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. Author manuscript; available in PMC: 2014 Jul 21.
Published in final edited form as: Kidney Int. 2011 Aug 3;80(8):822–831. doi: 10.1038/ki.2011.229

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 1,2,#, Ming-Chang Hu 1,2,#, Jianning Zhang 1, Victor Babich 1,2, I Alexandru Bobulescu 1,2, Mingjun Shi 2, Paul McLeroy 1, Thomas E Rogers 3, Orson W Moe 1,2,4
PMCID: PMC4104484  NIHMSID: NIHMS571283  PMID: 21814178

Abstract

Ischemic renal injury is a formidable clinical problem, the pathophysiology of which is incompletely understood. As the Na/H exchanger-3 (NHE3) mediates the bulk of apical sodium transport and a significant fraction of oxygen consumption in the proximal tubule, we examined mechanisms by which ischemia–reperfusion affects the expression of NHE3. Ischemia–reperfusion dramatically decreased NHE3 protein and mRNA (immunohistochemistry, immunoblot, and RNA blot) in rat kidney cortex and medulla. The decrease in NHE3 protein was uniform throughout all tubules, including those appearing morphologically intact. In the kidney cortex, a decrease in NHE3 surface protein preceded that of NHE3 total protein and mRNA. Kidney homogenates from rats exposed to mild renal ischemia-reduced cell surface NHE3 protein expression in opossum kidney cells in vitro, whereas homogenates from animals with moderate-to-severe ischemia reduced both total NHE3 protein and mRNA. The decrease in total NHE3 protein was dependent on the proteasomal degradation associated with NHE3 ubiquitylation measured by coimmunoprecipitation. The transferable factor(s) from the ischemic homogenate that reduce NHE3 expression were found to be heat sensitive and to be associated with a lipid-enriched fraction, and did not include regulatory RNAs. Thus, transferable factor(s) mediate the ischemia–reperfusion injury-induced decrease in NHE3 of the kidney.

Keywords: acute kidney injury, epithelial sodium transport, ischemia– reperfusion, Na transport, proximal tubule

Acute kidney injury (AKI) from ischemia is a common syndrome in clinical nephrology.1,2 Despite advances in multiple fronts in overall clinical care, the morbidity and mortality of this syndrome remain staggeringly high.1–4 Until recently, therapeutic options consisted of largely supportive measures,4,5 and numerous therapies designed to alter the course of the illness with proven efficacy in animal models have encountered rather disappointing results in subsequent human trials.6–8 On a positive note, considerable efforts are being devoted to understanding this syndrome and some recent interventional agents may appear more promising.8,9 A thorough understanding of the pathophysiology of acute ischemic renal injury is critical for further advances in the treatment of this disorder.

The effect of ischemia–reperfusion (IR) on Na/H exchanger-3 (NHE3), the principal renal apical membrane Na+ transporter, is the focus of the present study. NHE3 is a member of a mammalian gene family of transmembrane proteins that facilitate electroneutral exchange of Na+ for H+. NHE3 is expressed in the apical membrane of the mammalian proximal convoluted tubule and thick ascending limb.10 In the proximal tubule, NHE3 is responsible for 50% and 70% of volume and bicarbonate absorption, respectively.11,12 In the thick ascending limb, NHE3 mediates most of the bicarbonate absorption,13 and its role in NaCl absorption is more controversial. The functional role of NHE3 in maintenance of systemic volume and acid–base homeostasis is evident from the massively reduced proximal absorption, systemic hypotension, and metabolic acidosis seen in NHE3-null mice, despite distal compensation.14–16

Previous studies have described a reduction in renal NHE3 transcript and protein after IR injury.17–19 It was postulated that reduction of NHE3 expression can potentially explain the clinical findings of salt wasting and disordered acidification, which is occasionally encountered in AKI.20 The mechanisms by which NHE3 activity and expression are regulated are manifold, many of which are not well understood. In other physiological and pathophysiological models, NHE3 is regulated at the levels of protein phosphorylation, protein–protein interaction, trafficking, translation, and transcription.21 The current study shows that, after IR, NHE3 expression is reduced via changes in NHE3 surface, total protein, and transcript abundance because of transferable factor(s) present in ischemic renal tissue. These factor(s) are heat sensitive, may have a lipid component, or may associate with lipid fractions, but are not regulatory RNAs. These factor(s) induce destabilization of NHE3 protein by means of proteasomal degradation of ubiquitylated NHE3 and will reduce NHE3 transcript.

