Kallistatin (KS) levels are reduced in the kidney and blood vessels under oxidative stress conditions. To determine the function of endogenous KS in the renal and cardiovascular systems, KS levels were depleted by daily injection of anti-rat KS antibody into DOCA-salt hypertensive rats for 10 days. Administration of anti-KS antibody resulted in reduced KS levels in the circulation but increased levels of serum thiobarbituric acid reactive substances (an indicator of lipid peroxidation) as well as superoxide formation in the aorta. Moreover, anti-KS antibody injection resulted in increased NADH oxidase activity and superoxide production but decreased nitric oxide levels in the kidney and heart. Endogenous KS blockade aggravated renal dysfunction, damage, hypertrophy, inflammation, and fibrosis as evidenced by decreased creatinine clearance and increased serum creatinine, blood urea nitrogen and urinary protein levels, tubular dilation, protein cast formation, glomerulosclerosis, glomerular enlargement, inflammatory cell accumulation, and collagen deposition. In addition, rats receiving anti-KS antibody had enhanced cardiac injury as indicated by cardiomyocyte hypertrophy, inflammation, myofibroblast accumulation, and fibrosis. Renal and cardiac injury caused by endogenous KS depletion was accompanied by increases in the expression of the proinflammatory genes tumor necrosis factor-α and intercellular adhesion molecule-1 and the profibrotic genes collagen I and III, transforming growth factor-β, and tissue inhibitor of metalloproteinase-1. Taken together, these results implicate an important role for endogenous KS in protection against salt-induced renal and cardiovascular injury in rats by suppressing oxidative stress, inflammation, hypertrophy, and fibrosis.
kallistatin (ks) is a plasma protein that is also widely distributed in the kidney, heart, and blood vessels, implicating its role in renal and cardiovascular function. Indeed, we have shown that KS is capable of controlling a wide spectrum of biological actions in the heart and kidney, including reduction of oxidative stress, apoptosis, inflammation, hypertrophy, and fibrosis (8. 16, 37). KS gene delivery improved kidney function and decreased renal damage, inflammation, and collagen deposition in Dahl salt-sensitive (DSS) hypertensive rats (37). KS administration also enhanced cardiac function and reduced infarct size, cardiomyocyte apoptosis, inflammatory cell accumulation, and ventricular remodeling after acute myocardial ischemia/reperfusion and chronic myocardial infarction (8, 16). The effects of KS on both the heart and the kidney were associated with reduced oxidative stress and increased nitric oxide (NO) levels. Moreover, in vitro studies (16, 37) using cardiac and renal cells showed that KS suppressed intracellular reactive oxygen species (ROS) formation. Furthermore, we (7, 38) previously demonstrated that circulating KS levels are reduced in several animal models that exhibit oxidative stress, such as salt-induced and NO-deficient hypertensive rats. Similarly, KS expression is diminished by H2O2 in cultured endothelial cells (38). These results indicate that KS may play a protective role against cardiovascular and renal injury by inhibiting oxidative stress and increasing NO formation. Rats with DOCA-salt hypertension exhibit oxidative damage in the heart, kidney, and vasculature (2, 3, 5). Therefore, we employed the DOCA-salt hypertensive rat model for investigating the endogenous role of KS. In this study, depletion of KS by anti-KS antibody injection into DOCA-salt rats demonstrated that endogenous KS functions as a cardiovascular- and renal-protective agent.
MATERIALS AND METHODS
All procedures complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD). The protocol for our animal studies was approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. Male Wistar rats (Harlan Sprague-Dawley, Indianapolis, IN) initially weighing 200–220 g were housed in an approved animal care facility. Rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) before undergoing left unilateral nephrectomy. One week after surgery, rats in the sham group (n = 6) received weekly subcutaneous injections of sesame oil and were provided with tap water. Experimental animals received weekly subcutaneous injections of deoxycorticosterone acetate (DOCA; 25 mg/kg body wt; Sigma, St. Louis, MO) suspended in sesame oil and were provided with 1% NaCl drinking water. Ten days after surgery, DOCA-salt rats received daily intravenous injections of either 0.5 mg of polyclonal anti-rat KS antibody (DOCA/α-KS; n = 8) or 0.5 mg of normal rabbit IgG (DOCA/IgG; n = 6). Anti-rat KS antibody was purified by a protein A-affinity column as previously described (26). Eleven days after initial antibody treatment (i.e., 3 wk after surgery), rats were anesthetized with pentobarbital (50 mg/kg) and hearts, kidneys, and aortas were removed for morphological, histological, and biochemical analyses.
