Association/Dissociation of Caveolin-1 and Nitric Oxide Synthase in Preserving Renal Medulla Function during Hemorrhage and Aging

Background: Caveolin-1 is a negative regulator of endothelial nitric oxide synthase (eNOS), as its interaction and binding suppress the activity of eNOS by making caveolin–eNOS complex. The critical role of caveolin-1 and eNOS in the renal medulla during hypovolemic state and aging is unclear. Objective: To investigate changes in the renal medulla the activity and protein levels of the different NOS forms, changes in the abundance of caveolin-1 and caveolin-1/eNOS association in response to acute hemorrhage in young and middle-aged rats. Methods: In the present study we determined: i) NOS activity and protein levels of the different NOS isoforms, ii) the abundance of caveolin-1 and, iii) caveolin-1/eNOS association during hypovolemic state of young and middle-aged rats in the kidney. Results: We have demonstrated that caveolin-1 is involved in the regulation of renal NOS activity in response to acute hemorrhage and aging. Conclusion: Our data showed that association/dissociation of caveolin-1 and eNOS in renal medulla play a critical role in preserving the renal function during hypovolemic state in young and middle-aged rats.


Introduction
Many organ failures are a common consequence of a hypovolemic disorder, the kidney being one of the primary target organs [1]. It was reported that one of the most important physiological systems involved in hydrosaline homeostasis is nitric oxide (NO) pathway [2]. We recently demonstrated an involvement of NO and caveolins in the age-associated functional and structural changes in a heart under osmotic stress caused by a long-term liquid restriction [3].
Like the cardiac system, NO also plays a significant role during renal aging process [4]. Aging kidneys undergo structural and functional changes decreased autoregulatory capacity and increase susceptibility to acute stress [5]. There is a substantial literature indicating a progressive endothelial dysfunction with aging, which includes a reduction in NO-dependent responses [6,7]. This reduced peripheral NO activity could reflect a fall in NO synthesis, perhaps secondary to lack of NOS or essential cofactors and/or could involve increased breakdown of NO by oxidants [8]. Major pathways of NO release have been associated with caveolae [9]. We previously reported that alterations in the amount of caveolin-1 and caveolin-1/eNOS interaction are involved in aged-related binding suppress the activity of eNOS by making caveolin-eNOS complex [12]. In addition, there is evidence to show that caveolin-1 would be a prime candidate for such a role as a major determinant of the aging process [13,14]. Nonetheless, it remains unclear whether caveolin-1-mediated inhibition of activated eNOS plays a major role following hemorrhage and whether it contributes to the effectiveness of compensatory mechanisms to hypovolemic state with aging in the kidney. Therefore, the present study has been designed to investigate: i) NOS activity and protein levels of the different NOS isoforms, ii) the abundance of caveolin-1 and, iii) caveolin-1/eNOS association during hypovolemic state of young and middle-aged rats in the kidney.

Materials and Methods
The experiments were performed on male Sprague-Dawley rats of ages 2 months (young) and 12 months (middle-aged) years old. The rats were housed in separate cages in a temperature-

Animal preparation
All rats were anesthetized with urethane (1.0 g/kg, ip) and kept under anesthesia by additional small doses of urethane throughout the experiment. The rats were placed on a thermostatically controlled heated table. Body temperature was monitored with a rectal thermometer and maintained between 36 and 38°C. A tracheotomy was performed with use of polyethylene (PE-240) tubing to ensure an open airway. The left femoral artery was cannulated to facilitate blood withdrawal. After an equilibrium period of 15 min the rats were bled via a catheter using a syringe at a constant rate for 2 min. Hemorrhage (20% of the volemia) was induced by withdrawing blood (1.4 ml/100 g body weight). The total amount of blood withdrawn was kept constant (total blood volume was at about 7.0 ± 0.1 ml/100 g body weight).

Experimental protocols
Two experimental groups were used for the study of different

Western blotting analysis
In order to determine the effect of hypovolemic state on nNOS, eNOS, iNOS and caveolin-1 protein levels, freshly renal inner medulla were isolated from S and H groups (n=4 each group at each time). Rats of both groups were sacrificed at 120 min after the

Statistical analysis
Data are mean ± SEM. The Student's t test paired was used to compare changes in hemodynamic parameters. Analysis of variance of a variable (ANOVA) followed by a posteriori Bonferroni test for multiple comparisons were used for the analysis of other data. We checked the assumptions of normality, homogeneity of variances and independence of the data. The 5% probability level was used as a criterion for significance. The software Prism (Graph Pad Software, San Diego, CA, USA) was used for statistical analysis.

Serum creatinine
In clinical practice, the detection of impaired renal function, which is characterized by a rapid decline of the glomerular filtration rate, is based on an increase of serum creatinine [15].

NOS activity
The potential ability to produce NO by the renal inner medulla was assessed by measuring NOS activity by measuring the conversion of [14C (U)]-L-arginine to [14C (U)]-L-citrulline by in the kidney of young and middle-aged rats, as illustrated in Figure 1.
In S group, kidney NOS activity was significantly smaller in middleaged than in young animals. Hemorrhage increased NOS activity in young and middle-aged animals compared with S group (62% and 56%, respectively).

