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Klotho is a recently discovered antiaging gene. The objective of this study was to test the hypothesis that klotho gene delivery attenuates the progression of spontaneous hypertension and renal damage in spontaneous hypertensive rats (SHRs). An adeno-associated virus (AAV) carrying mouse klotho full-length cDNA (AAV.mKL) was constructed for in vivo expression of klotho. Four groups of male SHRs and 1 group of sex- and age-matched Wistar-Kyoto rats (5 rats per group) were used. Blood pressure was measured twice in all of the animals before gene delivery. Four groups of SHRs received an IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The Wistar-Kyoto group received PBS and served as a control. AAV.mKL stopped the further increase in blood pressure in SHRs, whereas blood pressures continued to increase in other SHR groups. One single dose of AAV.mKL prevented the progression of spontaneous hypertension for at least 12 weeks (length of the study). Klotho expression and production were suppressed in SHRs, which were reverted by AAV.mKL. AAV.mKL increased plasma interleukin 10 levels but decreased Nox2 expression, NADPH oxidase activity, and superoxide production in kidneys and aortas in SHRs. AAV.mKL abolished renal tubular atrophy and dilation, tubular deposition of proteinaceous material, glomerular collapse, and collagen deposition seen in SHRs, indicating that klotho gene delivery attenuated renal damage. Therefore, the suppressed klotho expression may play a role in the progression of spontaneous hypertension and renal damage in SHRs. AAV delivery of klotho may offer a new approach for the long-term control of hypertension and for renoprotection.
Klotho is a newly identified antiaging gene.1 It encodes a single-pass transmembrane protein with a long extracellular domain and a short cytoplasmic tail. Klotho protein is predominantly expressed in distal convoluted tubules in the kidneys and in the choroid plexus in the brain.2–4 There are 2 forms of klotho, transmembrane (130-kDa) and secreted (65-kDa) forms.1 Genetic deficiency of klotho causes extensive premature aging phenotypes, including a drastically shortened life span.5 Overexpression of the klotho gene extends the life span.3 Klotho can exert its effects in tissues or cells that do not express klotho, suggesting that it may function as an endocrine hormone.1 It was reported that the klotho gene suppresses the insulin/insulin-like growth factor 1 signaling, increases NO availability, and regulates ion channel activities.2,6,7 Klotho may protect against endothelial dysfunction, although it does not express in blood vessels.8–10 Saito et al10 reported that in vivo klotho gene delivery decreased blood pressure (BP) in Oktsuka Long-Evans Tokushima Fatty rats but did not affect angiotensin II–induced hypertension.11 However, it is critical to determine whether klotho plays a role in the progression of spontaneous hypertension and renal damage.
The prevalence of hypertension and related cardiovascular diseases increases with age.12,13 Indeed, the mortality from cardiovascular disease is higher in aged versus young people.12,13 Thus, hypertension and related cardiovascular diseases are aging disorders. Cardiovascular aging is an important aging process that determines the lifespan. It was reported recently that the plasma level of klotho decreases with age after 40 years.14 Therefore, it is important to determine whether klotho is involved in the pathogenesis of hypertension. The spontaneously hypertensive rat (SHR) is a well-characterized genetic model of hypertension.15,16 It has many features of human essential hypertension.16 The characteristic of this model is that hypertension progresses spontaneously with age. In addition, SHRs develop end-organ damages, including renal injury, cardiac hypertrophy, and heart failure.16 The purpose of this study was to test the hypothesis that klotho gene delivery attenuates the progression of spontaneous hypertension and renal damage in SHRs.
The procedure for constructing the recombinant adeno-associated virus (AAV)-2 carrying the mouse full-length cDNA (AAV.mKL) was described in details in the online Data Supplement (Figures S1 to S3). AAV carrying green fluorescent protein (GFP; AAV.GFP) and β-galactosidase (LacZ; AAV.LacZ) were constructed as reporter gene constructs.
Four groups of SHRs and 1 group of age-matched Wistar-Kyoto (WKY) rats were used (5 rats per group, all males, 12 weeks old). Systolic BP and body weight were measured twice before gene delivery. BP was measured using the tail-cuff method, as described in the online Data Supplement. Briefly, the 4 groups of SHRs received AAV.mKL, AAV.GFP, AAV.LacZ, and PBS, respectively. The viral particles were delivered intravenously via the tail vein at 2×108 particles per rat (0.5 mL). The WKY group received PBS and served as a control. BP and body weight were measured weekly after gene delivery. All of the animals were euthanized at the end of week 12. Serum creatinine and kidney collagen deposition were measured. The detailed protocols can be found in the online Data Supplement. The protocol was approved by the institutional animal care and use committee of the University of Oklahoma Health Sciences Center.
