Kallikrein gene transfer reduces renal fibrosis, hypertrophy, and proliferation

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Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina3 [9 w6 m/ H2 D' P
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ABSTRACT
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In DOCA-salt hypertension, renal kallikrein levels are increased and may play a protective role in renal injury. We investigated the effect of enhanced kallikrein levels on kidney remodeling of DOCA-salt hypertensive rats by systemic delivery of adenovirus containing human tissue kallikrein gene. Recombinant human kallikrein was detected in the urine and serum of rats after gene delivery. Kallikrein gene transfer significantly decreased DOCA- and salt-induced proteinuria, glomerular sclerosis, tubular dilatation, and luminal protein casts. Sirius red staining showed that kallikrein gene transfer reduced renal fibrosis, which was confirmed by decreased collagen I and fibronectin levels. Furthermore, kallikrein gene delivery diminished myofibroblast accumulation in the interstitium of the cortex and medulla, as well as transforming growth factor (TGF)-1 immunostaining in glomeruli. Western blot analysis and ELISA verified the decrease in immunoreactive TGF-1 levels. Kallikrein gene transfer also significantly reduced kidney weight, glomerular size, proliferating tubular epithelial cells, and macrophages/monocytes. Reduction of proliferation and hypertrophy was associated with reduced levels of the cyclin-dependent kinase inhibitor p27Kip1, and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). The protective effects of kallikrein were accompanied by increased urinary nitrate/nitrite and cGMP levels, and suppression of superoxide formation. These results indicate that kallikrein protects against mineralocorticoid-induced renal fibrosis glomerular hypertrophy, and renal cell proliferation via inhibition of oxidative stress, JNK/ERK activation, and p27Kip1 and TGF-1 expression.- h% [! W1 W7 o1 T+ a
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transforming growth factor-1; oxidative stress; nitric oxide; mitogen-activated protein kinase& W7 S% r  D8 }  |. D! |
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DOCA-SALT ADMINISTRATION has been reported to provoke a low-renin hypertension in rats (9). This volume-overload hypertension results in sodium and water retention in an angiotensin-independent manner. The renal kallikrein-kinin system (KKS) has been shown to play a role in suppressing the development of DOCA-salt hypertension (16, 22). The renal KKS is activated after the administration of mineralocorticoids in conscious rats and consequently enhances urinary excretion of prostaglandins (28). This compensatory augmentation of renal kallikrein found in DOCA-salt rats is most likely a response to sodium and volume retention (3). However, activation of the endogenous renal KKS may not be adequate to protect against hypertension, renal injury, and other organ damages caused by DOCA-salt treatment (31). It has been shown that potentiation of kinin formation in the renal tubules prevents the development of hypertension by inhibition of sodium retention (12, 15, 21). Moreover, in vivo transfer of antisense oligonucleotide against urinary kininase blunted DOCA-salt hypertension in rats (10). In addition, our previous study showed that adenovirus-mediated kallikrein gene delivery attenuates hypertension and protects against renal injury in DOCA-salt rats (7). Therefore, potentiation of the renal KKS may offer an intervention to protect against DOCA-salt-related renal damage.
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6 s3 R, g# A+ K" H8 w2 h0 QDOCA-salt treatment often triggers a malignant hypertension that gradually leads to damage of the kidney, heart, and vasculatures (18, 24, 34, 36). Glomerular sclerosis, tubular fibrosis, and cardiac hypertrophy and fibrosis are commonly observed in DOCA-salt-treated animals, along with activation of renal transforming growth factor (TGF)-1 expression and downregulation of endothelial nitric oxide synthase levels in the heart, kidney, and blood vessels (19, 37). Furthermore, administration of DOCA-salt induces oxidative stress by increasing the formation of superoxide (23), thereby contributing to organ injury. Based on these observations, we further evaluated DOCA-salt-induced renal injury by examining the effect and potential mechanism of human kallikrein gene delivery on renal fibrosis, hypertrophy, and cellular proliferation. In this study, we demonstrate that the KKS has protective effects on kidney damage in the DOCA-salt animal model by suppressing oxidative stress, extracellular matrix (ECM) protein expression, TGF-1 levels, and c-Jun NH2-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) activation.* H; n, W8 q- p$ v# ~8 `

; L' f5 O8 |3 g. `, AMATERIALS AND METHODS
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Preparation of adenovirus harboring the human tissue kallikrein gene. Adenoviral vectors harboring the luciferase or human tissue kallikrein cDNA under the control of the cytomegalovirus (CMV) enhancer/promoter (Ad.CMV-Luc, Ad.CMV-TK) were constructed and prepared as previously described (7).