Blood pressure and renal function measurements.
On the day of death, mean arterial blood pressure (MAP) was measured and serum was collected by cardiac puncture (25). Twenty-four-hour urine was collected from rats in metabolic cages 2 days before death. To eliminate contamination of urine samples, animals received only water during the 24-h collection period. Blood urea nitrogen, urinary protein levels, serum creatinine, and creatinine clearance were measured as previously described (25).
Superoxide measurement in aorta.
Superoxide levels in aortas were determined by in situ and chemiluminescent methods as previously described (39).
Histological and immunohistochemical staining.
Hearts, kidneys, and aortas were fixed in 4% formaldehyde, dehydrated, and paraffin-embedded. Four-micrometer-thick sections were subjected to hematoxylin and eosin, periodic acid-Schiff (PAS), silver, and Sirius red staining. Immunohistochemistry was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA) following the supplied instructions. Heart and kidney sections from paraffin-embedded tissue were incubated at 4°C overnight with primary antibodies against the monocyte/macrophage marker ED-1 (Chemicon, Temecula, CA) and the myofibroblast marker α-smooth muscle actin (α-SMA; Sigma). After development, tissue sections were moderately counterstained with hematoxylin.
Light microscopic morphological evaluation of glomeruli was conducted in a blinded fashion as previously reported (25). At least 30 glomeruli per section were examined for the evaluation of glomerular lesions and hypertrophy using PAS- and silver-stained slides, respectively. The severity of glomerulosclerosis and glomerular size was semiquantified using a 0 to 3 scale (0, normal or almost normal; 1, mild; 2, moderate; 3, severe) for each glomerulus. The number of monocytes/macrophages in the heart and kidney (including the interstitium and within glomeruli) was counted as positive staining for the monocyte/macrophage marker ED-1 (Chemicon, Temecula, CA) in a blinded manner from 10 different fields of each section at ×200 magnification. Heart sections and cortical areas of kidney sections stained with Sirius red, which stained collagen fibers red, were analyzed for collagen fraction volume (25). Twenty fields without large vessels were randomly selected from each heart and kidney section at a magnification of ×200. Collagen fraction volume was then calculated as percentage of stained area within a field. Positive staining for α-SMA in heart and kidney sections was quantified from 10 different fields of each section. For measurement of cardiomyocyte size, 100 cardiomyocytes in silver-stained left ventricular sections were chosen randomly at ×400 magnification. Collagen fraction volume, α-SMA-positive staining, and cardiomyocyte size were quantified using NIH image software (National Institutes of Health).
Levels of KS in serum were measured by ELISA specific for rat KS (26). Biochemical assays for measuring circulating thiobarbituric acid reactive substances (TBARS) levels as well as renal and cardiac NADH oxidase activity, superoxide, and nitrate/nitrite (NOx) were performed as previously described (25).
Quantitative real-time PCR.
Total RNA was extracted from heart and kidney tissue using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was transcribed from 2 μg of RNA using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster, CA) following the manufacturer's instructions. PCR was carried out using Taqman Gene Expression Assays for tumor necrosis factor (TNF)-α, intercellular adhesion molecule (ICAM)-1, collagen I, collagen III, transforming growth factor (TGF)-β, tissue inhibitor of metalloproteinase (TIMP)-1, and atrial natriuretic peptide (ANP), and a detection kit for GAPDH on a 7300 real-time PCR system (Applied Biosystems). Quantification was determined by Relative Quantification Software (Applied Biosystems).
Data were analyzed using standard statistical methods and ANOVA followed by Fisher's paired least significant difference. Group data are expressed as means ± SE. Values of all parameters were considered significantly different at a value of P < 0.05.
