidney tissues were excised for histological and immunohistochemical analyses. Severe vascular calcification was observed by von Kossa staining in the arcuate aorta of all the DS-IS rats, but hardly in DS or DR rats. Immunohistochemistry demonstrated that osteopontin, core binding factor 1 (Cbfal), alkaline phosphatase (ALP), osteocalcin, IS and organic anion transporter (OAT) 3 were colocalized in the cells embedded in the aortic calcification area of DS-IS rats. Wall thickness was significantly increased in arcuate, thoracic and abdominal aortas of DS-IS rats compared with DS and DR rats. DS-IS rats showed significantly increased extent of glomerular hypertrophy, mesangial expansion, Masson's trichrome-positive tubulointerstitial area and glomerular and tubulointerstitial expression of transforming growth factor-ssl as compared with DS and DR rats. IS induced aortic calcification with expression of osteoblast-specific proteins and aortic wall thickening. IS is not only a nephrotoxin but also a vascular toxin, and may contribute to the progression of aortic calcification in stage 5 CKD patients.

Implications of hyperhomocysteinemia in glomerular sclerosis in hypertension.

Hyperhomocysteinemia (hHcys) has been recognized as a new risk factor for cardiovascular diseases independent of plasma lipid levels or other factors. However, it remains unknown whether hHcys is implicated in the target organ damages associated with hypertension. The present study first examined the possible role of hHcys in the development of glomerulosclerosis in Dahl salt-sensitive (DS) hypertensive rats. High-performance liquid chromatography showed that plasma total homocysteine (tHcys) concentration was 7.64 +/- 0.29 micromol/L in conscious DS rats on a low salt (0.4% NaCl) diet, which was higher than 5.23 +/- 0.25 micromol/L in Dahl salt-resistant normotensive rats. When these rats were exposed to a high salt (4% NaCl) diet, plasma tHcys markedly increased in DS rats (14.7 +/-1.31 micromol/L) but not in Dahl salt-resistant rats (5.34 +/- 0.54 micromol/L). An iron chelater, desferrioxamine (0.3 mg/kg IV per day), completely normalized high salt--induced elevations of plasma tHcys and significantly attenuated the sclerotic changes in the glomeruli in DS rats. To further determine whether hHcys has an independent effect in the development of glomerulosclerosis, Sprague-Dawley rats were fed drinking water containing methionine (1 g/kg per day) for 6 weeks to produce hHcys. In these rats, plasma tHcys increased to 12.5 +/- 1.9 micromol/L (versus 6.1 +/- 2.6 micromol/L in control rats), and the aorta exhibited typical sclerotic changes, but arterial pressure was not altered. Urinary protein excretion increased to 52 +/- 2 mg/24 hours (versus 17 +/- 2 mg/24 hours in control rats), and the glomerular mesangium was expanded with glomerular hypercellularity, capillary collapse, and fibrous deposition in the rats with hHcys. These results suggest that elevated plasma homocysteine may be an important pathogenic factor for glomerular damage in hypertension independent of arterial pressure.

The interrelationship between chloride ions and endothelium on alpha(1)-adrenoceptor-mediated contractions in aortic rings from Dahl normotensive and hypertensive rats.

The effects of chloride-free buffer in the absence or presence of the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), or chloride channel antagonist, indanyloxyacetic acid 94 (IAA-94) on alpha(1)-adrenoceptor mediated contractions were investigated in aortic rings from Dahl salt-resistant normotensive (SRN) and salt-sensitive hypertensive (SSH) rats on a 4% salt diet. Systolic and diastolic blood pressure were measured via intra-arterial catheters under halothane anesthesia. Subsequently, the aorta was removed and cirazoline-induced contractions were recorded in normal Krebs and chloride-free buffer using ring preparation. Guanosine 3',5'-cyclic monophosphate (cyclic GMP) content of aortic rings was also determined by scintillation proximity assay kits. Systolic and diastolic blood pressure of SSH (180/130+/-1/1, mean+/-S.E.; n=14) were significantly higher than those of SRN (101/76+/-1/1, mean+/-S.E.; n=14) 7 weeks after they were placed on a salt diet. While the presence of L-NAME failed to have any impact on cirazoline-induced contractions in aortic rings from SSH rats, it significantly accentuated the effects of cirazoline in tissues from SRN rats. On the other hand, IAA-94 was able to inhibit cirazoline-mediated contractions in aortic rings from both SRN and SSH rats. The removal of chloride ions potentiated contractions produced by cirazoline in tissues from SRN but not SSH rats. Moreover, cirazoline-evoked responses in tissues from SRN were not further accentuated by the inclusion of L-NAME in chloride-free buffer. Cirazoline-mediated contractions in tissues from SSH rats were not influenced by absence of chloride ions, and the presence of L-NAME. It was also apparent that the inclusion of IAA-94 in absence of chloride ions did not prevent the potentiation of responses to cirazoline. Removal of chloride ions did not significantly decrease basal cyclic GMP levels in aortic rings from either strain. Basal release of nitric oxide seems to make a greater contribution in the suppression of cirazoline-evoked contractions in vessels from SRN as opposed to SSH rats. Chloride channels appear to contribute to cirazoline-evoked contractions in normal Krebs but not in chloride-free buffer.

