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Supplemental Methods Experimental Protocol. All experiments were performed on 6-8 week old female mice. A single subcutaneous dose of 8.14 mg/kg HgCl2 (Fisher Scientific) in normal saline causes significant AKI in mice on the Balb/c background . Balb/c mice were administered 8.14mg /kg HgCl2 and sacrificed 24 hours (1 day), 72 hours (3 days), 5 days or 10 days later and compared to mice not administered HgCl2, 0 day. To block transcriptional activity of all NFATc proteins in Balb/c mice, CsA was administered daily, beginning 7 days before treatment with HgCl2, until each end point. CsA prepared in polyoxyethylated castor oil and absolute alcohol (Bedford Labs) was diluted in normal saline and administered in the peritoneal cavity at a daily dose of 10mg/kg bodyweight. n=5 mice/dose/time point. In situ hybridization. Section in situ hybridization analysis was performed following standard protocols as previously described . qRT-PCR. RNA extraction and quantitative Real-Time PCR were performed as described in the text. Primer sequences used were NFATc2 5-CATCCGCGTGCCCGTGAAAGTC-3 and 5-CTGCCCCTGCCCCTGAGAACCA-3; NFATc3 5- GAGCCCATTATGAAACTGAAGGTA-3 and AAGTTAGCTGGCTGGAGATAGAGG-3; NFATc4 5-CTTTGGCCCTGACGTGGACTTCTC-3 and 5- TAGCCTGGCCCCACCTCATTGTAG-3; and GAPDH 5-CACTGGCATGGCCTTCCGTG-3 and 5-AGGAAATG AGCTTGACAAAG-3. Supplemental Figure 1. In situ hybridization with NFATc1 on cortical kidney segments before (A.) and 3 days (B.) after treatment with HgCl2. 1 Supplemental Figure 2. Transcription of other NFATc family members was not altered following HgCl2 induced AKI. The relative expression of A. Nfatc2, B. Nfatc3, and C. Nfatc4 in mRNA samples isolated from whole kidney cortex of adult mice was measured using quantitative Real-Time PCR as described in the text. Supplemental Figure 3. Pharmacologic attenuation of NFATc1 with 10mgCsA/kg/day causes increased serum creatinine concentrations, increased apoptosis and decreased PTC proliferation following HgCl2 injury. A. Acute kidney injury scores are significantly increased in mice treated with 10mgCsA/kg following HgCl2 induced injury (p<0.0001 comparing WT vs. 10mgCsA/kg treated mice by two-way ANOVA). B. Survival curve demonstrating high mortality in mice treated with 10mgCsA/kg. C. Serum creatinine concentrations are significantly (p<0.0001 comparing WT vs. 10mgCsA/kg treated mice by two-way ANOVA). D. Apoptotic PTCs are significantly increased in mice treated with CsA (p<0.0001 comparing WT vs. 10mgCsA/kg treated mice by two-way ANOVA). E. Proliferation is significantly decreased in mice treated with 10mgCsA/kg (p=0.0013 comparing WT vs. 10mgCsA/kg treated mice by two-way ANOVA). (A-E, *p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA with Bonferroni posttest verses day 0). 2 Supplemental References 1. Miyaji, T., X. Hu, and R.A. Star, alpha-Melanocyte-simulating hormone and interleukin- 10 do not protect the kidney against mercuric chloride-induced injury. Am J Physiol Renal Physiol, 2002. 282(5): p. F795-801. 2. Jiao, K., et al., An essential role of Bmp4 in the atrioventricular septation of the mouse heart. Genes Dev, 2003. 17(19): p. 2362-7. 3
"Supplemental Methods - JASN"