Blockade of endogenous KS increases oxidative stress in the aorta, kidney, and heart.
The role of endogenous KS on oxidative stress parameters and organ injury was investigated by injecting anti-KS antibody into DOCA-salt hypertensive rats. KS levels were measured in serum collected 11 days after the start of antibody administration. DOCA-salt rats receiving control IgG had significantly lower circulating KS levels compared with rats in the sham group, whereas administration of the anti-KS antibody caused a further reduction in KS levels in the serum compared with the DOCA/IgG group (Table 1). Anti-KS antibody injection caused an increase in aortic superoxide formation compared with DOCA-salt rats receiving control IgG (Fig. 1, A and B). Moreover, KS depletion resulted in a significant rise in circulating TBARS levels, an indicator of lipid peroxidation (Fig. 1C). Superoxide levels and NADH oxidase activity in the kidney were markedly increased in DOCA-salt rats receiving anti-KS antibody compared with the DOCA/IgG group (Fig. 2, A and B). Furthermore, anti-KS antibody injection induced a dramatic reduction in renal NOx levels compared with DOCA-salt rats receiving control IgG (Fig. 2C). Likewise, rats in the DOCA/anti-KS group exhibited elevated superoxide production and NADH oxidase activity and markedly reduced NOx formation in the heart compared with the DOCA/IgG group (Fig. 2, D–F).
Blockade of endogenous KS on physiological and morphological parameters in the kidney and heart.
The effects of anti-KS antibody injection on the physiological and morphological parameters in the kidney and heart in DOCA-salt rats are shown in Table 1. No significant difference in MAP was observed between both DOCA-salt groups, although MAP was higher in the DOCA-salt rats compared with rats in the sham group. Anti-KS antibody administration increased blood urea nitrogen, urinary protein, and serum creatinine levels but decreased creatinine clearance, indicating that the depletion of KS adversely affects renal function. DOCA-salt rats had lower body weights and greater kidney weights and ratios of kidney weight to body weight compared with rats in the sham group. Moreover, rats in the DOCA/anti-KS group had markedly higher kidney weights and kidney weight-to-body weight ratios than those given control IgG. Furthermore, DOCA-salt rats given control IgG had higher heart weights than sham rats, although not to a significant extent. Anti-KS antibody injection, however, markedly increased heart weight compared with the DOCA/IgG group. In addition, the heart weight-to-body weight ratio was increased in rats in the DOCA/IgG group compared with the sham group, and anti-KS antibody further increased this ratio. The kidney and heart morphological data suggest that blockade of endogenous KS promotes hypertrophic responses in these organs.
Blockade of endogenous KS accelerates renal injury and inflammation.
Kidneys of DOCA-salt rats exhibited tubular dilation, brush border loss, protein cast formation, glomerular sclerosis, and glomerular hypertrophy, as determined by examination of PAS- and silver-stained tissue sections (Fig. 3A). Renal damage was exacerbated by blockade of endogenous KS. Semiquantitative analysis showed that the severity of glomerular sclerosis and hypertrophy was significantly greater in DOCA/anti-KS rats compared with DOCA-salt rats given control IgG (Fig. 3, B and C). Administration of anti-KS antibody also aggravated renal inflammation in DOCA-salt rats, as determined by ED-1 immunohistochemistry (Fig. 3A). This observation was confirmed by quantitation of ED-1-positive cells in the renal interstitium and within glomeruli (Fig. 3D). Renal inflammation was also assessed by measuring the expression of proinflammatory genes. DOCA-salt rats receiving control IgG had markedly greater expression of TNF-α and ICAM-1 in the kidney than rats in the sham group (Table 2). Moreover, anti-KS antibody administration further stimulated the expression of these genes compared with the DOCA/IgG group.
Blockade of endogenous KS exacerbates renal fibrosis.
