Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. epithelial cell apoptosis and EMT. = 3) showed that silencing was obvious at 24, 48, and 72 h after transfection. Therefore, 24 h after transfection, cells were stimulated with TGF-1. The effect of HSP72 silencing on EMT was observed at 72 h after TGF-1 exposure. For determination of the efficiency of HSP72 knockdown, Western blot analysis for HSP72 Milciclib was performed. Several Milciclib concentrations of HSP72 siRNA (10C50 nM) were used to determine the optimal knockdown concentration. Animals The experiments were performed with male Sprague-Dawley rats weighing 200C250 g managed with free access to water and standard rat food. UUO was performed by using the protocol approved by Animal Care and Use Committee of the Sun Yat-sen University or college (Guangzhou, China). Rats were anesthetized by injection of chloral hydrate (400 mg/kg ip) and allowed to breathe spontaneously. The left ureter of each animal was Milciclib located and double-ligated with 4-0 silk thread after a midline abdominal incision. Sham-operated rats experienced their ureters uncovered but not ligated. To induce optimal renal HSP72 expression, the GGA protocol explained by Suzuki et al. (35) was altered by our laboratory, and additional studies on dose- and time-dependent induction of HSP72 were performed using doses of 0, 200, and 400 to 800 mg/kg daily and time points of 12, 24, and 48 h after a signal dose of GGA (400 mg/kg). Rats received daily oral administration with optimal 400 mg/kg GGA, starting 1 day before the operation and continuing throughout UUO or sham surgery. Rats in the control group were given the same dose of vehicle. Rats (= 5 each) were killed on after UUO, and both kidneys were harvested and were then subjected to the studies explained below. Analysis of Morphology and TIF Kidney tissues were fixed in 10% phosphate buffer formalin, dehydrated through graded alcohol and xylene, embedded in paraffin, sectioned at 3-m thickness, and then stained with periodic acid-Schiff or Masson’s trichrome. Histological examinations were performed in a blinded manner. A point-counting method was used to evaluate chronic tubular injury and TIF (40). Briefly, under high magnification (400), 10 Sav1 nonoverlapping fields from each section of the renal cortex were photographed. A grid made up of 117 (13 9) sampling points was superimposed on each photograph. The number of points overlying chronic tubular injury and TIF was counted and expressed as a percentage of all sampling points (40). Chronic tubular injury was defined as tubular atrophy with interstitial fibrosis and infiltrate with/without tubular dilation, tubular cast formation, flattening of tubular epithelial cells, and thickening of the tubular basement membrane (25). Milciclib Immunohistochemical and Immunofluorescence Stainings All immunostainings were performed on 4-m paraffin-embedded kidney sections. Antigen retrieval was performed by microwave treatment. Unfavorable controls were performed with nonimmune mouse serum substituted for the specific main antibodies. In immunohistochemical staining, sections were exposed to 3% H2O2 for 20 min to destroy Milciclib endogenous peroxidase activity, rinsed with PBS once, blocked with 10% nonimmune sheep serum in PBS for 1 h, incubated with monoclonal mouse anti-HSP72 or PCNA, respectively, at 4C overnight, rinsed with PBS three times for 5 min each, and then incubated for 1 h with either sheep or horse horseradish peroxidase-conjugated anti-mouse IgG. The reaction was halted by rinsing with PBS when the color was fully developed using diaminobenzidine for 5 min. The PCNA-positive tubular and interstitial cells in the cortex were calculated in 10 high-power fields at 400 magnification within the same section of kidney from an individual animal. Results were expressed as percentage of the total nuclei in cortex. To detect E-cadherin, -SMA, or collagen I, cells or tissue sections were incubated with monoclonal mouse anti-E-cadherin, anti–SMA, and anti-collagen I at 4C overnight followed by Cy3-conjugated anti-mouse IgG. To identify nuclei, cells or sections were counterstained with 4,6-diamidino-2-phenylindole for 2 min. The positive stainings were detected using a laser-scanning confocal microscopy (Zeiss LSM 510 META, Carl Zeiss). For semiquantitative analysis of collagen I expression, all cortical fields were scored from 0 to 4 for each field, with an average for each kidney.