RESULTS

Time course of NHE3 protein and transcript

IR leads to severe reduction in NHE3 protein in both the cortex and medulla. In the renal cortex (Figure 1a and b), NHE3 reduction is evident by 16 h and is maximal at 1 day. Recovery begins at 7 days and is complete by 10–14 days (Figure 1b). The trend is similar for both apical and total membranes; however, changes in apical membrane NHE3 are more prominent at 16 h, compared with that of NHE3 total membrane. Figure 2a and b shows the corresponding results in total membranes from the medulla. The reduction is evident by 16 h, maximal by 3 days, and recovery is complete by 10–14 days. Decrease in NHE3 protein is associated with a decrease in NHE3 transcript in both the cortex and medulla (Figures 3 and 4). Reduction in NHE3 transcript in the cortex is evident by 16 h and reaches its maximum on day 1. Recovery begins at 3 days and is complete by 10–14 days. In the medulla, reduction in NHE3 transcript is significant by 16 h, maximal by 1 day, and recovery is complete by 10–14 days. Decreases in NHE3 protein (in particular, in the NHE3 apical membrane of the renal cortex) precede that of and are more severe than decreases in NHE3 transcript early post-ischemia. This suggests that there are translational and/or post-translational events involved in the reduction of NHE3 protein after injury from IR. Furthermore, reduction in NHE3 protein is dramatic with loss of up to 80% of total cellular NHE3 on day 1. Considering that this model usually leads to only patchy areas of tubular necrosis, loss of NHE3 protein must also occur in morphologically normal tubules.

Figure 1. Immunoblot of Na/H exchanger-3 (NHE3) antigen in apical and total membranes from the renal cortex.

Figure 1

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Rats were subjected to either 40 min 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 16 h 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): 16 h 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): 16 h 59%, Po0.05; 1 day 22%, P<0.01; 3 days 17%, P<0.01; 7 days 19%, P<0.01; 10 days 74%, Po0.05; 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).

Figure 2. Immunoblot of Na/H exchanger-3 (NHE3) antigen in total membranes from the renal medulla.

Figure 2

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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 16 h 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): 16 h 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 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).

Figure 3. RNA blot of Na/H exchanger-3 (NHE3) transcript in the renal cortex.

Figure 3

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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 16 h 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): 16 h 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).

Figure 4. RNA blot of Na/H exchanger-3 (NHE3) transcript in the renal medulla.

Figure 4

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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 16 h and 1, 3, 7, 10, and 14 days (d) after the experimental or sham manipulation. NHE3 transcript (ischemia as a percentage of sham, significance): 16 h 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).

Immunohistochemistry of NHE3 protein

As expected, IR resulted in patchy areas of tubular necrosis. In contrast, loss of NHE3 expression was quite global in distribution (Figures 5 and 6). We counterstained the sections with Evans blue to visualize the apical brush border. Figure 6a and b shows a representative tubule where the apical brush border is intact and yet is largely devoid of NHE3 protein. In sham-operated animals, NHE3 protein expression was robust at the brush border (Figure 6c), as expected.10 These findings indicate that decrease in NHE3 expression is not a result of destruction of the apical brush-border membrane.

Figure 5. Immunohistochemistry of Na/H exchanger-3 (NHE3) antigen in the renal cortex.

Figure 5

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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.

Figure 6. Immunohistochemistry of Na/H exchanger-3 (NHE3) antigen counterstained with Evans Blue in the renal cortex.

Figure 6

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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.

Effect of ischemic extract on NHE3 in opossum kidney cells

Considering that disappearance of NHE3 protein extends far beyond morphological tubular lesions, we postulated that diffusible factor(s) might be responsible for this effect.