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# [4 x4 m- \3 o0 @9 F0 hAnimal treatment. Left unilateral nephrectomy was performed on male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) at 4 wk of age. After surgery (1 wk), experimental animals received weekly subcutaneous injections of DOCA (30 mg/kg body wt; Sigma) suspended in sesame oil and were provided with 1% NaCl drinking water. Each rat was injected with 1 x 1010 plaque-forming units of either Ad.CMV-Luc or Ad.CMV-TK (n = 10 each) one time via the tail vein 2 wk after the start of steroid/salt treatment. For the control group, seven rats were subcutaneously injected with sesame oil and provided with tap water. 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 were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.
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# z* N; e% q& E8 ]- v7 ]Blood pressure measurement. Systolic blood pressure was measured with DASYlab 5.5 software (Kent Scientific, Turrington, CT) by the tail-cuff method. Unanesthetized rats were placed in a plastic holder resting on a warm pad maintained at 37°C during the measurements. Average readings were taken for each animal after the animals had become acclimated to the environment.
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3 E$ e. ^0 z1 F. c; y  w, ZBlood and urine collection. For blood collection, unanesthetized rats were placed in a 37°C incubator for 10 min. Rats were then transferred to a plastic holder, and an insulin syringe was used to withdraw blood from the tail vein. Serum was collected at days 3, 7, and 12 after virus injection. Twenty-four-hour urine was collected from rats in metabolic cages 5 days after virus delivery. To eliminate contamination of urine samples, animals received only water during the 24-h collection period. Urine was collected and centrifuged at 1,000 g to remove particles. Serum and urine were stored at –20°C until analysis.
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Morphological and histological analyses. After gene transfer (17 days), rats were anesthetized with ketamine/xylazine (90 mg﹞10 mg–1﹞1 kg body wt–1). Kidneys were removed, washed in saline, blotted, and weighed. Kidney sections were preserved in 4% formaldehyde solution, paraffin-embedded, and cut to a thickness of 4 μm. Kidney sections were subjected to immunohistochemistry and Sirius red staining (7), which stains collagen fibers red and cytoplasm yellow. Kidney damage was identified by Sirius red staining by counting the damaged and sclerotic glomeruli and tubular protein casts in each section. The glomerular area of 25 glomeruli of each kidney section was measured using NIH Image software under blind conditions without prior knowledge as to which section belonged to which rat. For immunohistochemistry, the following antibodies were used: mouse anti-collagen I (1:400 dilution; Sigma-Aldrich), mouse anti--smooth muscle actin (-SMA, 1:2 dilution; Sigma-Aldrich), rabbit anti-TGF-1 (1:100 dilution; Santa Cruz), mouse anti- p27Kip1 (1:500 dilution; BD Transduction), and mouse anti-proliferating cell nuclear antigen (PCNA, 1:3,000 dilution; Sigma-Aldrich). Immunohistochemistry was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories). Collagen I immunostaining was evaluated in a score of 0–3, based upon the amount of staining observed in a gridded area of the kidney at low magnification (x40) as follows: 0 = no staining, 1 = 1–3 areas, 2 = 4–7 areas, 3 = 8 or more areas. PCNA-positive cells were quantified in 15 fields per cross-section in the cortex, excluding glomeruli. Double immunolabeling was performed by incubating a mixture of rabbit anti-PCNA antibody (1:100 dilution; Santa Cruz) and mouse anti-ED-1 antibody (a marker for macrophages/monocytes, at 1:20 dilution; Chemicon). A mixture of anti-mouse IgG-TRITC antibody (1:200 dilution; Sigma-Aldrich) and anti-rabbit IgG-FITC antibody (1:200 dilution; Sigma-Aldrich) was used as a secondary antibody.' T0 B' A3 m3 |. h2 T: P5 @  n
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Renal cytoplasmic and nuclear protein extraction. Renal tissues were homogenized in lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 20 mM Na4P2O7, 2 mM Na3VO4, and 1% Triton X-100) containing 1:100 protease inhibitor cocktail (Sigma) and centrifuged at 14,000 rpm at 4°C for 40 min. After centrifugation, the supernatant (the cytosolic fraction) was removed and stored at –80°C. To extract the nuclear proteins, pellets were resuspended in an equal volume of lysis buffer, and NaCl was added to a final concentration of 0.6 M. After incubation on ice for 1 h, the lysates were centrifuged at 14,000 rpm at 4°C for 30 min, and the nuclear proteins in the supernatant were removed and stored at –80°C.
