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We examined whether and how peritubular capillary (PTC) loss in the renal cortex contributes to the development of deoxycorticosterone acetate (DOCA)/salt-induced tubulointerstitial fibrosis. Uninephrectomized rats provided with 0.9% NaCl/0.3% KCl drinking solution ad libitum were divided into control, DOCA, and spironolactone groups, which were administered vehicle, DOCA alone, and DOCA plus spironolactone for 1 (initial phase) and 4 weeks (delayed phase), respectively. Exposure to DOCA initiated a sequence of events that initially involved reduced PTC density, followed by a delayed response that involved further reduced PTC density, development of tubulointerstitial fibrosis and hypertension, enhanced expression of transforming growth factor-β1 and connective tissue growth factor, and impaired renal function. Concomitant with the reduced PTC density, the 2 hypoxia-responsive angiogenic factors (vascular endothelial growth factor and hypoxia-inducible factor-1α) and the antiangiogenic factor (thrombospondin-1) were upregulated in cortical tubular cells of the DOCA group during the 2 phases and only in the delayed phase, respectively. In the DOCA group, PTC endothelial cell apoptosis was enhanced during the 2 phases, and PTC endothelial cell proliferation was inhibited in the delayed phase. In accordance with upregulation of thrombospondin-1, p53 expression was enhanced in the DOCA group in the delayed phase. The initial and delayed effects of DOCA were blocked in the spironolactone group. We conclude that exposure to DOCA initially caused the reduced PTC density associated with enhanced apoptosis independent of thrombospondin-1, which induced tubulointerstitial fibrosis via p53-mediated thrombospondin-1 activation, and spironolactone conversely corrected the effects of DOCA to prevent fibrosis.

A 66-year-old woman was admitted to our hospital because of vomiting and appetite loss. For the 2 days prior to admission, she had a cold, which had developed into acute viral bronchitis on admission. Because laboratory data on admission showed hyponatremia, intravenous infusion of Ringer's lactate solution was started. However, generalized seizures appeared, and she developed a coma on the day of admission. Her plasma antidiuretic hormone (ADH) level was high in the context of a low serum osmolality on the second hospital day. The infusion rate was increased, and the patient's consciousness level returned to normal. However, her normalized serum Na level declined again as she drank much water to reduce throat discomfort. As the throat discomfort caused by the throat inflammation improved with azulene gargling, her water intake was reduced, and the serum Na concentration returned to normal. Thus, polydipsia caused by a throat inflammation partially contributed to hyponatremia in this patient. We note that increased ADH secretion has been reported in adults with acute respiratory infection. Therefore, we concluded that polydipsia caused by the throat inflammation, plus increased ADH secretion, resulted in hyponatremia in this patient. We should pay attention to the behavior of drinking extra fluid in patients with acute respiratory infections.

Mineralocorticoid receptor (MR) antagonist spironolactone (SPL) is an effective agent for prevention of cardiovascular injury. However, whether and how SPL ameliorates hepatic fibrosis in rats is unknown. Pig serum (PS) (0.5 mL, twice a week, ip) or vehicle-administered rats for 12 weeks were used as rats with hepatic fibrosis or control rats, respectively. Rats given PS were treated with SPL (50 mg/kg/day, sc) for 12 weeks. Hepatic fibrosis, using picro-sirius red staining and determination of hydroxyproline content, immunohistochemistries of alpha-smooth muscle actin (alpha-SMA)-positive hepatic stellate cells (HSCs), Na/H exchange isoform-1 (NHE-1) protein, CYP11B2 aldosterone synthase protein for liver tissues, and plasma aldosterone concentrations were compared among the 3 groups of rats. Rats given PS alone exhibited hepatic fibrosis as well as increases in the number of the alpha-SMA-positive HSCs and NHE-1 protein expression in HSCs and hepatocytes, all of which were suppressed by SPL. Rats given PS alone revealed increased CYP11B2 protein expression in HSCs and hepatocytes, which was not inhibited by SPL. Plasma aldosterone concentrations were significantly greater in rats given PS and SPL than in control rats and rats given PS alone, although they were not different between control rats and rats given PS alone. PS-induced hepatic fibrosis together with HSC activation and NHE-1 protein expression occurs via MRs, and SPL ameliorates hepatic fibrosis presumably via the inhibition of HSC activation and NHE-1 protein expression in PS-induced liver injuries. The aldosterone produced in the injured liver contributes to the PS-induced hepatic fibrosis.