We used a proximal tubule-like cell that expresses native NHE3, opossum kidney cells (OK cells), to test the hypothesis in vitro. Homogenates from ischemic kidneys rapidly induced loss of NHE3 total protein in OK cells in a dose- and time-dependent manner, whereas renal homogenates from sham-operated animals had no effect on NHE3 protein (Figure 7a and b). Interestingly, reduction of NHE3 protein is associated with a reduction in the NHE3 transcript and depends on the duration of ischemia and on the severity of renal dysfunction (Figure 7c). We measured NHE3 surface, total protein, and transcript expression in OK cells after addition of ischemic renal homogenates obtained after varying durations (30–60 min) of ischemia. Homogenates from mild ischemia (plasma creatinine (PCr) = 0.9–2.1 mg/ml 48 h post IR) induced a decrease of NHE3 surface protein but not of total protein and transcript expression (Figure 7c). Homogenates obtained from moderate (PCr = 2.2–3.0 mg/ml) and severe (PCr43.0 mg/ml) ischemia decreased NHE3 surface and total protein and transcript expression (Figure 7c). Generally, 30, 40, or 60 min of ischemia leads to mild, moderate, or severe ischemia, respectively, although this is not universally true because of some animal-to-animal variations. This is why we chose PCr values to distinguish between severities of ischemia.

Figure 7. Effect of kidney homogenates on Na/H exchanger-3 (NHE3) expression in opossum kidney (OK) cells: time-, dose-, and kidney damage-dependence.

Figure 7

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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 4 h. (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 1 mg/ml of kidney homogenates was applied to OK cells. Data were arbitrarily divided into mild (plasma creatinine (PCr) = 0.9– 2.1 mg/ml, 48 h post IR), moderate (PCr = 2.2– 3.0 mg/ml, 48 h post IR), and severe (PCr> 3.0 mg/ml, 48 h post IR) ischemia. PCr from sham animals was 0.4–0.5 mg/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.

Variation in degrees of renal damage affects NHE3 regulation differently in vitro. We retrospectively looked at whole-animal data and found that degrees of ischemia also affect NHE3 regulation differentially in vivo (Supplementary Table S1 online).

Next, we evaluated the mechanisms that decrease NHE3 total protein in vitro. The rapidity of the effect of the ischemic extract on NHE3 protein suggests that NHE3 is targeted for degradation. Inhibition of the lysosomal pathway had little effect on ischemic homogenate-induced decrease in NHE3 protein in OK cells (Figure 8a and b). In contrast, inhibition of the proteasomal pathway abrogated the decrease in NHE3 expression in response to ischemic homogenates over the 4 h period (Figure 8a and b). Ubiquitylation is a prerequisite for identifying proteins for proteasomal degradation. Figure 8c shows that NHE3 is acutely ubiquitinated upon exposure to ischemic renal homogenates. In concert, these results support the notion that factor(s) in the ischemic tissue target NHE3 for destruction through ligation with ubiquitin and degrada tion in the proteasome.

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.

Figure 8

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Two days after either ischemia or sham manipulation, animals were nephrectomized and 0.1 mg/ml of kidney homogenates was applied to confluent quiescent OK cells for 4 h, either alone or in combination with inhibitors of lysosomal (leupeptin 0.5 μg/ml) or proteasomal pathways (lactacystin 10 μmol/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 10 μmol/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).

Characterization of transferable factor(s) in the ischemic renal homogenate

We next initiated the characterization of transferable factor(s) in the ischemic homogenate, which is a formidable task because the effect on NHE3 could be, and likely is, mediated by a combination of factors of different natures. Heating the homogenates to 95 °C significantly reversed the reduction in NHE3 total protein induced by the ischemic renal homogenate (Figure 9a and b). This reversal could be because of heat denaturation of protein structure, suggesting that the transferable factor(s) may be proteins or protein-bound molecules that are destabilized by protein denaturation. We next evaluated whether the transferable factor(s) are lipids or lipid associated. Removal of the lipid fraction from the ischemic renal homogenate significantly blunted the reduction of NHE3 total protein by the ischemic renal extract. Lipid-enriched kidney homogenates from IR rats induced a significant decrease in NHE3 protein compared with lipid-enriched extract from sham-operated rats. Finally, we tested whether components of the transferable factor(s) may be small protein-bound RNA molecules. Large RNAs will not survive endogenous ribonuclease (RNAse) in our preparation, but small RNA molecules (for example, micro-RNAs) associated with binding proteins will be relatively resistant. Renal cortical homogenates were subjected to proteinase to remove RNA-binding proteins, followed by RNAse treatment. Pretreatment with proteinase K is an effective method of exposing small protein-bound RNA molecules to degradation.22 Heating the homogenates to 95 °C significantly blunted the inhibitory effect of ischemic homogenates on OK cell NHE3 abundance, and no further change was detectable after proteinase and RNAse treatment, which rules out regulatory RNAs as candidates (Figure 9c).