The in situ superoxide production was measured in aortas and kidneys using the oxidation-sensitive dye dihydroethidium (DHE), as described in the online Data Supplement.
Klotho and NADPH oxidase protein expressions were measured by Western blot, as described in the online Data Supplement.
Mouse klotho mRNA was analyzed by RT-PCR using mouse-specific primers as described in the online Data Supplement.
The method for GFP expression was provided in the online Data Supplement.
The data for BP and BW were analyzed by a 2-way ANOVA (temperature and treatment) followed by a 1-way ANOVA (repeated in time). The remaining data were analyzed by a 2-way ANOVA. The Newman-Keuls procedure was used to assess the significance of the difference between means. The significance was set at the 95% confidence limit.
The baseline BP of 12-week–old SHRs was significantly higher than that of the WKY rats (Figure 1). BP of the SHR-GFP, SHR-LacZ, and SHR-PBS groups continued to increase and reached ≈177±7 mm Hg at 12 weeks after gene delivery. In contrast, BP of the AAV.mKL group did not increase compared with its pretreatment level (Figure 1). One single dose of AAV.mKL prevented progression of hypertension for ≤12 weeks (length of the study). AAV.mKL did not affect food and water intakes or body weight gain (Figure S4).
Aortic segments of SHRs showed an increase in superoxide production (red fluorescence or DHE staining) compared with those of WKY rats (Figure 2A). In contrast, vascular superoxide production was markedly decreased in SHRs treated with AAV.mKL. A quantitative analysis confirmed that klotho gene delivery significantly attenuated the in situ vascular superoxide production (Figure 2B).
Nox2 protein expression was increased in aortas of SHRs compared with that of the WKY rats (Figure 3A and 3B). Klotho gene delivery decreased aortic Nox2 expression in SHRs to that of the WKY rats. Nox1, Nox4, and endothelial NO synthase were not affected by klotho gene delivery (data not shown). NADPH oxidase activity was increased significantly in aortas of SHRs, which could be abolished by klotho gene delivery (Figure 3C).
Plasma levels of membrane and secreted forms of klotho were decreased significantly in SHRs compared with WKY rats (Figure 3D and 3E). AAV.mKL increased plasma levels of klotho protein in SHRs to those of WKY rats, indicating that klotho gene delivery increased klotho production. The urine levels of klotho were also decreased in SHRs over a time course (weeks 5 and 8) and were reverted by klotho gene delivery (Figure S5).
The plasma level of interleukin (IL) 10 was decreased significantly in SHRs, which was reverted by klotho gene delivery (Figure 3D and 3F). The IL-10 level was not different between SHR-GFP/SHR-LacZ and SHR-PBS, indicating that the AAV vector may not alter IL-10 expression.
Tissue sections of kidneys of SHRs showed increased superoxide production compared with those of WKY rats (Figure 4A). In contrast, superoxide production was markedly attenuated in SHRs treated with AAV.mKL. A quantitative analysis confirmed that klotho gene delivery significantly attenuated superoxide production in kidneys (Figure 4B).
Renal klotho protein expression was decreased significantly in SHRs compared with WKY rats (Figure 5A and 5B). Klotho gene delivery increased renal klotho expression in SHRs to the control (WKY) level. Renal Nox2 expression was upregulated in SHRs, which was reverted by klotho gene delivery (Figure 5A and 5C). Renal expression of endothelial NO synthase, Nox1, and Nox4 was not altered in SHRs or affected by klotho gene delivery (Figure S6).
Immunohistochemical analysis indicated that klotho protein expression (brown staining) was localized in the renal tubule epithelial cells (Figure 5D). Renal klotho expression was decreased in SHRs compared with WKY rats. Klotho gene delivery increased klotho protein expression (heavy brown staining) in the renal tubule epithelial cells of SHRs (Figure 5D). Klotho staining was not found in the aorta (photos not shown).