Spironolactone prevents early renal injury in streptozotocin-induced diabetic rats. Background Glomerular and tubulointerstitial injury leads to chronic impairment of renal function, and thus, reversal of the injury may improve renal function and survival. The present study investigated whether and how mineralocorticoid receptor antagonist spironolactone ameliorates early renal injury in streptozotocin-induced diabetic rats. Methods Streptozotocin (65 mg/kg, single intraperitoneal injection)- or vehicle-administered rats were used as diabetic or control rats, respectively. The streptozotocin-administered rats were treated with spironolactone (50 mg/kg/day sc) for 3 weeks. Among the 3 groups of rats, we compared renal fibrosis and renal hypertrophy, using picro-sirius red staining and immunohistochemistry of ED-1 macrophage marker, plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor (TGF)-β1. Results Three weeks after administration of streptozotocin, rats exhibited increased collagen deposition in glomerular, tubulointerstitial, and perivascular areas in the kidney, which was completely attenuated by spironolactone treatment. In rats given streptozotocin alone, there were increases in ED-1–positive cell, PAI-1 expression, and TGF-β1 expression in glomeruli and tubulointerstitiums, which were also suppressed by spironolactone treatment. Maximal glomerular and proximal tubular areas were not significantly different among the 3 groups. Rats given streptozotocin alone revealed an increase in proximal tubule wall-to-lumen ratio that was not influenced by treatment with spironolactone. Conclusion Streptozotocin-induced renal fibrosis, PAI-1 expression, TGF-β1 expression, and macrophage infiltration occur via mineralocorticoid receptor, and spironolactone ameliorates renal fibrosis presumably via the inhibition of macrophage infiltration, PAI-1 expression, and TGF-β1 expression in streptozotocin-induced early diabetic injury.

The rapid (1-4 h) responses of epithelial target tissues to mineralocorticoids contrast with the days/weeks apparently required for responses in the cardiovascular system. The present study explores the time course and pattern of early events leading to cardiac fibrosis in the mineralocorticoid-salt rat model. Uninephrectomized rats were given deoxycorticosterone (20 mg, sc, weekly) plus 0.9% NaCl/0.3% KCl to drink and were killed at 2, 4, 8, 16, and 32 d. Type III collagen increased progressively from d 2, and blood pressure from d 4, with 4 and 8 d rats showing marked perivascular inflammatory cell infiltration. Apoptosis was also noted in perivascular areas at 4 and 8 d and in scar areas at 8, 16, and 32 d. Elevation of mineralocorticoid hormone levels inappropriate for salt status thus provokes a series of changes in cardiac vessels and myocytes leading to increased collagen deposition. When mineralocorticoid levels are elevated acutely by bolus injection, changes are discernible after 2 d, in contrast with previous infusion studies in which 3-4 wk were required for measurable changes.

We demonstrated the functional evaluation of adrenal incidentaloma in 8 patients who had no abnormal finding associated with Cushing's syndrome. Adrenal tumors were incidentally discovered by abdominal echogram in 5 patients and by computed tomography (CT) in 3 patients. Serum cortisol levels and urinary excretion of 17-hydroxycorticosteroids (17-OHCS) were within normal limits in four of 8 patients. Urinary excretion of free cortisol was also within normal limits except for patient 8. Urinary excretion of 17-OHCS, however, was not suppressed by dexamethasone administration in five of 8 patients. Excretion of urinary 17-OHCS did not increase in response to metyrapone in 3 of 4 dexamethasone-insuppressible patients, but increased in 3 dexamethasone-suppressible ones. Serum cortisol increased in response to exogenous ACTH in all 6 patients examined. 131I-Adosterol accumulation was found in only the tumor side in 7 patients and bilaterally in one patient. Adrenalectomy was done in 7 patients, and microscopic findings showed adrenocortical adenoma. Serum cortisol was significantly suppressed in response to dexamethasone in the post-operative stage in all 7 patients examined. These results indicate that these adrenal incidentalomas seem to have a cortisol over-production which is dexamethasone-insuppressible and ACTH-dependent, and that they can be classified as "Pre-Cushing's Syndrome."

