Third, recent studies have demonstrated that the genetic deficiency of HO-2 does not accentuate the vasoconstrictive effects of ANG II on renal blood flow (38). a stressed organ (by ANG II), but not that of the unstressed organ, requires intact renal HO activity, even when the imposed stress neither induces HO-1 nor HO activity. These findings may be germane to conditions attended by heightened ANG II levels, ineffective renal perfusion, and susceptibility to acute kidney injury. of the National Institutes of Health. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) were maintained on standard rat chow with water ad libitum and were used in all protocols. Administration of ANG II by Osmotic Minipumps ANG II (50 ngkg?1min?1 iv) or saline vehicle was administered for 2 wk to rats via an osmotic minipump (model 2ML2; Durect, Cupertino, CA) as detailed in our prior study (27). Briefly, under intramuscular ketamine and xylazine (100 and 10 mg/kg, respectively) anesthesia, incisions were made in the midscapular region and in the ventral neck. Osmotic minipumps were implanted in a subcutaneous pocket created in the midscapular region, and a catheter connected to the minipumps was tunneled through the subcutaneous space to the ventral neck and implanted into the external jugular vein. Assessment of Renal Hemodynamics Two CX-4945 sodium salt weeks after the start of ANG II or saline vehicle infusion by minipump, renal hemodynamics were assessed in rats using methods described in detail in our prior studies (26, 27). Briefly, rats were anesthetized with Inactin (100 mg/kg ip; BYK-Gudden, Konstanz, Germany) and placed on a heated table to maintain body temperature at 37C. A tracheotomy was performed using polyethylene (PE)-240 tubing. The femoral artery and vein were cannulated with PE-50 tubing for monitoring blood pressure and for infusions, respectively. Euvolemia was achieved and maintained by infusion of 4% albumin in 0.9% saline solution, initially at 1% of body wt over 30 min and subsequently at the rate of 0.5 ml/h. Additionally, a 1% inulin solution in 0.9% saline was given as a 1-ml bolus infused over 5 min and thereafter at a rate of 1 1.5 ml/h for clearance studies. The urinary bladder was catheterized with PE-160 tubing for urine collection. Whole kidney blood flow of the left kidney was measured with a 0.7 mm-diameter perivascular flow probe (Transonic Systems, Ithaca, NY) placed around the renal artery. Intrarenal distribution of renal blood flow was measured using a laser Doppler needle flow probe (25 gauge; Transonic Systems) set on a micromanipulator; one probe was placed on the superficial cortex, and CX-4945 sodium salt a second probe was advanced CX-4945 sodium salt into the renal medulla (visually verified at the end of the experiment). ANG II-treated and saline vehicle-treated rats were administered an inhibitor of HO activity (40 mol/kg iv SnMP, given as a bolus) or vehicle, exactly as previously described by Rodriguez and colleagues (32, 33). After 60 min of equilibration, CX-4945 sodium salt clearance studies were begun during which urine was collected for two consecutive periods with blood samples drawn in the middle of each period. Assessment of Renal mRNA Expression In additional groups of rats, ANG II (50 ngkg?1min?1 iv) or saline vehicle was administered by osmotic minipump as described above, and after 2 wk, renal mRNA expression was assessed. Renal mRNA expression was also assessed in studies in which SnMP or vehicle was administered to ANG II-treated rats. Total RNA was extracted from rat kidney tissue using the Trizol method (Invitrogen, Carlsbad, CA) and further purified with an RNeasy Mini Kit (Qiagen, Valencia, CA), according to each manufacturer’s protocol. Two hundred nanograms of total RNA were used in reverse transcription reactions (Transcriptor First Strand cDNA Synthesis Kit; Roche Applied Science, Indianapolis, IN) by using random hexamers. The resulting cDNA was used in quantitative real-time PCR analysis as in our earlier study (31). Reactions were performed on an ABI Prism 7900HT (Applied Biosystems, Foster City, CA) using TaqMan Mastermix reagent (part no. 4324020; Applied Biosystems). Probes and primers used for TUBB3 quantification were obtained as assay.