[PubMed] [Google Scholar] 22. 0.3% Na+Apo (= 7). Apo was administered in the drinking water at 1.5 mmol/l (4) starting 4 days before the 5-wk period. Catheters were implanted into the femoral artery and vein after 3 wk on the diets using isoflurane anesthesia (1%). Rats were allowed a 4-day recovery period after catheter surgery, and a 10-day period of collection of arterial pressure, GK921 heart rate, and renal hemodynamic data followed (23, 37, 39, 41). Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious rats on for 10 min at 4C to remove large molecular-weight DNA and insoluble structural proteins, and the supernatant was processed to isolate RNA. Aliquots of 5 g of RNA isolate were treated with DNase I (DNA-free RNA kit; Zymo Research). The concentration of nucleic acid was assessed using a UV spectrophotometer (SmartSpec 3000; Bio-Rad Laboratories). RNA quality was assessed by A260-to-A280 ratio and by electrophoresis of 0.9C1.0 g aliquot on a 1.2% agarose gel using 1 TBE buffer, with ethidium bromide staining. RNA was judged to be intact if the sample lane showed prominent discrete bands for 18S and 28S rRNA with no smearing. DNase-treated RNA was then used as a template for cDNA synthesis (iScript cDNA synthesis kit; Bio-Rad Laboratories) following the manufacturer’s protocol. The cDNA samples were diluted 1:10 with nuclease-free water before being used as a template for real-time RT-PCR. Real-time RT-PCR. Real-time RT-PCR assays were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) on an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories). Specific oligonucleotide primers for gp91phox, p22phox, p47phox, and p67phox were used for PCR amplifications. Undiluted (?)RT products were used as templates in negative control reactions to check for genomic DNA contamination within the cDNA. Other negative control reactions included NT reactions (which contained SYBR Green Supermix, forward and reverse primers, and nuclease-free water) as well as a blank (which contained SYBR Green Supermix and nuclease-free water). Relative fold expression of mRNA was quantified by using the 2?Ct mathematical model (7, 20). Measurement of renal glutathiones and renal monocytes/macrophages. Reduced (GSH) and oxidized glutathione (GSSG) were determined using the fluorescent detection of dansyl derivatives using HPLC according to the method of Jones (15) as we have done before (40, 41). Renal monocytes/macrophages GK921 from tissues collected at 5 wk of the various diets were measured by indirect immunoperoxidase methodology (26) using ED-1, a monoclonal antibody to monocytes/macrophages (Chemicon). Analysis of glomerular and tubulointerstitial injury. Kidney sections were examined for necrotic and sclerotic glomeruli at a 200 magnification using PAS-hematoxylin/eosin stains (22, 37). Tubulointerstitial renal injury was determined using a Masson trichrome-stained kidney section from each rat. Briefly, this injury was measured as the area of interstitial tissue with increased amounts of blue staining, dilated cast-containing tubules, or tubules showing GK921 acute injury divided by the area of nonglomerular and nonvascular cortex. Allopurinol study. Arterial and venous catheters were implanted as above in three 8% Na diet rats and three 8% Na+allopurinol (Allo) rats to block XO, and studies were run over a 5-wk period. Allo was administered in the drinking water at 10 mgkg?1day?1 starting 4 days before the 5-wk period began, and this dose of Allo has been shown to totally inactivate renal XO (17). Mean arterial pressure was measured continuously over the last 10 days of the experiment, and GFR was measured on as above. Statistical analysis. Comparisons of data from S rats on high or low sodium with and without apocynin treatment were performed using ANOVA followed by a Fisher least significant difference test for post hoc analysis. Variations were considered to be statistically GK921 significant if < 0.05. All data are indicated as means SE. RESULTS Renal cortical NAD(P)H oxidase subunit reactions to high-Na+Apo. Number 1 demonstrates S rats on high-Na-intake experienced significant raises in renal cortical gp91phox, p22phox, p47phox, and p67phox mRNA compared with 0.3% Na rats. Apo treatment in high-Na rats resulted in marked decreases in all subunits of NAD(P)H oxidase. Consequently, transcription of NAD(P)H oxidase is definitely importantly affected by Na intake and by Apo treatment. Open in a separate windowpane Fig. DHCR24 1. Effects of apocynin (Apo) and Na diet within the mRNA manifestation of NADPH oxidase subunits in Dahl salt-sensitive (S) rats in 8% Na (= 9), 8% Na+Apo (= 8), 0.3% Na (= 6C7), and 0.3% Na+Apo organizations (= 6). ?< 0.05 when comparing 8% Na with 8% Na+Apo group. *< 0.05 compared with 0.3% Na alone group. Renal cortical GSH/GSSG and renal cortical O2?? launch reactions to high-Na+Apo Number.