To examine whether and how heart ANG II influences the coordination between cardiomyocyte hypertrophy and coronary angiogenesis and contributes to the pathogenesis of diabetic cardiomyopathy, we used Spontaneously Diabetic Torii (SDT) rats treated without and with olmesartan medoxomil (an ANG II receptor blocker). In SDT rats, left ventricular (LV) ANG II, but not circulating ANG II, increased at 8 and 16 wk after diabetes onset. SDT rats developed LV hypertrophy and diastolic dysfunction at 8 wk, followed by LV systolic dysfunction at 16 wk, without hypertension. The SDT rat LV exhibited cardiomyocyte hypertrophy and increased hypoxia-inducible factor-1α expression at 8 wk and to a greater degree at 16 wk and interstitial fibrosis at 16 wk only. In SDT rats, coronary angiogenesis increased with enhanced capillary proliferation and upregulation of the angiogenic factor VEGF at 8 wk but decreased VEGF with enhanced capillary apoptosis and suppressed capillary proliferation despite the upregulation of VEGF at 16 wk. In SDT rats, the phosphorylation of VEGF receptor-2 increased at 8 wk alone, whereas the expression of the antiangiogenic factor thrombospondin-1 increased at 16 wk alone. All these events, except for hyperglycemia or blood pressure, were reversed by olmesartan medoxomil. These results suggest that LV ANG II in SDT rats at 8 and 16 wk induces cardiomyocyte hypertrophy without affecting hyperglycemia or blood pressure, which promotes and suppresses coronary angiogenesis, respectively, via VEGF and thrombospondin-1 produced from hypertrophied cardiomyocytes under chronic hypoxia. Thrombospondin-1 may play an important role in the progression of diabetic cardiomyopathy in this model.

Therapeutic efficacy of the non-peptide AVP antagonist OPC-31260 in cirrhotic rats. The present study was undertaken to determine whether a non-peptide arginine vasopressin (AVP) antagonist [5-dimethylamino-1-{4-(2-methylbenzoylamino)benzoyl}-2,3,4,5-tetrahydro-1H-benzaze-pine] (OPC-31260) improves the impaired water excretion in rats with experimental liver cirrhosis. Male Wistar rats weighing 200 to 250g were injected in an equal volume (4 ml/kg) of carbon tetrachloride and olive oil at an interval of seven days for three months, causing liver cirrhosis with ascites. Control rats were injected with only olive oil. Body weight (body wt) and hematocrit (Hct) were lower in the cirrhotic rats than the control rats (body wt 360.7 vs. 238.5 g, P < 0.01; Hct 46.3 vs. 39.2%, P < 0.01). A water loading test (30 ml/kg) was carried out and 20-minute urine collections were made for three hours. The percent of water load excreted was 62.5% in the cirrhotic rats, a value significantly less than that of 102.1% in the control rats. However, its percent increased to 215.1% after the oral administration of 5 mg/kg OPC-31260 (P < 0.01). Minimal urinary osmolality (UOsm) was 185.5 mOsm/kg H2O in the cirrhotic rats receiving the vehicle, a value greater than the control rats of 125.5 mOsm/kg H2O (P < 0.01). The oral administration of 5 mg/kg OPC-31260 reduced minimal UOsm to 85.2 mOsm/kg H2O in the cirrhotic rats (P < 0.01). Urinary excretion of sodium was lower in the cirrhotic rats than the control rats (87.1 vs. 312.4 µEq/3 hr, P < 0.01). The cirrhotic rats had lower GFR than the controls (2.2 vs. 2.9 ml/min, P < 0.05). Plasma AVP levels in the cirrhotic rats were 4.2 pg/ml, a value greater than the control rats of 1.7 pg/ml (P < 0.01). Plasma AVP levels were reduced to 2.4 pg/ml in the cirrhotic rats 60 minutes after the water load, while they were suppressed to 0.9 pg/ml in the control rats. These results indicate that the unsuppressible secretion of AVP is involved in the impaired water excretion in the cirrhotic rats and that the orally effective, non-peptide AVP antagonist OPC-31260 is an effective therapeutic for the water retention in decompensated liver cirrhosis.