Figure 9. Characterization of extractable tissue factor(s).

Figure 9

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Opossum kidney (OK) cells were incubated with 1 mg/ml of kidney homogenate from ischemic or sham-operated rats (for 4 h) 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 10 min), 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.

DISCUSSION

Prominent among the functional effects induced by IR injury is reduced Na+ and water tubular reabsorption. The abundance of several major Na+ transporters, including NHE3, is drastically reduced after acute IR in rats.17–19 It was postulated that severe suppression of NHE3 represents, at least in part, the molecular basis for the natriuresis, acidosis, and volume depletion observed after IR injury.17,19 In this model, downregulation of NHE3 constituted a pathophysio-logical event that could potentially worsen the clinical syndrome. However, in most clinical settings, salt wasting is actually not a usual encounter in AKI. An alternative, but not necessarily mutually exclusive, view of downregulation of NHE3 in this scenario is that this is not a consequence of damage to the proximal tubule but is rather an adaptive defense mechanism. This notion frames the whole finding in a different light. First is conservation of energy in the proximal tubule as proposed by Blantz et al.23 As active Na+ transport constitutes the bulk of ATP consumption24 in this largely aerobic nephron segment,25,26 a rapid reduction in Na+ transport by downregulating the principal apical Na+ entry step achieves that purpose rapidly. Second is conservation of isotonic extracellular fluid as proposed by Thurau et al.27 If tubular transport is reduced, it is critical that glomerular filtration be curtailed to prevent massive volume loss. Increased distal solute delivery to the macula densa can be partially responsible for reduction of the glomerular filtration rate in acute tubular necrosis.28 Loss of NHE3 in NHE3 −/− animals results in severe reduction of glomerular filtration rate through activated tubulo-glomerular feedback, which is one of the mechanisms why these animals do not succumb to circulatory collapse.15 Third is the model that reduction in proximal tubule apical Na+ entry may in fact be cyto-protective to cells, as low cell Na+ accelerates Ca2+ efflux and ameliorates Ca2+-mediated cell injury.29 From these viewpoints, downregulation of NHE3 becomes a vital part, rather than an undesirable consequence, of the tubular response to IR injury. Understanding how NHE3 is down-regulated in this setting is critical to understanding the renal response to IR. The present study focused on studying some of the molecular mechanisms by which IR regulates NHE3 expression.

First, we found that IR induces a drastic reduction of NHE3 protein and transcript in the rat kidney. Our data are comparable to previously reported findings of drastic reduction of renal NHE3 in IR.17–19 However, urinary NHE3 protein abundance was found to be increased in patients with acute tubular necrosis compared with normal subjects.30 In the setting of tubular necrosis, it is unclear whether antigen in urinary exosomes actually mirrors renal levels on the basis of constitutive shedding or is more a reflection of discharge through necrosis. Both NHE3 protein and transcript in the kidney reverse back to control levels after about 7–10 days, which is congruent with the recovery of PCr in this rat model of acute IR.31 Furthermore, we found that decreased NHE3 expression starts in the apical membrane, followed by total protein and then transcript. This sequence suggests differential mechanisms that operate to downregulate NHE3-mediated transport.

Second, we determined that reduction in NHE3 expression is not necessarily associated with tubular necrosis or loss of apical membrane structure but can be globally distributed. These observations suggest that inhibition of NHE3 might not be a consequence of proximal tubule damage but is rather a generalized adaptive response mechanism to cope with low ATP supply. In support of this hypothesis, pretreatment with the NHE inhibitor EIPA (5-(N-ethyl-N-isopropyl) amiloride) attenuated Na+ fractional excretion and renal endothelin-1 released in response to IR injury.32 Pharmacological inhibition of NHE3 with S3226 decreased serum creatinine after IR and improved acute renal failure.33 Systemic administration of another specific inhibitor of NHE3 also improves tubular function in acute renal failure and provides protection from acute rejection in renal transplant in rats.34 The finding that changes in NHE3 function are not exclusively restricted to damaged tubules but are globally distributed may be key to understanding the pathophysiology of AKI, considering that tubular necrosis is extremely patchy in AKI in humans.35

