Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Clinical evidence for the effects of pharmacological interventions with angiotensin II (AngII) type 1 (AT1) receptor blockers (ARBs) indicates that AngII exerts a fundamental role in the pathogenesis of hypertension and renal injury [1]. It has also been made apparent that activation of the intrarenal renin–angiotensin system (RAS) is regulated in a manner distinct from the circulating RAS [2]. Reduced renal function and renal structural damage are associated with inappropriate activation of the intrarenal AngII [2]. Double transgenic mice, expressing human renin systemically and human angiotensinogen in proximal tubular cells, show increased intrarenal AngII levels with unchanged plasma AngII levels [3]. High blood pressure and renal inflammation or collagen accumulation were observed in these mice, suggesting that the selective augmentation of AngII production in proximal tubules causes hypertension and renal injury [3]. Selective inhibition of renal AT1 receptor expression by intrarenal infusion of AT1 receptor antisense oligodeoxynucleotides resulted in natriuresis in spontaneously hypertensive rats (SHR) [4]. Kidney cross-transplantation experiments showed that the mere presence of AT1a receptors in proximal tubules is sufficient to reproduce the phenotype of AngII-dependent hypertension [5]. It has also been observed that AngII infusion in mice lacking renal AT1a receptors enhanced natriuresis and resistance to hypertension, suggesting a critical role of intrarenal AT1 receptors in the development of hypertension [5]. In-vitro studies have also shown that AngII directly stimulates sodium handling through AT1 receptor-dependent pathways in proximal tubular cells [6]. Furthermore, AngII-mediated epithelial mesenchymal transition leads to the secretion of extracellular matrix in proximal tubular cells [7]. Collectively, these data indicate that the activation of AT1 receptors in proximal tubules by locally produced AngII plays an important role in the pathogenesis of hypertension and renal injury. Over the last decade, evidence has accumulated that the production of reactive oxygen species (ROS) is facilitated by NADPH oxidase during the development of hypertension and renal injury [8]. In particular, many of the effects of the AngII/AT1 receptor are dependent on ROS production stimulated by the activation of NADPH oxidase. ROS generated by NADPH oxidase can also act as signaling molecules that mediate AngII-dependent signal transduction pathways (redox-sensitive signaling) [8,9]. Data from in-vivo studies have indicated that NADPH oxidase-dependent ROS production contributes to AngII-dependent regulation of renal hemodynamics, sodium reabsorption and hypertension [10–12]. In-vitro studies also showed that NADPH oxidase inhibition attenuates AngII-induced superoxide production and cellular hypertrophy in cultured proximal tubular cells [13]. Similarly, treatment with an antioxidant, dimethylthiourea, abolished AngII-induced apoptosis in proximal tubules in rats [14]. More recently, Liu et al. [15] showed that treatment with a specific NADPH oxidase inhibitor, apocynin, decreased NADPH oxidase activity and attenuated proximal tubular apoptosis and tubulointerstitial fibrosis in mice with proximal tubular cell-specific overexpression of angiotensinogen. These data are consistent with the concept that locally generated AngII in proximal tubules induces renal injury through NADPH oxidase-dependent ROS production. NADPH oxidase is an enzyme composed of several membrane components, such as gp91phox homologues (Nox1 and Nox4) and p22phox, and intracellular components, such as p47phox, p67phox and rac1 [8,9]. Among the membrane components of NADPH oxidase, Nox4 is abundantly expressed in kidney epithelium, but also found in cultured mesangial cells and podocytes [8]. Nox4 plays a functional role in AngII-dependent signaling pathways in mesangial cells [16]. Furthermore, AngII increased Nox4 expression in podocytes [17]. However, the precise mechanisms by which AngII increases Nox4 in the kidney have not been determined. Caveolae are cell membrane invaginations and subsets of rafts in a variety of cells, including proximal tubular cells [18]. Several lines of studies indicate potential roles of the major structural protein of caveolae, caveolin, in AngII/AT1 receptor-dependent signal transduction [19]. AngII rapidly translocates the AT1 receptors from the general plasma membrane fraction to the caveolin-1-enriched fractions [19–21]. Caveolin acts as a molecular chaperone rather than a plasma membrane scaffold for AT1 receptors [21]. Recent studies have also suggested that caveolin plays an important role as a platform for the compartmentalization of redox signaling events through NADPH oxidase-dependent production of ROS [22]. Thus, it can be speculated that caveolin is involved in AngII-induced NADPH activation in the kidney. In this issue of Journal of Hypertension, Bocanegra et al. [23] investigated the possible contributions of caveolin-1 and heat shock protein 70 (Hsp70) to the regulation of Nox4 expression in the