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ELISA for human tissue kallikrein and TGF-1. The levels of immunoreactive tissue kallikrein in rat serum and urine were measured by an ELISA specific for human tissue kallikrein, as previously described (4). Total TGF-1 levels in renal extracts were determined by ELISA according to the manufacturer's instructions (R&D).; R; d2 U( I3 T; D
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Western blot analyses for fibronectin, TGF-1, p27Kip1, and MAPKs. Kidney extracts (100 μg) were resolved by SDS-PAGE for Western blot. Proteins were electrotransferred on nitrocellulose membrane in a transfer buffer containing 25 mM Tris base, 0.2 M glycine, and 20% methanol (pH 8.5). Membranes were incubated in blocking buffer (1x Tris-buffered saline, 0.1% Tween 20 with 5% wt/vol nonfat dry milk) for 1 h and then incubated with antibodies against the following: fibronectin, phospho-p42/p44 ERK (1:1,000 dilution; New England BioLabs), total p42/p44 ERK (1:1,000 dilution; Santa Cruz), TGF-1 (1:1,500 dilution; Santa Cruz), phospho- and total p46/p54 JNK (1:1,000 dilution; Cell Signaling), p27Kip1 (1:2,500 dilution; BD Transduction Laboratories), and -actin (1:5,000 dilution; Sigma) at 4°C overnight with gentle shaking. Membranes were incubated with secondary anti-rabbit or anti-mouse antibody conjugated to LumiGLO chemiluminescent reagent. Chemiluminescence of the blot was detected by an ECL-Plus kit (Perkin-Elmer) according to the manufacturer's instruction and exposed to Kodak X-ray films.1 Q  P" C  |5 @# M

- V. B2 C% l1 e0 {Assays for urinary protein, nitrate/nitrite, and cGMP levels. Total urinary protein levels were measured by MicroLowry assay. Urinary nitrate/nitrite (NOx) levels were measured by a fluorometric assay (26). Briefly, urine samples were incubated in the presence of 5 μl of 14 mU nitrate reductase (Sigma) and 5 μl of NADPH (Sigma) in 20 mM Tris buffer, pH 7.6. Next, 10 μl 2,3-diaminonaphthalene were added, resulting in the generation of a fluorescent product. Fluorescence was measured using a spectrofluorometer set at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Urinary cGMP levels were measured by RIA, as previously described (39).
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6 t4 w* i- a- R+ hSuperoxide measurements. Superoxide levels were quantified by a spectrophotometric assay based on the rapid reduction of ferricytochrome c to ferrocytochrome c. Non-superoxide-dependent reduction of cytochrome c was corrected for by deducting the activity not inhibited by superoxide dismutase (SOD).
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7 O/ u8 r; _, y" pStatistical analysis. Results are expressed as means ± SE. Comparisons among groups were made by ANOVA followed by Fisher's protected least-significant difference or by unpaired Student's t-test. Differences were considered significant at P
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' G! F1 I. `! F8 t. MRESULTS5 Y" G8 `  L1 P' U: m
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Expression of human kallikrein and its effects on blood pressure. Expression of recombinant human tissue kallikrein in rats was measured by an ELISA. After intravenous injection of Ad.CMV-TK, immunoreactive human kallikrein expression reached a level of 3.6 ± 0.5 μg/ml (n = 10) in rat serum at day 3 postgene delivery and declined thereafter (243.1 ± 82.6 ng/ml at day 7; 137.1 ± 55.8 ng/ml at day 12, n = 10). Human tissue kallikrein was also detected in rat urine at day 5 at a level of 16.5 ± 2.2 μg﹞100 g body wt–1﹞day–1 (n = 10). Moreover, kallikrein gene delivery significantly reduced systolic pressure compared with rats receiving the luciferase gene 5 days after virus injection (158.1 ± 4.7 vs. 177.4 ± 3.3 mmHg, n = 10, P 3 u- a$ Z  U: @0 T

