Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy.

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.

Matrix metalloproteinase 2 induces epithelial-mesenchymal transition in proximal tubules from the luminal side and progresses fibrosis in mineralocorticoid/salt-induced hypertensive rats.

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.

Spironolactone suppresses peritubular capillary loss and prevents deoxycorticosterone acetate/salt-induced tubulointerstitial fibrosis.

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-beta1 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-1alpha) 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.

Hyponatremic seizure associated with acute respiratory infection.

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 antagonist spironolactone prevents pig serum-induced hepatic fibrosis in rats.

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)-beta1. 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-beta1 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-beta1 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-beta1 expression in streptozotocin-induced early diabetic injury.

Na/H exchange isoform 1 is involved in mineralocorticoid/salt-induced cardiac injury.

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.

Hyperglycemia enhances VSMC proliferation with NF-kappaB activation by angiotensin II and E2F-1 augmentation by growth factors.

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.