proximal tubules of SHR. In particular, the authors tested the hypothesis that an ARB, losartan, exerts its protective effects against proximal tubular injury by decreasing ROS through Nox4 downregulation and associated NADPH oxidase inactivation. In their study, the SHR showed increased proximal tubular NADPH oxidase activity and Nox4 protein levels, which were prevented by treatment with losartan. However, the protein levels of other NADPH components, p22phox and p47phox, were unchanged in the microdissected proximal tubular membrane fraction. Caveolin-1 expression was also significantly decreased in both the renal cortical membrane fraction and the microdissected proximal tubules of SHR. Interestingly, losartan markedly increased proximal tubular caveolin-1, suggesting that intrarenal AngII is involved in the downregulation of caveolin-1 during the development of hypertension and renal injury in SHR. Furthermore, losartan significantly increased Hsp70 transmembrane translocation and its colocalization with caveolin-1 in the proximal tubules of SHR. Losartan also significantly decreased Nox4 protein levels in proximal tubules. The finding that the gene expression of Nox4 was not affected by losartan suggests that losartan induces degradation of Nox4 protein in the proximal tubules of SHR. Interestingly, treatment with an anti-Hsp70 antibody abolished the effect of losartan on NADPH oxidase, suggesting a potential role of Hsp70 in losartan-induced NADPH oxidase inactivation. Furthermore, all these effects of losartan were associated with reduced colocalization of Hsp70 and Nox4 in the renal cortical membrane fraction of SHR. Based on these data, the authors suggest that the ARB exerted protective effects on proximal tubules in SHR by decreasing ROS through inactivation of NADPH oxidase. They also indicate that membranous translocation of Hsp70 and its colocalization with caveolin-1 may induce Nox4 protein degradation, which may be involved in the cytoprotective effects of losartan (Fig. 1).Fig. 1Bocanegra et al. [23] proposed a new concept that ARBs exert their renoprotective effects by promoting degradation of Nox4 protein through the interaction of caveolin and a chaperone protein, Hsp70, in the proximal tubules of hypertensive study participants. However, the mechanisms by which treatment with ARBs increase caveolin-1 protein expression and membranous translocation of Hsp70 in proximal tubules have remained unclear. Based on their current data, it is possible that the ARB-induced membranous translocation of Hsp70 leads to translocation of Nox4 from the cell membrane into small intracellular vesicles, where it is degraded. It is important to note that the ARB did not affect protein levels of other NADPH oxidase components, p22phox and p47phox, in the proximal tubules of SHR. Hilenski et al. [24] showed that Nox1 is colocalized with caveolin in punctuate patches on the surface, whereas Nox4 is colocalized with vinculin in focal adhesions in vascular smooth muscle cells. Thus, it can be speculated that the different effects of the ARB on NADPH oxidase components in proximal tubules may be dependent on where and how each component is located in caveolae. It is also not clear why these changes were not observed in normotensive rats, although the authors suggest that these could be explained by a different intracellular distribution of AT1 receptors in the proximal tubular cells [25]. It is also difficult to exclude the possibility that the above-mentioned effects of the ARB are merely exerted by a reduction in blood pressure. Thus, further studies are needed to clarify the precise molecular mechanisms involved in the potential roles of caveolin and Hsp70 in the ARB-induced reduction in NADPH oxidase activity during the development of hypertension. Previous studies showed that renal cortical tissue Nox4 gene expression was not different between normotensive rats and SHR, but it was lower in AngII-infused hypertensive rats than in normotensive rats [26,27]. Bocanegra et al. [23] also showed that Nox4 gene expression is not increased in the proximal tubules of SHR. However, the authors showed that the protein levels of Nox4 are significantly increased in SHR proximal tubules, suggesting that Nox4 protein degradation is decreased in proximal tubules during the development of hypertension. Therefore, chronic treatment with ARBs may enhance the degradation of Nox4 protein, which may reduce NADPH activity and ROS levels in the kidney. These effects of ARBs may contribute to some of their hypotensive and renoprotective effects because locally generated ROS in the kidney play critical roles in regulating sodium transport, renal hemodynamics and renal injury [8]. In conclusion, the current findings by Bocanegra et al. [23] may open a new field of research focusing on the roles of caveolin and Hsp70 in AngII-dependent renal injury and hypertension through the regulation of NADPH oxidase activity. They also provide new insights into the mechanisms responsible for renoprotective effects of ARBs. However, further studies are needed to clarify these precise molecular mechanisms (Fig. 1).