DOCA-salt rats exhibited an increase in collagen deposition in the kidney compared with sham rats, as shown by Sirius red staining (Fig. 4A). Administration of anti-KS antibody caused a significant rise in collagen accumulation compared with rats in the DOCA/IgG group. Similarly, the numbers of myofibroblasts, as identified by positive α-SMA immunostaining, were elevated in the DOCA/IgG group compared rats in the sham group, and anti-KS antibody injection further increased the amount of myofibroblasts (Fig. 4A). Quantitative analysis of collagen fraction volume and α-SMA-positive cells confirmed these observations (Fig. 4, B and C). Analysis of profibrotic gene expression profiles showed that DOCA-salt rats given control IgG had significantly higher expression of collagen types I and III, TGF-β, and TIMP-1 compared with rats in the sham group (Table 2). Anti-KS antibody injection caused a dramatic elevation in the expression of these genes compared with rats in the DOCA/IgG group.
Blockade of endogenous KS aggravates cardiac damage and inflammation.
As was observed in the kidney, anti-KS antibody administration induced a detrimental effect on the heart. Histological evaluation of hematoxylin and eosin-stained heart sections indicated that anti-KS antibody administration exacerbated the formation of myocardial lesions compared with rats in the DOCA/IgG group (Fig. 5A). Moreover, cardiomyocyte size was significantly increased in DOCA/IgG rats, and further enlarged in the DOCA/anti-KS group, as determined by analysis of silver-stained slides and cardiomyocyte size quantitation (Fig. 5, A and B). Expression levels of the hypertrophic marker ANP corroborated the injurious effect of anti-KS antibody injection on cardiac hypertrophy (Fig. 5C). Cardiac inflammation was also found to be increased in the DOCA/anti-KS group compared with DOCA-salt rats given control IgG, as determined by ED-1 immunohistochemistry (Fig. 5A). Quantitation of monocytes/macrophages confirmed this elevation in inflammatory cells in the heart (Fig. 5D). In addition, expression levels of TNF-α and ICAM-1 in the heart were significantly increased in the DOCA/anti-KS group compared with rats in the DOCA/IgG group (Table 2).
Blockade of endogenous KS worsens cardiac fibrosis.
Analysis of Sirius red-stained heart sections showed that anti-KS antibody administration exacerbated collagen accumulation compared with DOCA-salt rats given control IgG (Fig. 6A). The extent of positive α-SMA immunostaining was also dramatically increased in the DOCA/anti-KS group compared with rats in the DOCA/IgG group, indicating an increase in myofibroblast numbers in the hearts of DOCA/anti-KS rats (Fig. 6A). Quantitative analysis of collagen fraction volume and α-SMA-positive cells supported these observations (Fig. 6, B and C). Furthermore, DOCA/anti-KS rats exhibited elevated expression of collagen types I and III, TGF-β, and TIMP-1 in the heart compared with DOCA-salt rats given control IgG (Table 2).
Our study demonstrated that depletion of endogenous KS via antibody injection exacerbates oxidative renal and cardiovascular injury induced in DOCA-salt hypertensive rats. Administration of anti-rat KS antibody caused a significant reduction of circulating rat KS levels in association with aggravated renal dysfunction. Blockade of KS also elevated oxidative stress and lowered NO levels in renal and cardiovascular tissues. Moreover, KS depletion worsened tissue injury, inflammation, hypertrophy, and fibrosis in the heart and kidney. The effects of KS blockade were accompanied by increased expression of proinflammatory and profibrotic genes. Interestingly, blood pressure was not increased by anti-KS antibody injection compared with DOCA-salt rats given control IgG. It is possible that a high blood pressure state may have already been established in the DOCA/IgG group, preventing a further rise in blood pressure by anti-KS antibody administration. In addition, the lack of a blood pressure rise along with a parallel increase in heart and kidney damage may be due to the time point in which the animals were killed. That is, 11 days of anti-KS antibody administration may not allow sufficient time to observe an increase in blood pressure yet is adequate for a tissue-damaging effect. Indeed, we (8) have shown that KS exerts beneficial effects in the cardiovascular system independent of its vasodilating property. Collectively, these observations are consistent with studies (8, 16, 37) showing that KS administration is protective in salt-induced kidney damage and ischemic heart disease. Our findings reveal a role for KS as an endogenous renal and cardiovascular-protective agent by inhibiting oxidative stress, inflammation, and tissue remodeling.