9 b3 h. D  z7 L9 `% jEffect of kallikrein gene transfer on proteinuria and renal injury in DOCA-salt rats. Delivery of the kallikrein gene significantly reduced urinary protein levels compared with the luciferase group (35.7 ± 2.8 vs. 64.3 ± 13.1 mg﹞day–1﹞100 g body wt–1, n = 7–9, P ; d- K, q0 x' L& e$ L0 v4 x
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Glomerular sclerosis was quantified in DOCA-salt hypertensive rats, as shown in Fig. 1B. It was observed that DOCA-salt hypertensive rats injected with the kallikrein gene had a significant reduction in glomerular sclerosis compared with DOCA-salt rats receiving the luciferase gene (1.44 ± 0.56 vs. 14.57 ± 7.15 damaged and sclerotic glomeruli/cross section, n = 7–9, P
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% @: x$ ~, Z& `$ K3 e: W% VEffect of kallikrein gene transfer on renal fibrosis in DOCA-salt rats. The protective effect of kallikrein on DOCA-salt-induced renal fibrosis was further verified by immunostaining of kidney sections for collagen I. Figure 2A shows representative images of collagen I immunostaining. Positive staining was found only in connective tissue around blood vessels in the control (Fig. 2A, left). After DOCA-salt treatment, many damaged tubules were stained in the Ad.CMV-Luc group (Fig. 2A, middle), and kallikrein gene transfer (Ad.CMV-TK) significantly reduced collagen accumulation (Fig. 2A, right) to the level comparable to the control group. Kallikrein gene transfer markedly decreased the quantitative score of collagen type I deposition in the cortex compared with the luciferase group (0.5 ± 0.39 vs. 1.88 ± 0.66, n = 5, P
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Figure 3A shows immunostaining of -SMA, a marker of myofibroblasts, in the renal cortex and medulla. Positive staining of -SMA was found only in the blood vessels in the control group. In DOCA-salt rats receiving the luciferase gene, -SMA was mainly expressed in the interstitial space. However, the expression of -SMA was attenuated with kallikrein gene transfer. As shown in Fig. 3B, immunoreactive TGF-1 was mainly found in the glomeruli of the luciferase group, whereas kallikrein gene transfer markedly decreased TGF-1 expression in these regions. Western blot analysis showed that kallikrein gene delivery significantly reduced relative TGF-1 levels normalized by -actin in the kidney of DOCA-salt rats compared with those in rats receiving the luciferase gene (Fig. 3C). This observation was consistent with the TGF-1 ELISA results (Fig. 3D) in that kallikrein gene transfer markedly reduced total renal TGF-1 levels compared with that of the luciferase group (257.3 ± 28.3 vs. 515.6 ± 69.0 pg/mg protein, n = 5–6, P 5 W) T( N7 n/ o* q
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Effect of kallikrein gene transfer on renal hypertrophy and cellular proliferation in DOCA-salt rats. The ratio of right kidney weight to body weight was significantly reduced after kallikrein gene transfer compared with the luciferase group (0.75 ± 0.03 vs. 0.88 ± 0.06 g/100 g body wt, n = 6–10, P ) B* ?' V# A6 c/ t* r' k

! w3 p% ]$ r  p0 j6 h7 W  P' n4 }9 SEffect of kallikrein gene transfer on p27Kip1 levels and MAPK activation. Positive immunostaining of the cyclin-dependent kinase inhibitor p27Kip1 was mainly observed in glomeruli in DOCA-salt rats receiving Ad.CMV-Luc, but not in control or kallikrein groups (Fig. 6A). Representative Western blot of p27Kip1 in renal nuclear extracts is shown in Fig. 6B, bottom. Expression of renal p27Kip1 protein was markedly decreased after kallikrein gene transfer compared with that of the luciferase group. Figure 6, C and D, shows Western blot analyses of phospho- and total JNK and ERK and the quantitative result. Kallikrein gene delivery markedly reduced phosphorylation of JNK and ERK in the kidney of DOCA-salt rats compared with rats injected with the luciferase gene.
