Toru Aizawa

In Vivo klotho Gene Transfer Ameliorates Angiotensin II-Induced Renal Damage

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3×1010 plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.

Renoprotective Properties of Angiotensin Receptor Blockers beyond Blood Pressure Lowering

Clinical studies have demonstrated that some antihypertensive agents provide renoprotection independent of BP lowering. Recent in vitro and in vivo studies evaluated the mechanisms involved in this protection. First, the in vitro effects of several angiotensin II type 1 receptor blockers (ARB), calcium channel blockers (CCB), and beta blockers (BB) on various mediators were compared: Formation of pentosidine (an advanced glycation end product), hydroxyl radical-induced formation of o-tyrosine, and transition metals-induced oxidation of ascorbic acid (the Fenton reaction). All of the six tested ARB but neither the six CCB nor the nine BB inhibited pentosidine formation. ARB, as well as BB but not CCB, inhibited hydroxyl radicals-mediated o-tyrosine formation. ARB but neither BB nor CCB inhibited efficiently transition metals-catalyzed oxidation of ascorbic acid. Second, the in vivo consequences for the kidney of these various in vitro effects were evaluated. Hypertensive, type 2 diabetic rats with nephropathy, SHR/NDmcr-cp, were given for 20 wk either olmesartan (ARB) or nifedipine (CCB), or atenolol (BB). Despite similar BP reduction, only ARB significantly reduced proteinuria and prevented glomerular and tubulointerstitial damage (mesangial activation, podocyte injury, tubulointerstitial injury, and inflammatory cell infiltration). It is interesting that only ARB prevented abnormal iron deposition in the interstitium, corrected chronic hypoxia, reduced expressions of heme oxygenase and p47phox (a subunit of NADPHoxidase), and inhibited pentosidine formation (which correlates well with proteinuria). These observations confirm unique renoprotective properties of ARB, independent of BP lowering but related to decreased oxidative stress (hydroxyl radicals scavenging and inhibition of the Fenton reaction), correction of chronic hypoxia, and inhibition of advanced glycation end product formation and of abnormal iron deposition. These benefits of ARB may contribute to the renoprotection observed beyond BP lowering.

Heme Oxygenase-1 Is Upregulated in the Kidney of Angiotensin II–Induced Hypertensive Rats: Possible Role in Renoprotection

In this study, we investigated the regulation and physiological role of heme oxygenase-1 (HO-1) in the kidney of rats with hypertension. Rats were continuously administered either angiotensin II (Ang II) or norepinephrine with an osmotic minipump for up to 7 days. Ang II infusion decreased the glomerular filtration rate (GFR) as determined through creatinine clearance (3.2+/-0.2 versus 1.2+/-0.2 mL/min with Ang II infusion, P<0.01) and increased proteinuria (9. 7+/-1.3 versus 28.1+/-7.2 mg/d with Ang II infusion, P<0.01). In contrast, norepinephrine did not alter these laboratory values. Ang II infusion significantly increased HO-1 expression in mRNA (442+/-98% of control at day 5, P<0.01) and protein levels (314+/-49% of control at day 5, P<0.01). Immunohistochemistry showed that in the kidney of normotensive rats, HO-1 was expressed mainly in the basal side in the renal tubules. After Ang II infusion, HO-1 staining was more extensively dispersed in the tubular epithelial cells. The intraperitoneal administration of zinc protoporphyrin, an HO inhibitor, to Ang II-infused rats further decreased GFR (0.8+/-0. 1 mL/min) and increased proteinuria (52.5+/-13.0 mg/d). In contrast, the administration of hemin, an HO inducer, ameliorated the Ang II-induced decrease in GFR (2.4+/-0.2 mL/min) and increase in proteinuria (9.3+/-4.5 mg/d). These data suggest that HO-1 upregulation in the kidney of Ang II-induced hypertensive rats may exert a renoprotective effect against Ang II-induced renal injury.

Abnormal Iron Deposition in Renal Cells in the Rat with Chronic Angiotensin II Administration

Acute experimental iron loading causes iron to accumulate in the renal tissue. The accumulation of iron may play a role in enhancing oxidant-induced tubular injury by producing increased amounts of reactive oxygen species. From findings in cells from heme oxygenase-1 (HO-1)-deficient mice, HO-1 is postulated to prevent abnormal intracellular iron accumulation. Recently, it has been reported that HO-1 is induced in the renal tubular epithelial cells, in which iron is deposited after iron loading, and that this HO-1 induction may be involved in ameliorating iron-induced renal toxicity. We previously showed that chronic administration of angiotensin II to rats induces HO-1 expression in the tubular epithelial cells. These observations led us to investigate whether there is a link between iron deposition and HO-1 induction in renal tubular cells in rats undergoing angiotensin II infusion. In the present study, rats were given angiotensin II for continuously 7 days. Prussian blue staining revealed the distinct deposits of iron in the proximal tubular epithelial cells after angiotensin II administration. Electron microscopy demonstrated that iron particles were present in the lysosomes of these cells. Histologic and immunohistochemical analyses showed that stainable iron and immunoreactive ferritin and HO-1 were colocalized in the tubular epithelial cells. Treatment of angiotensin II-infused rats with an iron chelator, deferoxamine, blocked the abnormal iron deposition in kidneys and also the induced expression of HO-1 and ferritin expression. Furthermore, deferoxamine treatment suppressed the angiotensin II-induced increase in the urinary excretion of protein and N-acetyl-β-d-glucosaminidase, a marker of tubular injury; however, deferoxamine did not affect the angiotensin II-induced decrease in glomerular filtration rate. These results suggest that angiotensin II causes renal injury, in part, by inducing the deposition of iron in the kidney.

Increased leukotriene A4 hydrolase expression in the heart of angiotensin II‐induced hypertensive rat

Leukotriene A4 (LTA4) hydrolase is essential for the conversion of LTA4 to LTB4, an inflammatory lipid mediator. We investigated whether LTA4 hydrolase was regulated in the heart by angiotensin II (ang II) infusion. Continuous ang II infusion via an osmotic minipump for up to 7 days upregulated mRNA and protein levels of LTA4 hydrolase (∼3.5‐fold of control) in the heart in a pressor‐dependent manner. Immunohistochemistry demonstrated intense LTA4 hydrolase staining in the myofibroblast as well as migrated monocytes/macrophages. These data suggest that the cardiac LTA4 hydrolase‐LTB4 system plays a positive role in the promotion of cardiac inflammation in hypertension.