Oxidative stress is a state of redox imbalance caused by increased ROS generation and decreased NO bioavailability (30). Oxidative stress can result in the pathological development of hypertension-related conditions, such as endothelial dysfunction, inflammation, hypertrophy, and fibrosis (30). DOCA-salt administration induces significant NAD(P)H oxidase-mediated ROS production in aorta, kidney, and heart (2, 5, 18) as well as downregulation of endothelial NO synthase in the glomerulus (41), leading to hypertension and organ damage. Our previous studies (8, 16, 37, 38) have conclusively shown that KS functions as an antioxidant. However, in the current study we show that depletion of endogenous KS in DOCA-salt hypertensive rats increased oxidative stress in the circulation, blood vessels, kidney, and heart and decreased renal and cardiac NO levels. Taken together, these findings indicate a novel role of KS as an endogenous antioxidant in protection against renal and cardiovascular injury.
Oxidative stress plays an important role in the development of DOCA-salt-induced renal inflammation (3) and may also lead to inflammatory cell infiltration in the heart (15). ROS trigger cell signaling processes and transcription factors to induce the expression of cytokines, such as TNF-α, which in turn upregulate the synthesis of cell adhesion molecules, including ICAM-1 (17, 32). Also, by binding to and inactivating NO, ROS counteract the ability of NO to decrease the expression of cytokines, chemokines, and leukocyte adhesion molecules (35). In DOCA-salt hypertensive rats, ROS production as well as NF-κB expression and activation is increased (4, 34), subsequently upregulating the synthesis of proinflammatory genes. Monocytes/macrophages recruited to sites of tissue damage release additional ROS into the local environment, leading to a more severe inflammatory state. DOCA-salt hypertensive rats exhibit renal inflammation in association with increased oxidative stress, whereas treatment with anti-oxidants was shown to decrease blood pressure, aortic superoxide formation, and renal monocyte/macrophage accumulation (3). Furthermore, macrophage infiltration and expression of cell adhesion molecules are increased in the hearts of DOCA-salt rats (1, 6). In our study, anti-KS antibody administration caused a dramatic increase in monocyte/macrophage accumulation as well as TNF-α and ICAM-1 expression in both the kidney and heart. The inflammatory condition occurred in conjunction with elevtaed ROS and decreased NO production. The anti-inflammatory role of KS is in agreement with our previous studies (9, 42, 44) of animal models of endotoxic shock, arthritis and vascular permeability. Thus KS is purported to be an endogenous anti-inflammatory factor.
Renal tubulointerstitial lesions, glomerular sclerosis, and enlargement, as well as cardiac hypertrophy and fibrosis, are common features associated with DOCA-salt treatment (13, 43) and are accompanied by increased expression of TGF-β1, a major contributor to tissue remodeling (14, 24). As a profibrotic factor, TGF-β enhances the expression of extracellular matrix proteins, including collagens I and III, and prevents the degradation of extracellular matrix proteins by inducing the synthesis of TIMPs in renal and cardiac cells (14, 24, 31). TGF-β also promotes the accumulation of myofibroblasts via epithelial-mesenchymal transition of proximal tubular cells and mesangial cells in the kidney (14, 19) and by phenotypic conversion of cardiac fibroblasts (24, 31). Myofibroblasts, identified by positive α-SMA staining, are a cell type characterized by excessive secretion of profibrotic factors (19, 24, 31). The traits ascribed to DOCA-salt-induced tissue remodeling were observed in the present study. However, endogenous KS depletion dramatically increased myofibroblast and collagen accumulation in cardiac and renal tissue. Moreover, anti-KS antibody administration markedly elevated expression of collagens I and III, TGF-β1, and TIMP-1. Upregulation of these profibrotic factors were accompanied by a decrease in NO levels. Our previous studies (16, 37) have shown that KS gene transfer attenuates renal and cardiac tissue remodeling. KS administration has also been observed to increase renal endothelial NO synthase expression and reduce TGF-β1 levels in kidneys of DSS rats and in cultured mesangial cells (37). This is significant, as NO is known to inhibit TGF-β1 and collagen expression in renal cells (12, 45). Collectively, our data suggest that the fibrotic effects of KS blockade may, in part, be due to the reduction in NO production, thus leading to the inability of KS to prevent the rise in TGF-β1 levels.