A partial loss of kidney medulla was found in some SHRs (SHR-PBS, SHR-GFP, and SHR-LacZ; Figure 6A). Histological examination (staining) indicated that some cortical tubules were atrophic, dilated, and filled with proteinaceous material in SHRs (Figure 6B). Renal damage also included glomerular collapse in SHRs (Figure 6C). These pathological changes disappeared in the AAV.mKL-treated SHRs (SHR-mKL), indicating that klotho gene delivery abolished renal injury in SHRs (Figure 6). AAV.mKL also significantly decreased urinary output of protein (Figure S7), suggesting that klotho gene delivery improved renal function. Thus, klotho gene delivery attenuated kidney damage in SHRs.
There was a significantly increase collagen deposition (blue staining) in kidneys of SHR-PBS, SHR-GFP, and SHR-LacZ groups (Figure 7A and 7B). Renal interstitial fibrosis was found in these groups. Serum level of creatinine was increased in SHRs (Figure 7C), indicating a decrease in renal clearance function. Klotho gene delivery significantly decreased serum creatinine in SHRs, suggesting improvement of renal function.
GFP expression was found in livers and kidneys (Figure 8A and 8B), but not in aortas (photos not shown), in the SHR-GFP groups. The overlap (merge) of GFP and 4′,6-diamidino-2-phenylindole (DAPI) further disclosed that GFP expression was localized in the nuclei. The result indicated that the GFP reporter gene was still expressed in rats at 12 weeks after delivery of AAV.GFP. No GFP was found in livers or kidneys in the SHR-mKL groups (negative staining). In addition, strong X-galactosidase staining was found in kidneys of AAV.LacZ-treated rats (Figure S8), indicating that the LacZ reporter gene expression was active at the time of animal sacrifice. These results suggest that AAV achieved a long-term expression of transgenes.
Mouse klotho mRNA was strongly expressed in the kidney and liver in AAV.mKL-treated rats at 12 weeks after gene delivery (Figure 8C and 8D), indicating successful delivery of the mouse klotho gene. Mouse klotho mRNA was not found in any other groups. Mouse klotho mRNA expression was not detectable in the aorta or mesenteric arteries of the SHR-mKL group (data not shown).
The present study revealed that klotho expression and production were markedly suppressed in SHRs and were reverted by AAV.mKL. Notably, klotho gene delivery stopped further increases in BP in SHRs. To our knowledge, this is the first study showing that AAV delivery of the klotho gene prevented the progression of spontaneous hypertension. Thus, the suppression of the klotho gene in SHRs may play a role in the pathogenesis of the progression of spontaneous hypertension. It was reported that the level of the circulating klotho decreases with age,14 whereas the prevalence of hypertension increases with age in humans.12,13 Therefore, it will be interesting to evaluate whether klotho is involved in the progression of human essential hypertension. It is noted, however, that klotho gene delivery did not decrease the BP of SHRs to the control level. A separate study is required to determine whether klotho is involved in the initiation of spontaneous hypertension by klotho gene delivery before the elevation of BP.
It is notable that a single dose of AAV.mKL controlled hypertension for ≤12 weeks (length of the study). The prolonged antihypertensive effect of AAV.mKL was probably attributed to the long-lasting AAV vector.24–26 Indeed, AAV.mKL was still expressed at 12 weeks after gene delivery, as evidenced by the strong expression of mouse klotho mRNA in the kidneys of SHRs. Consequently, klotho gene delivery resulted in prolonged upregulation of klotho protein expression. In addition, AAV.GFP expression was found in the nuclei, suggesting that the transgene was expressed in the cells. It was reported that AAV-based transgene can integrate into the host genome.24,26,27 AAV has an advantage over other viral vectors, because it produces no or minimal inflammatory and immune responses in vivo.17,25 AAV did not change IL-10 expression, which is consistent with the reports of us and other that AAV does not induce inflammation.17,18,24,26 Indeed, no signs of inflammation were seen during autopsy, and no viral effect was observed in the data analysis. AAV also exhibited low vector toxicity both in animal experiments and clinical trials.17,18,28 Thus, AAV delivery of klotho may offer a new approach for long-term control of hypertension.