Structure/function relationship of CYP11B1 associated with Dahl's salt-resistant rats--expression of rat CYP11B1 and CYP11B2 in Escherichia coli.

Dahl's salt-resistant normotensive rats (DR rats) have been previously reported to express cytochrome P-450 (CYP11B1) containing five missense mutations [Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami H., Ogihara, T. & Okamoto, M. (1993) J. Biol. Chem. 268, 9117-9121]. To investigate structure-function relationships of CYP11B, wild-type rat CYP11B1 and CYP11B2 and DR-CYP11B1 (mutant CYP11B1 in Dahl's salt-resistant rats) have been successfully expressed in Escherichia coli. Steroid 11beta-hydroxylase (11beta-OHase) activity observed with DR-CYP11B1 was similar to that of wild-type CYP11B1, while 18-hydroxylase (18-OHase) activity of DR-CYP11B1 was lower than that of wild-type CYP11B1. Mutant CYP11B1s containing a single or a double amino acid substitution associated with DR-CYP11B1 have been also expressed in E. coli to investigate effects of the substitutions on enzymatic activity. Each of the single mutant enzymes showed lower 18-OHase activity than wild-type CYP11B1, but not as low as DR-CYP11B1. A double mutant CYP11B1 with V381L and I384L showed 18-OHase activity at a similar low level to that of DR-CYP11B1. The 19-hydroxylation (19-OHase) activity of DR-CYP11B1 was about one-third of that of the wild-type enzyme and this low activity appeared due to the V443M mutation. These results suggest that three of five amino acid substitutions present in DR-CYP11B1 account for the decreased 18-OHase and 19-OHase activities. A decrease in these enzyme activities may be responsible for the normotension of the DR rats when fed a high-salt diet.

Dahl's salt-resistant normotensive rat has mutations in cytochrome P450(11 beta), but the salt-sensitive hypertensive rat does not.

Molecular cloning of cytochrome P450(11 beta) cDNAs from the adrenal glands of Dahl's salt-sensitive hypertensive (DS) and salt-resistant normotensive (DR) rats was performed using a combined technique of the first strand cDNA synthesis by reverse transcriptase followed by polymerase chain reaction. The cDNA sequence of P450(11 beta)-DS was identical to that of wild type P450(11 beta). In contrast, the clone obtained from the DR rat contained six nucleotide substitutions causing five amino acid alterations (Arg-127-->Cys, Val-351-->Ala, Val-381-->Leu, Ile-384-->Leu, and Val-443-->Met). When the two cDNAs were expressed in COS-7 cells and steroid conversion rates of the transformed cells were determined, a ratio of 18-hydroxylation to 11 beta-hydroxylation of 11-deoxycorticosterone by P450(11 beta)-DS-expressed cells was 0.58, whereas that by P450(11 beta)-DR-expressed cells was 0.23. Plasma levels of 18-hydroxy-11-deoxycorticosterone and corticosterone (the 11 beta-hydroxylation product of 11-deoxycorticosterone) in DS and DR rats well reflected the steroidogenic activities of the two P450s. These results suggest that the characteristic plasma steroid level of the DR rat is caused by the mutations in P450(11 beta) gene and may act to maintain the normotensive blood pressure in this rat strain during sodium loading.