We studied the changes in serum sodium (Na) and potassium (K) levels in seventeen patients in diabetic ketoacidosis and nine patients in non-ketotic hyperosmolar coma, who had marked hyperglycemia (707.4 +/- 75.6 mg/dl, mean +/- SEM) and dehydration. The disorder characterized two types of alteration. The one group was hyponatremia with hyperkalemia in 17 patients in diabetic ketoacidosis (132.9 +/- 2.0 and 5.7 +/- 0.2 mEq/l), and 4 patients in non-ketotic hyperosmolar coma (125.8 +/- 4.3 and 5.2 +/- 0.5 mEq/l). The other was hypernatremia (162.5 +/- 1.8 mEq/l) with hypokalemia (3.4 +/- 0.2 mEq/l) in 5 patients in non-ketotic hyperosmolar coma. Intensive therapy with insulin and fluid administration improved the diabetic hyperglycemia and associated abnormalities. The vectors showing the normalization of serum Na and K levels was in quite opposite directions between the patients with hyponatremia with hyperkalemia and those with hypernatremia with hypokalemia. The amounts of loss of circulatory blood volume exceeded 20% in three groups of patients, a loss greater in the hypernatremic patients than in the hyponatremic ones. These results indicate that serious body water depletion produces hypernatremia instead of hyponatremia in patients in diabetic coma. The disorder may be caused by the altered distribution of electrolytes between the intra- and extra-cellular spaces.

Objectives Excess mineralocorticoids such as deoxycorticosterone acetate (DOCA) together with salt are known to cause tubulointerstitial fibrosis, but the mechanisms underlying fibrosis progression are unclear. Therefore, we investigated the role of matrix metalloproteinase 2 (MMP2) in the epithelial-mesenchymal transition and fibrosis progression. Methods Uninephrectomized rats drank 0.9% NaCl and 0.3% KCl solution and were treated with DOCA alone, DOCA + spironolactone, or vehicle for 1, 4, or 8 weeks. SBP, kidney function and morphology, and kidney and urine MMP2 activity were compared among the groups. Results At week 4, the DOCA-treated group exhibited hypertension, tubulointerstitial fibrosis, increased MMP2 activity in the kidney and urine, and overexpression of MMP2 in proximal tubule cells and MMP14 in apical membranes; these results were more pronounced at week 8. At week 8, the proximal tubule cell apicolateral surface proteins villin, claudin 2, and E-cadherin were downregulated, and the mesenchymal marker α-smooth muscle actin was upregulated in the tubulointerstitium of DOCA-treated rats. These DOCA/salt-induced changes (except for hypertension) and fibrosis progression observed at week 8 were reversed by TISAM (a selective MMP2 inhibitor), which was administered from week 4 to week 8. All of the effects of DOCA/salt at week 8 were attenuated by spironolactone. Conclusion Eight weeks of treatment with DOCA/salt activated MMP2, primarily on the apical surface of proximal tubule cells, which induced epithelial-mesenchymal transition from the luminal side and promoted tubulointerstitial fibrosis progression. These MMP2-induced changes occurred via downstream processes regulated by mineralocorticoid receptors.