Third, we found an abrupt decrease in NHE3 in OK cells never exposed to hypoxia but treated with homogenates from ischemic animals. Release of diffusible factor(s) in IR injury may have a significant role in the reduction of NHE3 expression. Indeed, growing evidence indicates that inflammatory response has a major role in IR injury.36 Inflammatory cascades initiated by endothelial dysfunction can be augmented by the ischemic proximal tubule. These include proinflammatory (for example, tumor necrosis factor-α, interleukin (IL)-6, IL-1β, and transforming growth factor-β) and chemotactic cytokines (for example, monocyte chemoattractant protein-1, IL-8, RANTES).37 IL-1b and tumor necrosis factor-a, especially in combination, extensively downregulate the expression of several Na+ transporters, including NHE3.38 In contrast, a-MSH, an anti-inflammatory agent that inhibits neutrophil migration and production of neutrophil chemokines,39 reversed the reduced abundance of Na+ transporters (for example, NHE3) and the marked increase in FENa induced by IR injury.40 These data favor the hypothesis that inhibition of NHE3 constitutes a pathophysiological event secondary to damage in IR injury. Alternatively, one can also view that amelioration of injury necessitates less protection by downregulation of NHE3.

Intracellular mediators also allow cells to respond and adapt to low oxygen tension. Hypoxia-inducible factors facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating several cellular mechanisms such as intracellular pH through induction of NHE1 expression.41,42 Our findings support the view that diffusible factor(s) released or activated in the tubular epithelium are heat-labile and comprised probably lipid and protein or protein in lipid-enriched domains, but not regulatory RNA components. Changes in lipid content and microRNA expression, which is an important regulator of gene expression,43 have also been associated with kidney dysfunction in AKI.44,45

Fourth, we proposed that these diffusible factor(s) released during IR injury affect NHE3 surface, total protein, and transcript expression likely through different mechanisms. Concordant changes in NHE3 protein and transcript have been previously described for other regulators of NHE3.21,46,47 Acid incubation, for example, increases NHE3 membrane insertion, total protein, and transcript with different kinetics.48–51 Mildly ischemic extracts added to OK cells clearly reduce only surface NHE3 protein. This is compatible with the early time point (16 h post IR), in which the magnitude of decrease in surface NHE3 exceeds the decrease in total NHE3, and with milder ischemia, in which the decrease in surface NHE3 is greater than total cellular NHE3 (Supplementary Table S1 online). In extracts from more severe renal damage, total cellular NHE3 protein and NHE3 transcript are both decreased, although the reduced transcript may or may not contribute to reduced protein. The proteasomal and lysosomal pathways are the two major proteolytic systems described in eukaryotic cells.52,53 The rapid reduction of NHE3 antigen is largely mediated by NHE3 degradation through the ubiquitin-proteasome proteolytic system. Ubiquitin has a major role in protein degradation as it serves as a tag for recognition of proteins by the proteolytic pathway.54–56 There are multiple examples of transport proteins being degraded by the ubiquitin–proteasome system,54,57–61 including NHE1.62 We did not determine whether a decrease in NHE3 transcript is mediated by the same factors(s) or mechanisms that induce NHE3 protein degradation.

In summary, we found that IR injury induces reduction in NHE3 surface and total protein and in NHE3 transcript. Recruitment of each of these mechanisms may occur through different pathways and may vary with the degree of kidney damage. We propose that downregulation of NHE3 at diverse levels may initiate a rapid way of conservation of energy and protect the cell; however, this study does not prove this conjecture. Reduction of NHE3 protein and transcript was induced by addition of ischemic homogenates to OK cells, suggesting that release of diffusible factor(s), which are likely proteins or proteolipid complexes, has a significant role in the inhibition of NHE3.

MATERIALS AND METHODS

Animal model

IR injury was induced in rats using protocols approved by the Institutional Animal Care and Use Committee. Sprague–Dawley rats (200–250 g, Harlan, Indianapolis, IN) were anesthetized (100 mg/kg ketamine, 10 mg/kg xylazine, and 1 mg/kg acepromazine; intramuscular) and placed on a heating table (39 °C) to maintain constant body temperature. Through a midline incision, bilateral cross-clamps were applied across the dissected renal artery for a designated period of time. Sham-operated animals were subjected to identical treatment, except that no cross-clamps were applied. After releasing the cross-clamps, kidneys were visually inspected to ensure restoration of blood flow, and 1 ml of prewarmed (37 °C) saline was instilled into the abdominal cavity and the abdomen was closed in two layers. The animals were placed in a 29 °C recovery and observation incubator overnight before being returned to their regular facilities.

