Mechanisms of Renal Injury Induced Chronic Hypertension

Li E. Yang, S. H. Ye, P. K.K. Leong, V.M. Campese, A. A. McDonough
Department of Physiology and Biophysics
University of Southern California, Keck School of Medicine
Los Angeles, CA 90089-9142

Materials and Methods

1. Phenol-renal injury animal model. Male Sprague-Dawley rats are anesthetized with ketamine and xylazine. Renal injury is caused by injecting 50 µl of 10% phenol into the lower pole of the kidney. Sham rats receive 50 µl of saline in the lower pole of the kidney. Rats are then allowed to recover. After 5 weeks rats are anesthetized and carotid artery is canulated to monitor blood pressure. Systolic blood pressure increased to 132 ± 3 mmHg in phenol injected rats vs. 115 ± 3 in saline injected rats. After the in vivo operation kidneys are cooled in situ before excision by flushing the abdominal cavity with ice-cold PBS solution to block membrane trafficking. Cortices are dissected, homogenized, and low speed supernatants collected for crude total membranes (So), or processed for further subcellular fractionation.

2. Subcellular fractionation of renal cortex.

3. Immunoblot analysis and antibodies.
Used to assess relative abundance and dis- tribution of NHE3 and NaPi2. Polyclonal anti-NHE3 NHE3-C00 is made in the McDonough Lab. Polyclonal antisera to NaPi2 is generated by Biber and Murer (University of Zürich, Switzerland). Signals are detected by either Odyssey™ Infrared Imaging System (LI-COR) or ECL (Amersham).

4. Indirect immunofluorescence.
Kidneys were fixed in situ in PLP fixative (2% paraformaldehyde, 75 mM lysine and 10 mM Na-periodate, pH 7.4) for 20 min, removed and postfixed in PLP for another 4-6 hours, cryoprotected in 30% sucrose in PBS, then embedded in Tissue-Tek O.C.T. Compound and frozen in liquid nitrogen. Cryosections (5 mM) were cut and transferred to Fisher Superfrost Plus-charged glass slides. The sections were rehydrated in PBS 10 min, followed by washing with 50 mM NH4Cl in PBS 10 min, then with 1% SDS in PBS for 4 min for antigen retrieval and blocked with 1% bovine serum albumin in PBS. Double labeling was performed by incubating with polyclonal antiserum NHE3-C00 and monoclonal antibody against villin. A mixture of FITC-conjugat ed goat-anti-rabbit and Alexa 568-conjugated goat-anti-mouse secondary anti bodies were used. Slides were viewed with a Nikon PCM Quantitative Measuring High-Performance Confocal System.

5. Quantitation and statistical analysis.
Data are expressed as means ± SE. Two- way ANOVA was applied to determine whether there was a significant effect of treatment on the overall distribution pattern. If a significance was established (P < 0.05), the location of the difference in the pattern was assessed by two-tailed Student's t-test for paired samples, and the differences were regarded significant at P < 0.05.