The severity of glomerulosclerosis and glomerular size was calculated semiquantitatively using a 0 to 3 scale (0, normal or almost normal; 1, moderate; 2, moderate; 3, severe) for each glomerulus. expression of tumor necrosis factor-, intercellular adhesion molecule-1, tissue inhibitor of metalloproteinase-2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Moreover, the detrimental effect of TK blockade resulted in reduced nitric oxide (NO) levels as well as increased serum lipid peroxidation, renal NADH oxidase activity, and superoxide formation. In cultured proximal tubular cells, TK inhibited angiotensin II-induced superoxide production and NADH oxidase activity via NO OXF BD 02 formation. In addition, TK markedly increased matrix metalloproteinase-2 activity with a parallel reduction of TIMP-2 and PAI-1 synthesis. These findings show that endogenous TK has the propensity to preserve kidney structure and function in rats with chronic renal disease by inhibiting oxidative stress and activating matrix degradation pathways. Keywords: chronic kidney disease, inflammation, fibrosis tissue kallikrein (TK), a serine proteinase synthesized in many organs, specifically processes low-molecular-weight kininogen to produce potent vasoactive peptides known as kinins (25). Kinins are especially active on the vascular endothelium where they stimulate kinin B2 receptors, which in turn, trigger the release of nitric oxide (NO) and other endothelial mediators to promote vascular dilation and inhibit platelet adhesion and aggregation. TK has also been shown to directly activate the kinin B2 receptor, impartial of kinin formation (16). TK is usually synthesized in large amounts in the kidney, released in the peritubular interstitium, and excreted in the urine (27). It has been reported that renal TK excretion is usually significantly lower in patients with moderate chronic renal disease and more markedly reduced in patients with severe renal failure (8, OXF BD 02 20). Conversely, restriction of dietary sodium intake in humans leads to increased kallikrein excretion in the urine (1). Reduced urinary kallikrein levels have also been explained in hypertensive rat models, including Dahl salt-sensitive and spontaneously hypertensive rats (9, 33). In addition, TK expression was specifically diminished in the rat kidney after recovery from ischemia-reperfusion injury (2). Interestingly, the use of the potent TK inhibitor aprotinin in cardiac surgery has been shown to be associated with increased renal failure and mortality (29). These combined findings suggest that endogenous TK plays an important role in preserving renal function and that expression of the kallikrein gene may serve as a powerful marker for linkage analysis in populations with salt-sensitive hypertension and renal disease. OXF BD 02 Therefore, the purpose of this study was to determine the role of endogenous TK in chronic renal injury in a rat model of salt-induced hypertension. MATERIALS AND METHODS TK purification and antibody generation. TK was purified using DEAE-cellulose and aprotinin-affinity column chromatography as previously explained (6, 30). Polyclonal antibody to TK was raised in rabbits and purified with a protein A-affinity column. Neutralizing ability of anti-TK antibody was verified by an enzymatic activity assay using the chromogenic substrate S-2266 (diaPharma, West Chester, OH). Animal treatment. All procedures complied with the requirements for care and use of animal subjects as stated in the (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 or college of South Carolina. Male Wistar rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 200C220 g were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) before undergoing left OXF BD 02 unilateral nephrectomy. One week after surgery, rats in the sham group (= 6) received weekly subcutaneous injections of sesame oil and were provided with tap water. Experimental animals received weekly subcutaneous injections of PLCG2 DOCA (25 mg/kg body wt; Sigma, St. Louis, MO) suspended in sesame oil and were provided with 1% NaCl drinking.