Assays for NHE3 protein and transcript in rat kidney homogenates

NHE3 protein was measured in total membranes from the renal cortex and medulla and in apical membranes from the cortex. Kidneys were cooled and rinsed in ice-cold PBS. One kidney was harvested for protein, whereas the contralateral kidney was used for RNA preparation. The cortex and medulla were manually dissected from decapsulated kidneys. For protein preparation, tissue was homogenized (Brinkman Polytron, Beckman, Fullerton, CA) in membrane buffer (in mmol/l: 150 NaCl, 50 Tris-HCl pH 8.0, 5 EDTA; in μg/ml: 100 PMSF, 2 leupeptin, 2 aprotinin, 2 pepstatin A). Debris was pelleted by centrifugation (5000 g, 5 min, 4 °C) and total membranes were separated from cytosol (55,000 g, 40 min, 4 °C), resuspended in dye-free SDS loading buffer (5 mmol/l Tris-HCl, pH 6.8, 1% SDS, 10% glycol, 1% β-mercaptoethanol, and 0.004% bomophenolblue), and protein content was quantified by the colorimetric assay of Bradford (Bio-Rad, Hercules, CA). The remainder of the cortical homogenate was used to prepare renal cortical brush-border membrane vesicles by Mg2+ aggregation.49 Total and apical membranes (40 μg and 10 μg, respectively) were fractionated by SDS-PAGE. NHE3 protein was detected by primary antisera (1:1000 dilution) #1568,10 followed by horse-radish peroxidase-conjugated secondary antisera (Amersham Biosciences, Piscataway, NJ), and quantified by densitometry. For RNA studies, tissue samples were homogenized in guanidium thiocyanate solution (4 mol/l guanidium thiocyanate, 0.1 mol/l β-mercaptoethanol, 0.025 mol/l Na3Citrate pH 7.0, 0.5% (w:v) N-lauroylsarcosine). RNA was extracted using phenol/ chloroform, ethanol precipitated, size fractionated with formaldehyde gel electrophoresis, transferred to a nylon membrane, and hybridized with a 32P-labeled 1.2 kbp PstI fragment of the rat NHE3 cDNA. NHE3 RNA signal was detected by autoradiography and normalized to 18S rRNA as previously described.51

Immunohistochemistry

Immunohistochemistry was performed at 48 h after either cross-clamp or sham, as previously described.10 For NHE3 staining, sections were preincubated for 5 min at room temperature with 3% milk powder in PBS containing 0.05% Triton and overlaid overnight at 4 °C with an NHE3 antibody (#1568) diluted 1:500. Sections were rinsed three times with Tris-buffered saline before incubation (4 °C, 1 h) with secondary swine anti-rabbit Fluorescein isothiocyanateconjugated IgG (Dakopatts, Glostrup, Denmark). Slides were counterstained with 3% aqueous Evans Blue dye and treated with 2.5% 1,4-diazabicyclo-(2.2.2)octane (Sigma, St Louis, MO) as a fading retardant. Sections were imaged with a laser scanning confocal microscope (Zeiss LSM 410, Zeiss, Jena, Germany).

