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To evaluate immunoreactivity to p21, the volume ratios of positive areas of renal tissue (in %) (Lancas et?al. 2011), determined by the color limit, were obtained by image analysis with the program Image-Pro Plus, version 4.1 (Media Cybernetics, Silver Spring, MD) on a computer coupled to a microscope (Axioskop 40; Carl Zeiss) and a digital camera. Gene expression GS-7340 We performed real-time qPCR in frozen renal tissue, assessing the following gene: VDR (Rn00690616-m1). We extracted and prepared total RNA. For cDNA synthesis, we used total RNA and SuperScript VILO MasterMix (Invitrogen, Carlsbad, CA). We performed real-time PCR using TaqMan (Applied Biosystems, Foster City, CA) on Step One Plus (Applied Biosystems). Primers were purchased from Invitrogen. Adenylyl cyclase We evaluated relative gene?expression with the 2?����Ct method (Livak and Schmittgen 2001), using glyceraldehyde 3-phosphate dehydrogenase as the housekeeping gene. Statistical analysis All quantitative data are mean?��?SEM. We analyzed differences among the means of multiple parameters by one-way analysis of variance followed by the Student-Newman-Keuls test. Values of P?selleck chemicals and higher RVR. Table 1 Biochemical and hemodynamic parameters in rats fed a standard or vitamin D-free diet and subjected or not to renal ischemia/reperfusion Calcium and phosphorus Serum calcium was lower in both VDD groups than in control rats (Table?(Table1).1). Serum phosphorus was lower in VDD, IRI, and VDD?+?IRI rats than in control rats. Fractional excretion of calcium (FECa) was lower in VDD and VDD?+?IRI rats than in control and IRI rats, although it was higher in VDD?+?IRI rats than in VDD rats. In contrast, fractional excretion of phosphorus (FEP) was higher in IRI rats than in control and VDD rats.