In addition to pomoting fibrosis, TGF-β also induces hypertrophic responses. Studies have shown that TGF-β promotes glomerular hypertrophy in diabetic mice (36) and cardiomyocyte hypertrophy in cultured cells (33). Angiotensin II and endothelin-1, which are both expressed in DOCA-salt hypertension (20, 23), have also been demonstrated to stimulate glomerular enlargement (21, 40) and cardiomyocyte hypertrophy (22, 33). Although KS gene delivery has been shown to attenuate renal and cardiac tissue remodeling (16, 37), KS depletion by anti-KS antibody worsened both glomerular and cardiomyocyte hypertrophy. Expression of ANP, a marker of cardiac hypertrophy, confirmed that endogenous KS is protective against cardiac remodeling.
KS may have differential effects as a potential therapeutic agent depending on the pathological condition. We have demonstrated that KS increases the proliferation and migration of vascular smooth muscle cells, leading to neointimal hyperplasia (27). KS was also shown to inhibit VEGF-mediated endothelial cell proliferation, migration, and tube formation, indicating that it possesses anti-angiogenic properties (28, 29). Moreover, our previous studies (8, 16, 37, 39) in rodent models of salt-induced hypertension, myocardial infarction, and ischemia/reperfusion have established KS as an antioxidant and anti-inflammatory and antiapoptotic agent. We have shown that KS is a novel inhibitor of vascular inflammation by dual mechanisms: 1) antagonizing TNF-α-mediated NF-κB activation and inflammatory gene expression (44); and 2) directly stimulating endothelial nitric oxide synthase activation and expression, thereby increasing NO levels (39). KS, via its pleiotropic properties, protects against renal and cardiovascular injury.
It is worthwhile to consider the design of smaller KS proteins that exhibit therapeutic benefits. The smaller KS-like drugs may be more advantageous than intact KS due to the cost and inconvenience of chronic administration of large proteins in patients. Structural and functional analysis showed that KS contains two important elements, namely a heparin-binding region and an active site for the inhibition of tissue kallikrein (10, 11). We demonstrated that wild-type KS and its active-site mutant, but not the heparin-binding site mutant, have the ability to inhibit VEGF-induced angiogenesis (29) and/or TNF-α-induced inflammation (44). Thus smaller KS-like molecules containing essential structural elements may provide specific functions in preventing organ injury. Furthermore, infusion of KS-like drugs or intact KS protein would be more beneficial than a gene therapy approach. Indeed, infusion by osmotic mini-pump provides a constant supply of KS as opposed to gene delivery through a viral vector, which generates a large amount of protein over a short period of time and then gradually diminishes to undetectable levels. Viral gene therapy also poses a risk to the patient by possibly stimulating an immune reaction or inserting the gene into a region of DNA that could lead to harmful mutations.
In conclusion, the present study provides the first evidence that endogenous KS is a novel protective agent in renal and cardiovascular oxidative stress, inflammation, hypertrophy, and fibrosis in salt-induced hypertension. Depletion of KS by anti-KS antibody injection aggravated renal and cardiovascular injury, increased superoxide production, and decreased NO levels. We have previously reported that KS delivery elicits a wide range of beneficial biological actions in the heart and kidney in conjunction with reduced oxidative stress and increased NO bioavailability. Therefore, taken together with our current findings, our research suggests that KS administration may be a novel and potential treatment for renal and cardiovascular diseases.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-44083 and HL-29397 and Extramural Research Facilities Program of the National Center for Research Resources Grant C06-RR-015455.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: Y.L., G.B., and J.C. conception and design of research; Y.L., G.B., M.H., and B.S. performed experiments; Y.L., G.B., M.H., and B.S. analyzed data; Y.L., G.B., M.H., B.S., L.C., and J.C. interpreted results of experiments; Y.L. and B.S. prepared figures; Y.L. and G.B. drafted manuscript; Y.L., G.B., L.C., and J.C. approved final version of manuscript; G.B., L.C., and J.C. edited and revised manuscript.
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