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Effect of kallikrein gene transfer on urinary NOx and cGMP excretion and superoxide formation. DOCA-salt induced NOx excretion in urine and kallikrein gene delivery further elevated urinary NOx levels compared with rats receiving the luciferase gene (145.1 ± 18.2 vs. 65.1 ± 13.6 mmol﹞day–1﹞100 g body wt–1, n = 8, P ' u% W6 z, j* Q. t

4 U, v. H1 y; G# qDISCUSSION, N( K: u; ?! _' f) m$ n* g# e

' m( b& i5 H. m9 y- f9 L' F9 TIn the present study, we demonstrated that human tissue kallikrein gene delivery protects against DOCA-salt-induced renal fibrosis, cellular proliferation, and glomerular sclerosis and hypertrophy. After systemic delivery of adenovirus containing the kallikrein gene, immunoreactive human kallikrein was detected in the serum and urine of DOCA-salt rats, indicating that recombinant human kallikrein was secreted from the liver and kidney. In addition, we have previously shown that, after kallikrein gene delivery, human kallikrein mRNA is present in key tissues involved in cardiovascular and renal function, such as the heart, aorta, and kidney (7). Expression of recombinant kallikrein in DOCA-salt rats was capable of eliciting renal protective actions via modulation of the profibrotic factor TGF-1, JNK/ERK activation, cyclin-dependent kinase inhibitor, and oxidative stress, leading to a reduction in kidney injury in these animals. The current study provides significant insights regarding the role of the KKS in mediating protective actions in volume-overload hypertension.
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2 ]5 Z+ q; G' |DOCA-salt hypertensive rats receiving the control virus exhibited apparent renal damage, including massive tubular protein cast accumulation, collagen deposition, and glomerular sclerosis. These observations were accompanied by a rise in urinary protein excretion. Increased urinary protein levels were most likely a result of kidney injury, secondary to increased glomerular pressure in the setting of volume-overload hypertension. After kallikrein gene transfer, however, urinary protein levels were markedly reduced in the DOCA-salt-treated animals, depicting renal protection by kallikrein./ @9 k/ c3 Y. c' P- l, k
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Low-renin hypertension is an outcome of DOCA-salt administration, yet accumulating evidence indicates that a local renin-angiotensin system (RAS) exists in the kidney and contributes to renal injury of DOCA-salt hypertension by stimulating gene expression of TGF-1 and ECM components (17). TGF-1 is a key factor in fibrosis by promoting ECM protein synthesis and myofibroblast formation (14). In the present study, renal TGF-1 levels, in conjunction with fibronectin and collagen type I protein expression, were upregulated after DOCA-salt administration, whereas kallikrein gene transfer markedly attenuated their increased expression. We also observed that DOCA-salt-induced myofibroblast accumulation was reduced after kallikrein gene delivery. We found that expression of recombinant kallikrein resulted in a significant increase in urinary NOx and cGMP excretion over that observed in rats receiving the luciferase gene. It is possible that, through an NO-cGMP pathway, kallikrein is able to downregulate the production of TGF-1 and ECM components. This is supported by the fact that NO can inhibit TGF-1 and collagen expression in mesangial cells (6). Thus, by reducing TGF-1 production, tissue kallikrein could decrease collagen deposition and myofibroblast accumulation, consequently preventing the development of renal fibrosis.' m- K4 B& g, l5 E- \
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The cell cycle inhibitory protein p27Kip1 functions as a regulator of cellular proliferation and hypertrophy (32). TGF-1 promotes glomerular hypertrophy in diabetic mice through prohypertrophic mechanisms in the mesangial cell (33), and p27Kip1 has been shown to be required for the development of glomerular hypertrophy induced by TGF-1 (27). In this study, we observed that DOCA-salt rats receiving the luciferase gene had a larger kidney weight and a significant increase in glomerular size. Increased immunostaining of p27Kip1 in the glomeruli and a notable rise in renal nuclear protein levels of p27Kip1 would explain these observations. Kallikrein gene delivery, on the other hand, dramatically attenuated the development of glomerular hypertrophy and the expression of p27Kip1 in the kidney. Because p27Kip1 is necessary for TGF-1-induced glomerular hypertrophy, downregulation of TGF-1 by kallikrein may be the cause for the decrease in glomerular size. TGF-1 and p27Kip1 were primarily expressed in the glomeruli of DOCA-salt rats, signifying that both play a role in the development of glomerular hypertrophy. JNK activity has been shown to be involved in cardiac hypertrophy (13) and, as we have observed in this study, may also play a part in the enlargement in glomerular size. Cellular proliferation, localized in distal tubules and collecting ducts, was also observed in the DOCA-salt rats receiving control virus. Proliferating cells, however, were not prevalent in the glomeruli, indicating that p27Kip1 may be regulating the cell cycle in addition to promoting hypertrophy in mesangial cells of the glomeruli. This finding is also in agreement with the observation that p27Kip1 was not localized in tubular and collecting duct cells. Although both elevated proliferation and hypertrophy were observed in our study, our results showed that increased levels of p27Kip1 accompanied glomerular hypertrophy. This suggests that p27Kip1 contributes to the development of hypertrophy, whereas if a relationship between p27Kip1 and proliferation existed, then the levels of these proteins would be reduced or undetectable in rats treated with DOCA-salt.