Cell culture studies

OK cells were maintained as previously described.63,64 Confluent monolayers were rendered quiescent by serum removal for 48 h before experimentation. Renal cortical homogenates were prepared from kidneys 48 h post either IR or sham operation. Varying amounts of the homogenate were added to postconfluent OK cells. At indicated times, cells were sonicated in membrane buffer and membrane fractions were pelleted (109,000 g, 25 min, 4 °C). A volume of 20 μg of total OK cell membrane protein was immuno-blotted with anti-OK NHE3 antiserum (1:1000 dilution) #5683.65 Parallel experiments were performed using inhibitors of lysosomal (leupeptin, 0.5 μg/ml) or proteasomal (lactacystin, 10 μmol/l) pathways applied simultaneously with the renal homogenates. To examine the effect of ischemic extract on NHE3 ubiquitylation, we measured ubiquitylation by epitope-tagged ubiquitin because different commercial anti-ubiquitin antibodies failed to generate reproducible and reliable data in OK cells. OK cells were transfected with hemagglutinin (HA)-tagged ubiquitin (HA-Ub), which was provided by Dr Bohmann (University of Rochester, Rochester, NY). Efficiency of transfection was about 70–80% by staining cells with anti-HA (not shown). Extracts from ischemic- or sham-operated animals were added for 4 h in the presence of 10 μmol/l lactacystin to arrest endogenous proteasomal degradation of ubiquitinated NHE3. Cells were then lysed in buffer (in mmol/l: 150 NaCl, 50 Tris-HCl, pH 8, 5 EDTA, 1 EGTA, 1% Triton X-100; in μg/ml: 100 PMSF, 2 leupeptin, 2 aprotinin, 2 pepstatin A)63 supplemented with 2 mmol/l ubiquitin-aldehyde to inhibit in vitro ubiquitin cleavage. Ubiquitin was immunoprecipitated with anti-HA and immunoblotted with anti-NHE3 antibody to detect NHE3 ubiquitination.

A volume of 1 μg/ml of renal cortical homogenates from post-IR or -sham operation was added to OK cells for 4 h for characterization of transferable factor(s). To examine the heat stability of the transferable factor(s), homogenates were heated for 10 min at 95 °C. Denatured proteins were removed by centrifugation (20,000 g, 10 min, 4 °C) and cleared supernatant was added to OK cells.66 For proteinase K and RNAse treatment, the homogenates were incubated with 100 μg/ml Proteinase K (Promega, Madison, WI) for 90 min at 37 °C, followed by Proteinase K inactivation at 95 °C for 10 min. Next, homogenates were incubated with 50 μg/ml of RNAse mix from bovine pancreas (Roche Applied Science, Indianapolis, IN) for 60 min at 37 °C, heated at 95 °C for 10 min, cooled to 37 °C, and added to OK cells. Control homogenates without proteinase or RNAse treatment were subjected to the same heating and cooling steps. To examine whether the transferable factor(s) are enriched in a lipid fraction, homogenates were centrifuged (40,000 g, 30 min, 4 °C), resulting in a sedimented membrane pellet, cytosolic fraction, and floating lipid fraction. Floating lipids were separated from the cytosolic fraction.67 The cytosolic fraction was filtered to remove residual lipids.68 Collected floating lipids and filtered cytosolic fractions were added separately to OK cells.

Quantification of surface NHE3 antigen by biotinylation and of NHE3 transcript by quantitative real-time PCR was performed for OK cells incubated with 1 μg/ml of homogenate for 4 h as previously described.63,64,69,70 RNA isolation was carried out using an RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse transcription was carried out using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). In the summary of data of total NHE3 antigen, the antigen signal was normalized to b-actin mouse monoclonal antibody (Sigma).

Statistical analyses

Results are presented as mean±s.e. Quantitative differences between control and test conditions were assessed statistically by Student’s t-test and analysis of variance. A probability (P) o0.05 was considered statistically significant.

Supplementary Material

Supplementary Table S1

ACKNOWLEDGMENTS

We thank Robert A Star (NIDDK, National Institutes of Health, Bethesda, MD) for helpful discussions and Dirk P Bohmann (University of Rochester, Rochester, NY) for kindly providing us with the HA-tagged ubiquitin. The authors acknowledge the technical expertise of Ladonna A Crowder, Komal Vadnagara, Tara Rosenthal, and Anthony Nguyen. This study was supported by the National Institutes of Health (DK-54396, DK-48482, AI-041612, and DK-078596), the Department of Veterans Affairs Research Service, the American Heart Association Western Affiliate (0325098Y, 98G-052, and 0865235F), the O’Brien Kidney Research Center (P30 DK-079328), and the Simmons Family Foundation. Di Sole and Bobulescu were each supported by a Carl W Gottschalk Research Scholar Award from the American Society of Nephrology.

Footnotes

DISCLOSURE All the authors declared no competing interests.

SUPPLEMENTARY MATERIAL Table S1. Patterns of changes in NHE3 in relation to changes in PCr (plasma creatinine) concentration.

Supplementary material is linked to the online version of the paper at http://www.nature.com/ki

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Supplementary Materials

Supplementary Table S1
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