NADPH oxidases play a critical role in the hemodynamic responses to angiotensin II and in the pathogenesis of hypertension.21,29–31 Vascular and renal superoxide production was increased in SHRs. It is well established that an increase in the reactive oxygen species level is involved in the pathogenesis of hypertension.21,29–35 Klotho gene delivery decreased vascular and renal superoxide production in SHRs, which may contribute to the mechanism of the antihypertensive effect of klotho. A decrease in superoxide production may be attributed to the suppression of Nox2 by klotho gene delivery, which did not affect other isoforms of NADPH oxidases or endothelial NO synthase. The NADPH oxidase is the primary source of reactive oxygen species in the vasculature,36 and it can act as a mediator of vascular injury and inflammation in many cardiovascular diseases.30,32,33 The suppressing effect of AAV.mKL on Nox2 and superoxide production was not attributed to a drop in BP, because AAV.mKL did not decrease BP compared with the pretreatment level. It is interesting that mouse klotho did not express in vasculatures, but klotho gene delivery suppressed vascular Nox2 and NADPH oxidase activity, supporting the notion that klotho functions as a hormone.1 Indeed, klotho gene delivery increased the level of the circulating klotho, providing a basis for the direct action of klotho in the vascular system that does not express klotho.1 Although the klotho receptor is unknown,1 our most recent study in the cell culture indicated that the selective inhibition of Nox2 expression by klotho in rat aorta smooth muscle cells may be mediated by the cAMP-protein kinase A pathway.37
Mouse klotho was expressed in kidneys and livers in SHRs after gene delivery. In kidneys, klotho protein expression was localized in the tubule epithelial cells. Only the transmembrane form of the klotho protein was found in the kidneys, whereas both transmembrane (130 kDa) and secreted (65 kDa) forms of klotho were identified in the circulation. Klotho gene delivery increased klotho protein levels in both kidneys and the circulation. The major sources of circulating klotho protein are alternative RNA splicing (klotho gene directly generates secreted form of klotho, which is liberated into the circulation) and proteolytic cleavage (transmembrane form of klotho protein is cleaved by enzymes and released into the circulation).1 The circulating klotho is important in cardiovascular regulation because it has direct access to the vascular endothelial and smooth muscle cells.
This study demonstrated, for the first time, that klotho gene delivery may upregulate anti-inflammatory cytokine IL-10 in SHRs, although the underlying mechanism remains to be discovered. The increased level of circulating IL-10 may contribute to the antihypertensive effect of klotho, because an increase in IL-10 expression attenuated pulmonary and systemic hypertension.38,39 It was reported that IL-10 inhibits vascular smooth muscle cell proliferation, macrophage activation, T-cell proliferation, and inflammation38,40–43 that play important roles in the pathogenesis of hypertension and end-organ damage.44
Importantly, klotho gene delivery abolished tubular atrophy and dilation and tubular deposition of proteinaceous material in SHRs, the signs of the end-stage kidneys. The tubular deposition of proteinaceous material reflects impaired renal function (unable to reabsorb small proteins). It is even more interesting that klotho gene delivery attenuated glomerular collapse and interstitial collagen staining in SHRs, which could eventually result in a loss of glomerular filtration. These findings are unexpected, because the implication of klotho in kidney protection has never been reported. These results suggest that AAV delivery of klotho may offer a new approach for prolonged and effective renoprotection.
Hypertension could contribute to the worsening of kidney function and accelerate kidney damage.45 However, the renoprotective effect of AAV.mKL cannot be fully explained by its antihypertensive effect, because klotho gene delivery did not decrease BP compared with the pretreatment level. Therefore, the renoprotective effect of AAV.mKL may be partially attributed to the direct effect of the increased klotho expression in the kidneys. The kidney is a target organ for klotho action, as well as a major site of klotho production.5 SHRs would undergo vascular changes and develop cortical ischemia that are accompanied by glomerular lesions and tubular changes.12,16 The present data suggest that klotho deficiency may contribute to renal damage in SHRs and that klotho gene delivery could provide renoprotection. The renoprotective effect of klotho may be attributed, at least in part, to its suppressing effect on Nox2 and superoxide production, although the precise mechanism remains to be found.
This is the first study showing that AAV delivery of the klotho gene prevented the progression of spontaneous hypertension and renal damage in SHRs. A single dose of AAV.mKL controlled hypertension and renal damage for ≤12 weeks. This finding may offer a new and effective approach for long-term control of hypertension and renal damage. The antihypertensive and renoprotective effects of klotho may be attributed, at least in part, to the downregulation of Nox2 expression and superoxide production and the upregulation of IL-10. The present finding also suggests that the suppressed klotho expression may be involved in the progression of spontaneous hypertension and renal damage in SHRs. Additional studies are required to determine the receptors that mediate the effects of klotho.
Sources of Funding
This work was supported by National Institutes of Health grant R01-NHLBI-077490 and the Reynolds Oklahoma Center on Aging.