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4 E0 A" [, m2 y: C' NAmple evidence has shown that, in cultured cell lines, mitogenic stimulation by various extracellular agonists correlates with activation of ERK (2). Dominant-negative mutants of Ras or Raf-1, components upstream of mitogen/extracellular cell-regulated kinase in the ERK signaling cascade, were shown to inhibit growth factor-induced cell proliferation, whereas constitutively activated Raf-1 induces cell proliferation (25, 30). Moreover, catalytically inactive mutants of ERK and its antisense cDNA inhibit proliferation (29). As shown in the present study, activation of ERK in the kidney of DOCA-salt rats is a novel finding and points to ERK as a putative regulator of the proliferative response to mineralocorticoid injury in vivo. More intriguingly, we found that phosphorylation of ERK was completely blocked in the kidney after kallikrein gene transfer. Because ERK plays a pivotal role in cellular proliferation, inhibition of ERK activation by kallikrein gene transfer may account for the reduction of cell proliferation in the kidney, thereby protecting against DOCA- salt-induced renal fibrosis and glomerular hypertrophy.
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; w! l# t6 F, d0 d5 i6 LIt is known that, after administration of DOCA-salt, oxidative stress is mainly induced in the vasculature (35), although the local RAS has been implicated in stimulating NADH/NADPH oxidase activity and superoxide formation in the kidney as well (1). Reduced Cu/Zn SOD activity is also considered to contribute to oxidative stress in the aorta of DOCA-salt rats (40). In the present study, increased superoxide formation in the kidney was markedly reduced after kallikrein gene transfer. This observation may be because of an increase in NO production. NO can directly react with superoxide to form peroxynitrite, thereby inactivating superoxide (5). Moreover, NO can inhibit the assembly of NADH/NADPH oxidase subunits, thus reducing superoxide formation (8). A previous study also showed that NO blockade enhances renal responses to SOD inhibition in dogs, suggesting that NO serves a renoprotective effect against these actions of superoxide (20). It would also be interesting to investigate whether kallikrein gene delivery could partly restore SOD activity in DOCA-salt rats and subsequently facilitate the protective effect of kallikrein/kinin on oxidative stress-induced renal damage.
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Our present study showed that unilateral nephrectomized rats after DOCA administration and high-salt loading for 19 days and injected with control virus for 5 days had an average systolic blood pressure value of 177 mmHg, whereas rats receiving the kallikrein gene had an average blood pressure value of 158 mmHg, which was 40 mmHg higher than control rats. The renoprotective effect of kallikrein gene transfer cannot be explained solely based on blood pressure reduction. In support of this notion, it has been shown that a long-term infusion of rat urinary kallikrein in Dahl salt-sensitive rats on high-salt intake was able to attenuate renal injury without affecting systolic blood pressure (38). Furthermore, infusion of the B2 receptor antagonist icatibant (HOE-140) in salt-loaded Dahl salt-sensitive rats abolished kallikrein's protective effect in the kidney but had no effect on the time-dependent rise in blood pressure (11). The mechanism by which kallikrein exerts renal protection in DOCA-salt-induced renal damage and the role of kinin B2 receptor in mediating this protective effect awaits further investigation./ G9 h" C  m* u' U$ p" v' h3 X8 F
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In summary, we have shown that adenovirus-mediated gene delivery of human kallikrein protected against oxidative stress-induced renal damage, fibrosis, and glomerular hypertrophy in volume-overload hypertensive rats. The ability of kallikrein gene delivery to produce these beneficial effects could be mediated by NO production, subsequently leading to attenuation of oxidative stress, reduction of renal TGF-1 expression, and inhibition of MAPK activation in DOCA-salt hypertension.
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GRANTS
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3 _1 j" K6 r$ G$ L2 p' ?This work was supported by National Institutes of Health Grant HL-29397 and DK-066350.
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0 I( G5 D8 G. IACKNOWLEDGMENTS
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We thank Dr. Jo Anne Simson for critical evaluation of tissue sections with histological and immunostaining analyses.$ h# Y' g9 k8 j( T! @% B  ?& F2 \

) f3 S  Q" T2 [* oFOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.4 o3 C  J/ u0 V8 ~- J2 u

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