Therapeutic efficacy of non-peptide ADH antagonist OPC-31260 in SIADH rats. The present study was undertaken to determine whether the non-peptide V2 antidiuretic hormone (ADH) antagonist 5-dimethylamino-1{4-(2-methylbenzoylamino)benzoyl}-2,3,4,5-tetrahydro-1H-benzazepine (OPC-31260) normalized hyponatremia in rats with an experimental syndrome of inappropriate secretion of ADH (SIADH). Rats were administered V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) subcutaneously at a rate of 5 ng/hr using an osmotic minipump and a 40 ml/day liquid diet. Serum sodium levels (SNa) and serum osmolality (SOsm) markedly decreased to 119 mEq/liter and 249 mOsm/kg H2O, respectively, 48 hours after the start of dDAVP administration. Hyponatremia persisted in a similar magnitude during the observation period of 14 days. On days 7 to 13 OPC-31260, administered 5 mg/kg per day orally, promptly raised SNa and SOsm to 134 mEq/liter and 282 mOsm/kg H2O in half a day, respectively, followed by the normalization of SNa and SOsm during the rest of the observation period. The cease of administration of OPC-31260 again decreased SNa and SOsm in rats receiving dDAVP. In contrast, SNa and SOsm were within the normal values in rats receiving 0.15 M NaCl, a vehicle for dDAVP, in the presence or absence of OPC-31260. The administration of OPC-31260 promptly caused marked water diuresis on day 7 in the hyponatremic rats receiving dDAVP, namely 5 mg/kg OPC-31260 markedly increased urinary volume and decreased UOsm. These results indicate that there is dilutional hyponatremia in rats receiving dDAVP and 40 ml/day liquid diets, and that OPC-31260 is an effective therapeutic for hyponatremia associated with dDAVP-induced SIADH.

Long-term exposure of uninephrectomized rats to desoxycorticosterone acetate (DOCA)/salt induces cardiac fibrosis and hypertrophy through mineralocorticoid receptors (MRs). However, the underlying cellular mechanisms remain unclear. To determine whether Na/H exchange isoform 1 (NHE1) is involved in the cellular mechanisms, we examined the effects of a specific NHE1 inhibitor, cariporide, and an MR antagonist, spironolactone, on DOCA/salt-induced cardiac fibrosis and hypertrophy. Uninephrectomized rats were given 20 mg of DOCA (single subcutaneous injection) plus 0.9% NaCl/0.3% KCl to drink and were killed at 8 days. Two groups of rats given DOCA/salt were treated with either spironolactone (50 mg/kg per day SC) or cariporide (30 mg/kg per day PO) for 8 days. Control rats were treated with only high salt after the operation. The DOCA/salt-induced perivascular collagen deposition was completely abolished by cariporide and spironolactone. DOCA/salt-induced interstitial collagen deposition was partially and completely suppressed by spironolactone and cariporide, respectively. The rats exposed to DOCA/salt had cardiocyte hypertrophy in the subendocardial and subepicardial regions, a finding that was completely inhibited by cariporide but not by spironolactone. In rats given DOCA/salt, NHE1 protein expression was markedly increased. This was partially and completely reversed by spironolactone and cariporide, respectively. We concluded that cardiac NHE1 contributes to DOCA/salt-induced cardiac fibrosis and hypertrophy and that the NHE1 inhibitor cariporide completely prevents the detrimental effects of DOCA/salt on the heart. We also demonstrated that DOCA/salt-induced cardiac injury through the MRs partly occurs through NHE1 activation.

To clarify the mechanisms of hyperglycemia-induced proliferation of vascular smooth muscle cells (VSMC), we examined the effects of high glucose (HG) on nuclear factor (NF)-kappaB and E2F-1. Angiotensin II (Ang II) significantly enhanced DNA binding activity of NF-kappaB under HG (25.6 mM) conditions with an increase in p65 subunit of NF-kappaB, and did it slightly under normal glucose (NG; 5.6 mM) conditions. Ang II failed to induce E2F-1 expression, or its binding to the cdc2 promoter, even under HG conditions. HG greatly augmented the cdc2 inducibility of fetal calf serum (FCS), through the increase in E2F-1 activity. These data indicate that hyperglycemia contributes to abnormal proliferation of VSMC by two mechanisms; the induction of NF-kappaB activation by Ang II, which facilitates transcription of certain growth factors, and the augmentation of E2F-1 in response to growth factors.