Western blot of NOS isoforms and caveolin-1
To assess the specificity of the expressional regulation of

Immunoprecipitation assay for e-NOS and caveolin-1 interaction
To assess the potential for the association of eNOS with the inhibitory protein caveolin-1 in situ, eNOS was immunoprecipitated with anti-caveolin-1 antibodies from whole renal inner medulla extracts of young and middle-aged rats. The presence of caveolin-1 and eNOS in renal tubular cells showed in Figure 3. The study showed a pattern of colocalization of eNOS localization and caveolin-1 with an increased association of these proteins in middle-aged animals compared to young one in S Group. In H Group, after 120 min of hemorrhage, it sees dissociation between caveolin-1 and eNOS in respect of S Group. This effect is evident in both, young and middleaged animals.

Discussion
Our results provide further support new evidence that caveolin-1 is involved in the regulation of renal NOS activity in response to acute hemorrhage and aging. The advancement of the current study is that association/dissociation of caveolin-1 and eNOS in renal medulla play a critical role in preserving the renal function during hypovolemic state in young and middle-aged rats. NO plays a pivotal role in regulation of renal hemodynamics by different NOS isoforms in the kidney [16][17][18][19]. In addition, studies in vivo and in vitro show that NO pathway is one of several modulators involved in the aging process [20,21]. The present study revealed a significant reduction of renal medulla NOS activity Erdely et al. [8] who reported that renal NO synthase activity is decreased with advancing age. Moreover, it was reported that loss of NO availability in the aged kidney (16 and 24 months old) would be expected to result in several adverse effects, including decreased renal perfusion, increased renal vasoconstriction, and enhancement of fibrosis [22]. Different circulating endocrine and local paracrine factors such as NO have been postulated to modulate the hemodynamic responses to hypovolemia [23]. There is good evidence that NO overproduction importantly contributes to vascular hyporeactivity and vascular decompensation following acute hemorrhage [24,25]. On the other hand, a small amount of NO is pivotal for the preservation of microvascular perfusion [26]. We previously demonstrated that hypovolemic state induced by acute hemorrhage (20% of the volemia) triggered a heterogeneous and dynamic NOS activation modulating the cardiovascular response [27]. We report here that hypovolemic state led to upregulation of renal medulla NOS activity in both, young and middle-aged rats.
Our findings support the hypothesis that acute hemorrhage induces an increase of availability of renal NO, which targets a pivotal protective function by maintaining renal homeostasis against organ injury and dysfunction associated with hypovolemic state. It is proved that NOS activity was affected by its gene expression and (reversible) post-translational modification of the enzyme, such as methylation of the eNOS promoter, NOS phosphorylation, and protein-protein interactions and so on [28]. Studies have suggested that caveolin-1 was expressed and localized in endothelial cells of arteries, arterioles, and glomeruli. Caveolin-1 immunoreactivity was also apparent in the vascular smooth muscle cells of glomerular arterioles and in basolateral aspects of distal tubules [28]. Binding of eNOS to caveolin-1 on plasma membrane inhibits the activity of this NOS isoform. However, caveolin-1 has no effect on cytosolic NOS isoforms (iNOS, nNOS) [29]. Since caveolin-1 play a central role in regulation of NO production by eNOS, we sought to examine the effect of acute hemorrhage and aging on abundance of this protein in the kidney. Our present results showed that the abundance of caveolin-1did not change after 120 min of bleeding or with aging. However, in addition to the absolute changes in the abundance of caveolin, it is also significant to consider whether the change in the tissue and subcellular distribution of this protein may influence the activity of eNOS. This issue is of potential importance, because it could explain the maintaining physiological regulation of renal function during hypovolemic state and age by NO unexpected from unchanged abundance of eNOS (or other NOS isoforms). Consistently, we found that aging induces a significant increase of colocalization of eNOS with caveolin-1, suggesting that this association limits renal NOS activity under physiological conditions in response to aging. There have been several reports that organs, such as brain and spleen, expressed caveolin-1 protein in significantly higher amounts only in the aged state [30]. In addition, the up-regulation of caveolin with aging is suggested to relate to insulin resistance, resulting in susceptibility to diabetes mellitus [31]. In this work, we have demonstrated that caveolin -1 would play an important role in the regulation of renal NOS activity in the middle-aged rats. The organ-specific regulation of caveolins in an age-dependent manner would explain the characteristic aging process of the respective organ. Although the precise mechanisms by which acute hemorrhage induces an increase of NOS activity in the kidney are not fully clear, the decreased in eNOS/ caveolin-1 association might be a major factor.
Immunoprecipitation experiments showed that acute hemorrhage decreased the association of caveolin-1 and eNOS suggesting that acute hemorrhage could activate eNOS in renal medulla in both, young and middle-aged animals. To our knowledge, this is novel study to provide direct evidence to demonstrate the relationship between increased NOS activity and dissociation of eNOS from caveolin-1 in the kidney and their link to hypovolemic state induced by acute bleeding during aging process.

Conclusion
Our findings further highlight the pivotal role of renal caveolin-1/eNOS/NO pathway to preserve renal alterations triggered by hypovolemic state during chronologic advance of age. This observation would be consistent with data suggesting the dependence of renal hemodynamics on the NO bioavailability [32,33]. The underlying molecular mechanisms are yet to be elucidated; therefore, further studies are required in the future.

Conflict of Interest
None.