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A link between hypertension and long-term bone health has been suggested. The aim of this study was to investigate the effects of chronic angiotensin II administration on urinary calcium/phosphate excretion, bone mineral density, bone remodeling and osteoblast population in a well-established experimental model of hypertension, in the absence of possible confounding factors that could affect bone metabolism. Male Sprague–Dawley rats, divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis, n = 8); (b) Ang II+losartan (Los, 50 mg/kg/day, per os, n = 6); (c) control group (physiological saline, sub cutis, n = 9); and (d) control+losartan (n = 6) were treated for four weeks. During the experimental period, 24-hour diuresis, urinary calcium, phosphate and sodium excretion were measured prior to the treatment, at two weeks of treatment, and at the end of the treatment. Systolic blood pressure was measured by plethysmography technique (tail cuff method). At the end of the experimental protocol, the rats were euthanized and peripheral quantitative computed tomography at the proximal metaphysis and at the diaphysis of the tibiae and quantitative bone histomorphometry on distal femora were performed. Angiotensin II-dependent hypertension is associated with increased calcium and phosphate excretion. AT1 receptor blockade prevented the increase of blood pressure and phosphate excretion but did not affect the increase of calcium excretion. These changes took place without significantly affecting bone density, bone histology or osteoblast population. In conclusion, in our experimental conditions, angiotensin II-dependent hypertension gave rise to an increased urinary excretion of calcium and phosphate without affecting bone density.

Background: Clinical trials have shown that empagliflozin (Empa), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, promotes nephroprotective effects in diabetic patients. The mechanisms underlying nephroprotection are not completely known and it is not known whether the renal beneficial action is present even in non-diabetic kidney disease. The aim of this study was to evaluate the effect of Empa administration on the development of renal fibrosis in an experimental model of angiotensin II (Ang II)-dependent hypertension. Methods: Sprague Dawley rats (n = 31) were divided into 4 experimental groups. Ang II (200 ng/kg/min, osmotic minipumps, s.c., n = 9) or Ang II + Empa (10 mg/kg/day, per os, n = 10) were administered for 2 weeks. Control rats were treated with placebo (physiological saline, n = 6), and another group was treated with placebo plus Empa (n = 6) for the same period. Blood pressure (plethysmographic method) was measured at the beginning and at the end of the experimental protocol. After 2 weeks, the rats were euthanized and the kidneys were excised for histomorphometric evaluation of glomerular and tubulo-interstitial fibrosis and for the immunohistochemical evaluation of inflammatory infiltrates (monocytes/macrophages) and types I and IV collagen expression. Results: The administration of Ang II resulted in an increase in blood pressure (p < 0.01), glomerular (p < 0.05) and tubulo-interstitial (p < 0.01) fibrosis, renal inflammatory infiltrates (p < 0.01) and type I (p < 0.01) and type IV collagen expression (p < 0.05) compared to the control group. Treatment with Empa did not significantly modify the increase in blood pressure due to Ang II, but prevented the development of renal glomerular and tubulo-interstitial fibrosis, and the increase in inflammatory infiltrates and types I and IV collagen expression in Ang II-treated rats (p < 0.01). Conclusions: These data demonstrate that the treatment with Empa prevents the development of renal fibrosis in Ang II-dependent hypertension. In Ang II-dependent hypertension, the anti-fibrotic effect due to SGLT2 inhibition is caused by the reduction of inflammatory infiltrates and it is independent on the modulation of blood pressure increase.

Compound 21 (C21), an AT2 receptor agonist, and Angiotensin 1-7 (Ang 1-7), through Mas receptor, play an important role in the modulation of the protective arm of the renin-angiotensin system. The aim of this study was to investigate in an experimental model of angiotensin II-dependent hypertension whether the activation of the potentially protective arm of the renin-angiotensin system, through AT2 or Mas receptor stimulation, counteracts the onset of myocardial fibrosis and hypertrophy, and whether these effects are mediated by inflammatory mechanism and/or sympathetic activation. Sprague Dawley rats (n = 67) were treated for 1 (n = 25) and 4 (n = 42) weeks and divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis); (b) Ang II+Compound 21 (C21, 0.3 mg/kg/day, intraperitoneal); (c) Ang II+Ang 1-7 (576 µg/kg/day, intraperitoneal); (d) Ang II+Losartan (50 mg/kg/day, per os); (e) control group (physiological saline, sub cutis). Systolic blood pressure was measured by tail cuff method and, at the end of the experimental period, the rats were euthanized and the heart was excised to evaluate myocardial fibrosis, hypertrophy, inflammatory cell infiltration and tyrosine hydroxylase expression, used as marker of sympathetic activity. Ang II caused a significant increase of blood pressure, myocardial interstitial and perivascular fibrosis and myocardial hypertrophy, as compared to control groups. C21 or Ang 1-7 administration did not modify the increase in blood pressure in Ang II treated rats, but both prevented the development of myocardial fibrosis and hypertrophy. Treatment with losartan blocked the onset of hypertension and myocardial fibrosis and hypertrophy in Ang II treated rats. Activation of AT2 receptors or Mas receptors prevents the onset of myocardial fibrosis and hypertrophy in Ang II-dependent hypertension through the reduction of myocardial inflammatory cell infiltration and tyrosine hydroxylase expression. Unlike what happens in case of treatment with losartan, the antifibrotic and antihypertrophic effects that follow the activation of the AT2 or Mas receptors are independent on the modulation of blood pressure.

Summary: The aim of this study was to evaluate the role of the angiotensin II (Ang II) induced-differential miRNA expression in renal glomerular and tubulo-interstitial fibrosis in an experimental model of Ang II-dependent hypertension. To clarify this issue, Sprague Dawley rats were treated with Ang II (200 ng/kg per minute, n = 15) or physiological saline (n = 14) for 4 weeks. Systolic blood pressure and albuminuria were measured every 2 weeks. At the end of the experimental period, renal glomerular and tubulo-interstitial fibrosis was evaluated by histomorphometric analysis, after Sirius-Red and Masson's trichrome staining. Ang II increased systolic blood pressure (P < 0.0001), albuminuria (P < 0.01) and both glomerular and tubulo-interstitial fibrosis (P < 0.01). Using laser capture microdissection and miRNA microarray analysis this study showed that miR-29a-3p was down-regulated in renal tubules and up-regulated in glomeruli. Real-time polymerase chain reaction (PCR) experiments confirmed in Ang II-treated rats a down-regulation of miR-29a-3p in tubules (P < 0.01), while no significant changes were observed in glomeruli. Matrix metalloproteinase-2 (MMP-2) was identified as putative miR-29a-3p target (by TargetScan, miRanda, Tarbase software) and functionally confirmed by luciferase activity assay. These data demonstrate that the effects of Ang II on miR-29a-3p expression in renal tubules is different from the one exerted in the glomeruli and that miR-29a-3p targets MMP-2. These results suggest that the development of renal fibrosis at glomerular and tubulo-interstitial level depends on different molecular mechanisms.