OBJECTIVE Abnormal bone metabolism in patients with Graves' thyrotoxicosis is well documented, but the precise time-course of its recovery remains poorly understood. The present study was undertaken to clarify longitudinal improvement in bony manifestations, especially in cortical bone, and bone metabolic markers in thyrotoxicosis. DESIGN Two year prospective follow-up study in patients with Graves' disease. PATIENTS Ten consecutive patients with Graves' disease (seven males and three females, of mean (±SEM) age 39.3 ± 3.9 years) were enrolled in the study and treated with antithyroid drugs. Thirteen sex- and age-matched patients with the disease in remission served as controls. MEASUREMENTS Bony manifestations were evaluated both by fine cortical bone striations in the metacarpals on magnified roentgenograms and lumbar bone mineral density (BMD) measurement. Urinary deoxypyridinoline (dPYR) and serum pyridinoline cross-linked telopeptide domain of type I collagen (ICTP) were monitored as markers of bone resorption, as well as serum osteocalcin (OC), carboxy-terminal propeptide of type I procollagen (PICP) and alkaline-phosphatase (ALP) as markers of bone formation. RESULTS Initial elevated free thyroid hormone levels were normalized within a month of starting therapy. Striation indices of the metacarpals were 1.89 ± 0.16 before therapy, higher than those of 0.49 ± 0.12 in the controls (P

The present study was undertaken to determine the pathophysiological role of arginine vasopressin (AVP) in elderly patients with hyponatremia, and the efficacy of fludrocortisone acetate in treating their hyponatremia. Eleven hospitalized patients aged 65 years or older whose serum sodium levels were less than 130 mEq/l were examined. The hyponatremic patients included two groups of patients: syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and central salt-wasting syndrome. And 24 healthy, young subjects aged 20 to 34 years, and 24 healthy, elderly subjects age 65 to 80 years were recruited by community announcement. The elderly subjects had decreased urinary concentrating ability and exaggerated response of AVP secretion to osmotic and nonosmotic stimuli, as compared to the young subjects. All the patients had hyponatremia, with the exaggerated urinary loss of Na. Plasma AVP levels were elevated despite hypoosmolality in all the 2 groups of hyponatremic, elderly patients. Plasma renin activity and plasma aldosterone concentrations were low in the patients with SIADH and central salt-wasting syndrome. Fludrocortisone acetate therapy was effective in the patients with central salt-wasting syndrome and 3 patients with SIADH whose hyponatremia remained unchanged after water restriction. Water restriction therapy normalized serum Na levels in only 3 patients with SIADH. These results indicate that AVP is involved in the mechanism for hyponatremia in the elderly patients with SIADH and central salt-wasting syndrome. Severe hyponatremia associated with SIADH and central salt-wasting syndrome responds well to mineralocorticoid therapy. Both the secretion of AVP and renal sodium handling may be involved in the mechanism of action of the disorders. The diagnostic criteria for SIADH in the elderly patients may have to be reevaluated and should be considered to indicate fludrocortisone acetate therapy.

We found symptomatic hyponatremia in four elderly patients in which serum sodium (Na) levels ranged from 101 to 122 mEq/l. All 4 patients had low levels of plasma adrenocorticotropic hormone (ACTH), serum cortisol, and urinary excretion of 17-OHCS, and poor responses of ACTH to exogenous insulin and antidiuretic hormone (ADH). Other pituitary hormones were all normal. They were therefore diagnosed as having isolated ACTH deficiency. Plasma ADH was relatively high despite hypoosmolality which was associated with the hyponatremia. Water loading test revealed impaired water excretion and poor suppression of plasma ADH. Replacement with 20-30 mg hydrocortisone completely restored the serum Na level and restored the plasma ADH level to the normal range in all 4 patients. Other factors such as decreased glomerular filtration, enhanced urinary Na loss and decreased Na intake were also included. These results indicate that there is marked hyponatremia and that in the presence of hypoosmolality the sustained secretion of ADH is the key factor in causing the impaired water excretion and hyponatremia in isolated ACTH deficiency.