1. Volume 80, Issue 8, Pages 822-831 (October 2011)
The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia– reperfusion injury is mediated by extractable tissue factor(s) 
Francesca Di Sole, Ming-Chang Hu, Jianning Zhang, Victor Babich, I. Alexandru Bobulescu, Mingjun Shi, Paul McLeroy, Thomas E. Rogers, Orson W. Moe 
Kidney International 
Volume 80, Issue 8, Pages (October 2011)
DOI: /ki
Copyright © 2011 International Society of Nephrology Terms and Conditions

2. Figure 1
Immunoblot of Na/H exchanger-3 (NHE3) antigen in apical and total membranes from the renal cortex. Rats were subjected to either 40min of bilateral renal artery cross-clamp (ischemia) or sham manipulation (sham). At indicated times, the animals were killed and total and apical membrane vesicles were prepared from the renal cortex and probed for NHE3 antigen by immunoblot. (a) Representative immunoblot. (b) Summary of all data collected at 16h and 1, 3, 7, 10, and 14 days (d) after the ischemia or sham manipulation. NHE3 antigen in apical membrane (ischemia as a percentage of sham, and relative statistical determination of significance): 16h 41%, P<0.01; 1 day 9%, P<0.01; 3 days 20%, P<0.01; 7 days 26%, P<0.01; 10 days 81% P=0.07; 14 days 94%, P=0.5. NHE3 antigen in the total cortex (ischemia as a percentage of sham, and relative statistical determination of significance): 16h 59%, P<0.05; 1 day 22%, P<0.01; 3 days 17%, P<0.01; 7 days 19%, P<0.01; 10 days 74%, P<0.05; 14 days 91%, P=0.7. Each data point represents the mean and s.e. from 3–5 independent animals. Asterisks indicate statistically significant difference between the sham and ischemia groups (P<0.05, unpaired t-test for each time point).
Kidney International  , DOI: ( /ki )
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3. Figure 2
Immunoblot of Na/H exchanger-3 (NHE3) antigen in total membranes from the renal medulla. At the indicated times post ischemia or sham manipulation, the animals were killed and membranes were prepared from the renal medulla and probed for NHE3 antigen by immunoblot. (a) Representative immunoblot. (b) Summary of all data collected at 16h and 1, 3, 7, 10, and 14 days (d) after the ischemia or sham manipulation. NHE3 antigen (ischemia as a percentage of sham, and significance): 16h 26%, P<0.01; 1 day 13%, P<0.01; 3 days 8%, P<0.01; 7 days 9%, P<0.01; 10 days 85% P=0.05; 14 days 88%, P=0.7. Each data point represents the mean and s.e. from 3–5 independent animals. Asterisks indicate statistically significant difference between the sham and ischemia groups (P<0.05, unpaired t-test for each time point).
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

4. Figure 3
RNA blot of Na/H exchanger-3 (NHE3) transcript in the renal cortex. At indicated times post ischemia or sham manipulation, the animals were killed and total RNA was prepared from the renal cortex and probed for NHE3 transcript by RNA blot. (a) Representative RNA blot. (b) Summary of all data collected at 16h and 1, 3, 7, 10, and 14 days (d) after the ischemia or sham manipulation. NHE3 transcript (ischemia as a percentage of sham, and significance): 16h 87.5%, P=0.10; 1 day 25%, P<0.001; 10 days 75%, P<0.01; 14 days 87%, P=0.16. Each data point represents the mean and s.e. from 3–5 independent animals. Asterisks indicate statistically significant difference between the sham and ischemia groups (P<0.05, unpaired t-test).
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

5. Figure 4
RNA blot of Na/H exchanger-3 (NHE3) transcript in the renal medulla. At indicated times post ischemia or sham manipulation, the animals were killed and total RNA was prepared from renal medulla and probed for NHE3 transcript by RNA blot. (a) Representative RNA blot. (b) Summary of all data collected at 16h and 1, 3, 7, 10, and 14 days (d) after the experimental or sham manipulation. NHE3 transcript (ischemia as a percentage of sham, significance): 16h 70%, P<0.001; 1 day 5%, P<0.001; 10 days 90%, P=0.1; 14 days 97%, P=0.8. Each data point represents the mean and s.e. from 3–5 independent animals. Asterisks indicate statistically significant difference between the sham and ischemia groups (P<0.05, unpaired t-test).
Kidney International  , DOI: ( /ki )
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6. Figure 5
Immunohistochemistry of Na/H exchanger-3 (NHE3) antigen in the renal cortex. Two days after either ischemia or sham manipulation, kidneys were perfusion-fixed and stained for NHE3 expression (NHE3 antibody #1568). Four independent animals from each group showed similar findings.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Immunohistochemistry of Na/H exchanger-3 (NHE3) antigen counterstained with Evans Blue in the renal cortex. Two days after either ischemia or sham manipulation, kidneys were perfusion-fixed and stained for NHE3 expression. NHE3 expression after ischemia manipulation visualized by Fluorescein isothiocyanate-conjugated secondary antiserum (green) can be seen against the background of brush-border staining by Evans Blue (blue). (a) Low power showing three tubules. (b) High power of one tubule. Arrowheads indicate the brush border. (c) A tubule from a sham-operated animal for comparison. Four independent animals from each group showed similar findings.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Effect of kidney homogenates on Na/H exchanger-3 (NHE3) expression in opossum kidney (OK) cells: time-, dose-, and kidney damage-dependence. Two days after either ischemia or sham manipulation, the animals were nephrectomized and kidney homogenates were applied to confluent quiescent OK cells in culture for 4h. (a) A representative blot showing a dose response. (b) Summary of all data for dose- and time-dependence. (c) Surface and total NHE3 protein and NHE3 transcript were measured after 1mg/ml of kidney homogenates was applied to OK cells. Data were arbitrarily divided into mild (plasma creatinine (PCr)=0.9–2.1mg/ml, 48h post IR), moderate (PCr=2.2–3.0mg/ml, 48h post IR), and severe (PCr>3.0mg/ml, 48h post IR) ischemia. PCr from sham animals was 0.4–0.5mg/ml. Typical blots and the summarized data are shown. Data points are expressed as mean and s.e. from 3–4 independent experiments. Asterisks denote statistically significant difference between cells treated with ischemic and those treated with sham homogenate (P<0.05, unpaired t-test). IR, ischemia– reperfusion.
Kidney International  , DOI: ( /ki )
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9. Figure 8
Effect of kidney homogenate on Na/H exchanger-3 (NHE3) expression in opossum kidney (OK) cells: inhibition of lysosomal versus proteasomal pathways, and NHE3 ubiquitylation. Two days after either ischemia or sham manipulation, animals were nephrectomized and 0.1mg/ml of kidney homogenates was applied to confluent quiescent OK cells for 4h, either alone or in combination with inhibitors of lysosomal (leupeptin 0.5μg/ml) or proteasomal pathways (lactacystin 10μmol/l). (a) Representative experiment. (b) Summary of all data. Data points are expressed as mean and s.e. from 3–4 independent experiments. Asterisks denote statistically significant difference between cells treated with ischemic homogenate and those treated with sham homogenate (P<0.05, unpaired t-test). (c) OK cells were transiently transfected with HA-ubiquitin (HA-Ub). Extracts from ischemic or sham-treated kidneys were added to OK cells, as described above, in the presence of 10μmol/l lactacystin to arrest endogenous proteasomal degradation of ubiquitinated NHE3. HA-ubiquitin was then immunoprecipitated with anti-HA antibody and immunoblotted with polyclonal anti-NHE3 (#5683) antibody to detect NHE3 ubiquitylation. One representative experiment is shown on the left and summary of all data is shown on the right. Data points are expressed as mean and s.e. from 3–4 independent experiments. Asterisks denote statistically significant difference between cells treated with ischemic homogenate and those treated with sham homogenate (P<0.05, unpaired t-test).
Kidney International  , DOI: ( /ki )
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10. Figure 9
Characterization of extractable tissue factor(s). Opossum kidney (OK) cells were incubated with 1mg/ml of kidney homogenate from ischemic or sham-operated rats (for 4h) and total Na/H exchanger-3 (NHE3) protein abundance was quantified by immunoblot. Data points are expressed as mean±s.e. from 3–4 independent experiments. Asterisks denote statistically significant difference between cells treated with ischemic and those treated with sham homogenate (P<0.05, unpaired t-test). #P<0.05 analysis of variance (ANOVA), OK cells treated with heated (or lipid deprived) kidney homogenates compared with OK cells treated with control kidney homogenates. $P<0.05 ANOVA, OK cells treated with lipid-enriched renal cortical homogenates compared with OK cells treated with lipid-deprived (post-lipid) kidney homogenates. (a) Representative experiment and (b) summary of all data of renal extract heated (95°C for 10min), lipid enriched (lipid enriched), or lipid deprived (post-lipid). (c) Representative experiment and summary of all data of heated, proteinase K and ribonuclease (RNAse) treatment of kidney homogenates. +: addition of indicated treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions