Despite the development of dialysis and other novel therapies in the critically ill patient, the mortality associated with acute renal failure (ARF) remains unacceptably high, between 40% and 60%1. Leading causes of death in ARF in technologically developed countries include cardiorespiratory failure, sepsis, and withdrawal of dialysis2. ARF is an independent, additional risk factor for mortality in the multiorgan dysfunction syndrome3. A strong association has been repeatedly demonstrated with ARF and noncardiogenic acute respiratory distress syndrome (ARDS)4. However, the links between ARF and ARDS are poorly understood. Capillary leakage and accumulation of salt and water in areas of gas exchange are among the hallmarks of ARDS5. We have recently demonstrated that renal ischemia/reperfusion (I/R) injury in rats leads to increased pulmonary vascular permeability, which is partially macrophage mediated6. In addition, impaired clearance of salt and water from alveoli may further compromise gas exchange in ARDS5. The importance of the epithelial sodium channel (ENaC) and aquaporins in pulmonary salt and water clearance has recently been highlighted7,8. We hypothesized that ARF could lead to down-regulation of ENaC, Na,K-ATPase and aquaporins, potentially contributing to deleterious salt and water accumulation in the lungs. We addressed this question using an established model of acute renal failure in the rat6,9 and examined effects on expression of pulmonary ENaC, Na,K-ATPase, and aquaporin-1 and aquaporin-5.
METHODS
Overview
Four groups of animals (with four or more animals in each group were studied: (1) those with bilateral I/R injury, (2) those who underwent sham surgery, (3) those who underwent bilateral nephrectomy, and (4) those with unilateral I/R injury. Animals were sacrificed at 24 or 48 hours following surgery with collection of blood and lung samples.
Surgical procedures
The model of renal I/R injury employed has been described in detail6,9. Briefly, male 200 to 300 g Sprague-Dawley rats were anesthetized with pentobarbital sodium (40 mg/kg intraperitoneally) and placed on a heating pad to maintain constant temperature (monitored with a rectal thermometer). The kidneys were exposed via midline abdominal incisions, and the renal pedicles were dissected free. Renal ischemia was induced by nontraumatic vascular clamps (Roboz Surgical Instruments, Washington, DC, USA) over either one or both pedicles (as appropriate for group assignment) for 30 minutes. After the clamp(s) were released, the incision was closed in two layers with 2-0 sutures. Sham animals underwent anesthesia, laparotomy, and renal pedicle dissection only. A final group underwent bilateral nephrectomy using standard techniques. All animals received warm saline solution instilled in the peritoneal cavity during the surgical procedure and were then allowed to recover with ad libitum access to food and water. Animals were sacrificed by exsanguination under surgical anesthesia 24 or 48 hours after experimental intervention. Blood samples were collected for measurement of serum creatinine.
RNA isolation
Lung tissue was snap frozen in liquid nitrogen and then stored at –70°C until processed. Total cellular RNA was extracted from lung tissues by the method of Chomczynski and Sacchi10. In brief, 0.5 to 1 g tissue was homogenized at room temperature in 10 mL Tri reagent (Molecular Research Center, Cincinnati, OH, USA). RNA was quantitated by spectrophotometry and stored at -80°C.
Northern hybridization
Total RNA samples were fractionated on a 1.2% agarose-formaldeyde gel. The samples were transferred to a nylon membrane, cross linked by ultraviolet light, and baked for 1 hour. Hybridization was performed according to Church and Gilbert11. The cDNA probes were labeled with 32P-deoxynucleotide, using the Rad Prime DNA labeling kit (Invitrogen, Carlsbad, CA, USA). Following hybridization, the membranes were washed, blotted dry, and exposed to a Phosphorimager cassette at room temperature for 24 to 48 hours, and read by Phosphorimager (Amersham Bioscience Co., Piscataway, NJ, USA). The following rat polymerase chain reaction (PCR) product fragments were used as probes for Northern blot analyses: Na channel beta subunit, nucleotides 1012–1848; aquaporin-1, nucleotides 494–736; and aquaporin-5, nucleotides nucleotides 308–678.
Electrophoresis and Western blotting
Semiquantitative immunoblotting experiments were carried out according to standard protocols. Briefly, the solubilized membrane proteins from lungs of control or I/R injury rats were size fractionated on polyacrylamide minigels (Novex, San Diego, CA, USA) and were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% milk proteins, and then probed with either an aquaporin-5 or an
Na,K-ATPase-specific antibody. For aquaporin-5, an affinity-purified polyclonal antibody (raised against a 17 aa synthetic peptide from the C-terminal portion of mouse; aquaporin-5, Alpha Diagnostic, San Antonio, TX, USA) was used at an immunoglobulin (IgG) concentration of 0.8
g/mL. The secondary antibody was donkey antirabbit IgG conjugated to horseradish peroxidase (Pierce). For Na,K-ATPase, a monoclonal antibody against the
subunit of the pump (a generous gift from Dr. Jerry Lingrel, Department of Molecular Genetics, University of Cincinnati) was used at 1/1000 dilution. The secondary antibody was a goat antimouse IgG antibody. The sites of antigen-antibody complexation on the nitrocellulose membranes were visualized using chemiluminescence method (SuperSignal Substrate, Pierce Biotechnology Inc., Rockford, IL, USA) and captured on light-sensitive imaging film (Kodak, Rochester, NY, USA). Aquaporin-5 was identified as a 27 to 30 kD band. Na,K-ATPase was identified as a 95 to 98 kD band. The equity in protein loading in all blots was first verified by gel staining using the coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA, USA).
Statistical methods
Results are expressed as mean
SEM. Statistical analysis was performed using ANOVA or unpaired Student t test, with a P value less than 0.05 considered significant.
RESULTS
Survival and weight
All animals survived until sacrifice. Sham I/R injury animals, despite undergoing operation, had little change in body weight (mean 0.7% increase over 48 hours). Animals with bilateral renal I/R injury tended to lose weight (6.3% loss in 48 hours), which has been well established and thought to be due to both cachexia and polyuria12. The group with bilateral nephrectomy and associated anuria tended to gain weight (14.8% increase over 48 hours). Animals with unilateral nephrectomy plus I/R injury lost weight (3.0% loss over 48 hours).
Renal function
Renal I/R injury led to a significant rise in serum creatinine compared to sham animals and unilateral ischemia animals Figure 1. Bilateral nephrectomy led to much higher serum creatinine levels.
Lung epithelial sodium channel expression
Bilateral renal I/R injury significantly decreased lung sodium channel mRNA expression at 48 hours postischemia compared to sham-operated animals (51%
5% vs. 100% in sham, P < 0.01, N = 4; Figure 2). Lung sodium channel mRNA expression was mildly decreased, but not significantly, at 24 hours (data not shown).
Figure 2.
Lung sodium channel expression 48 hours after surgery. (A) Northern blots for lung sodium channel mRNA and 28S RNA 48 hours after surgery. (B) Densitometry data for sodium channel expression 48 hours after surgery. IRI, ischemia/reperfusion injury.
Full figure and legend (50K)Lung aquaporin-5 expression
Bilateral renal I/R injury markedly decreased lung aquaporin-5 mRNA expression at 48 hours postischemia (decreased by 74%
5% 48 hours compared to sham-operated animals, P < 0.01, N = 4; Figure 3).
Figure 3.
Lung aquaporin (AQP-5) expression. (A) Northern blots for lung aquaporin-5 mRNA and 28S RNA 48 hours after surgery. (B) Densitometry data for aquaporin-5 expression 48 hours after surgery. IRI, ischemia/reperfusion injury.
Full figure and legend (45K)Lung aquaporin-1 expression
Renal I/R injury did not alter aquaporin-1 expression at either 24 or 48 hours Figure 4.
Figure 4.
Lung aquaporin-1 (AQP-1) expression. Northern blots for lung aquaporin-1 mRNA and 28S RNA 48 hours after surgery.
Full figure and legend (134K)Effect of azotemia on aquaporin-5 expression
To determine whether the reduction in the expression of aquaporin-5 was due to I/R products or renal failure itself, the experiments were repeated in animals subjected to either unilateral ischemia (I/R injury but no renal failure) or bilateral nephrectomy (renal failure but no I/R injury) As indicated in Figure 5, the aquaporin-5 mRNA expression significantly decreased in bilateral nephrectomy or bilateral I/R injury, but not with unilateral ischemia. The results of studies on four separate animals indicated that, at 48 hours, the expression of aquaporin-5 decreased by 78%
5% in bilateral ischemia, and by 71%
6% in bilateral nephrectomy (P < 0.01 vs. sham in both groups). The expression of aquaporin-5 in unilateral ischemia did not change (P> 0.05 vs. sham, N = 4).
Figure 5.
Results of lung aquaporin-5 (AQP-5) and 28S RNA surgery in animals subjected to sham surgery, bilateral ischemia reperfusion injury, bilateral nephrectomy, and unilateral ischemia. (A) Northern blots for lung aquaporin 5 mRNA and 28S RNA 48 hours after surgery. (B) Corresponding densitometry data 48 hours after surgery.
Full figure and legend (158K)Western blotting
In order to evaluate if the change in lung aquaporin-5 mRNA level was consistent with changes at the protein level, we used an antibody to aquaporin-5. Immunoblot of aquaporin-5 protein demonstrated a significant decrease during bilateral I/R injury compared to sham-I/R injury animals, consistent with results at the mRNA level Figure 6. Given the potential importance of Na,K-ATPase in pulmonary fluid clearance, immunoblotting was performed to detect this protein. There was a marked decrease in lung Na,K-ATPase protein content during bilateral I/R injury compared to sham-I/R injury animals Figure 7.
Figure 6.
Western blot for lung aquaporin-5 (AQP-5) 48 hours after surgery. Animals were subjected to sham surgery and bilateral renal ischemia/reperfusion injury (IRI). There is a marked decrease in AQP-5 in both IRI lung samples compared to sham-operated rat lungs.
Full figure and legend (20K)Figure 7.
Western blot for lung Na,K-ATPase 48 hours after surgery. Animals were subjected to sham surgery and bilateral renal ischemia/reperfusion injury (IRI). There is a marked decrease in Na,K-ATPase in both renal IRI lung samples compared to sham-operated rat lungs.
Full figure and legend (19K)DISCUSSION
Our results demonstrate that ARF resulting from either bilateral I/R injury or bilateral nephrectomy can down-regulate pulmonary ENaC, Na,K-ATPase and aquaporin-5 expression. The failure of unilateral renal I/R injury to induce similar down-regulation indicates that these changes are likely mediated by systemic effects of ARF rather than reperfusion products alone. Furthermore, the observation that bilateral renal I/R injury with weight loss yielded the same observations as bilateral nephrectomy with weight gain makes it unlikely that changes in fluid volume played an important role in these changes. We further demonstrated that in the case of aquaporin-5, changes at the protein level were consistent with those at the mRNA level. Our findings suggest that ARF may exert deleterious effects on lung physiology, effects that may be of particular importance in the settings of antecedent or concurrent lung injury or mechanical ventilation.
The down-regulation of ENaC we observed is temporally consistent with the pulmonary inflammation, increased albumin permeability, and interstitial edema seen in the same rat model after ARF6. The amiloride-sensitive epithelial sodium channel, ENaC, is a heterodimeric protein composed of three homologous subunits7. ENaC promotes sodium absorption in epithelial cells of the distal renal tubule, distal colon, and lung. ENaC is critical in airway fluid clearance, as demonstrated by the rapid death of ENaC-deficient mice after birth due to pulmonary edema and respiratory failure7. Further, inhibition of the amiloride-sensitive epithelial sodium channel augments edema formation in lungs subjected to pulmonary I/R injury13. Accordingly, our findings of depressed ENaC expression in rat lungs during ARF suggest that ARF may contribute to impaired pulmonary fluid clearance. Decreased edema clearance may be of particular importance in the setting of increased pulmonary permeability, a hallmark of ARDS.
Aquaporins are water-transporting proteins that are expressed in various tissues, including lung, kidney, brain, and salivary tissue14. To date, ten mammalian members of the aquaporin family have been identified15. Aquaporin-1 is expressed in pulmonary microvascular endothelia and is important for osmotically driven water transport across alveolar capillaries16. Aquaporin-5 is expressed in type 1 alveolar epithelial cells and is important in airspace-capillary osmotic water permeability8. Aquaporin-1 and aquaporin-5 are down-regulated after murine adenovirus infection, and thus may be involved in disease-related pulmonary edema17. However, recent studies in the aquaporin-5–deficient mice did not reveal any defect in active fluid absorption under normal conditions8 or acute lung injury18. Nonetheless, it is plausible that aquaporin-5 may serve other functions such as generation and maintenance of an aqueous surface layer, mediating carbon dioxide exchange, or maintaining volume and structure of the type I pneumocyte19. Recent findings suggest that aquaporin inhibition may increase susceptibility to ventilator-induced lung injury, consistent with this hypothesis20.
Impaired pulmonary fluid clearance arising from depressed ENaC expression, and perhaps derangements arising from decreased aquaporin-5 expression, may have significant deleterious effects in the context of increased pulmonary permeability, such as that observed in ARDS. To the extent that augmented edema retention predisposes to gross alveolar flooding (particularly in gravitationally dependent regions of the lung), apparent pulmonary compliance will be decreased and shunt fraction will increase. Less overt increases in alveolar fluid may inactivate surfactant, thereby decreasing lung compliance, increasing alveolar wall tension, and promoting further fluid filtration into the alveolar spaces. Increased alveolar surface tension may enhance stress-induced fracturing of the alveolar blood-air barrier21. Surfactant inactivation will also promote focal microatelectasis, increasing shear stresses during lung inflation and augmenting transmission of alveolar tension to interstitial structures (such as fragile pre- and post-alveolar capillary vessels)22,23,24. Damage to interstitial vessels appears be of importance in several models of lung injury24,25. These effects would be expected to increase susceptibility to ventilator-induced lung injury, an entity which is of increasing importance in the intensive care setting26,27,28,29. In addition, to the extent that the injured lung, acting via cytokine release, can "drive" the systemic inflammatory response, a "vicious cycle" may be initiated or perpetuated30.
The role of the Na,K-ATPase in clearance of lung salt and water has been the subject of considerable recent investigation in both normal and injured lungs and is the topic of recent reviews31,32. In brief, sodium enters the alveolar type II cell primarily through the luminal sodium channel, with a smaller contribution from other transporters, and is subsequently extruded via the Na, K-ATPase33,34. Water is thought to follow passively (isomotically).
2-adrenergic stimulation of the type 2 cell promotes incorporation of the Na,K-ATPase in the basolateral membrane, up-regulating the extrusion of sodium and augmenting sodium clearance; an effect which can be blocked by disruption of cytoskeletal microtubular elements34. This active transport mechanism continues to be operative in several models of lung injury, augmenting clearance of edema fluid35,36. The down-regulation of apical sodium-positive channel and basolateral Na,K-ATPase that we observed suggests that both the sodium entry across the apical membrane and the sodium exit across the basolateral membrane are impaired in the lungs during renal I/R injury. Whether the down-regulation of Na,K-ATPase is secondary to the down-regulation of sodium channel or is independent of that remains speculative. In addition, specific dopaminergic stimulation (DA-1) also increases edema clearance, albeit through a mechanism that appears distinct from that induced by
-adrenergic stimulation37.
Our findings indicate that ARF down-regulates pulmonary expression of ENaC, Na,K-ATPase and aquaporin-5. As noted, these molecular derangements may have detrimental effects on lung fluid balance, especially in the settings of antecedent or concurrent structural injury of the lung. Impaired pulmonary fluid handling may impair lung function and increase the susceptibility of the lung to injury, particularly during mechanical ventilation. It is important to note that the functional importance of the changes in the lung channels was not addressed in our study. Furthermore, observations in rats have to be cautiously interpreted for relevance in humans. The potential for "communication" between the injured kidney and the lung suggests that further study of kidney-lung interaction may prove both fruitful in the effort to reduce the high mortality associated with ARF.
References
REFERENCES
- Star, RA: Treatment of acute renal failure. Kidney Int 1998 54: 1817–1831, | Article | PubMed | ISI | ChemPort |
- Thadhani, R, Pascual, M, Bonventre, JV: Acute renal failure. N Engl J Med 1996 334: 1448–1460, | Article | PubMed | ISI | ChemPort |
- Chertow, GM, Levy, EM, Hammermeister, KE, et al: Independent association between acute renal failure and mortality following cardiac surgery. Am J Med 1998 104: 343–348, | Article | PubMed | ISI | ChemPort |
- Rabb, H, Chamoun, F, Hotchkiss, J: Molecular mechanisms underlying combined kidney-lung dysfunction during acute renal failure. Contrib Nephrol 2001 132: 41–52, | PubMed | ISI | ChemPort |
- Luce, JM: Acute lung injury and the acute respiratory distress syndrome. Crit Care Med 1998 26: 369–376, | PubMed | ISI | ChemPort |
- Kramer, AA, Postler, G, Salhab, KF, et al: Renal ischemia/reperfusion leads to macrophage-mediated increase in pulmonary vascular permeability. Kidney Int 1999 55: 2362–2367, | Article | PubMed | ISI | ChemPort |
- Hummler, E, Barker, P, Gatzy, J, et al: Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet 1996 12: 325–328, | Article | PubMed | ISI | ChemPort |
- Ma, T, Fukuda, N, Song, Y, et al: Lung fluid transport in aquaporin-5 knockout mice. J Clin Invest 2000 105: 93–100, | PubMed | ISI | ChemPort |
- Rabb, H, Mendiola, CC, Dietz, J, et al: Role of CD11a and CD11b in ischemic acute renal failure in rats. Am J Physiol 1994 267: F1052–F1058, | PubMed | ISI | ChemPort |
- Chomczynski, P, Sacchi, N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162: 156–159, | Article | PubMed | ISI | ChemPort |
- Church, GM, Gilbert, W: Genomic Sequencing. Proc Natl Acad Sci USA 1984 81: 1991–1995, | Article | PubMed | ChemPort |
- Wang, Z, Rabb, H, Haq, M, et al: A possible molecular basis of natriuresis during ischemic-reperfusion injury in the kidney. J Am Soc Nephrol 1998 9: 605–613, | PubMed | ISI | ChemPort |
- Khimenko, PL, Barnard, JW, Moore, TM, et al: Vascular permeability and epithelial transport effects on lung edema formation in ischemia and reperfusion. J Appl Physiol 1994 77: 1116–1121, | PubMed | ISI | ChemPort |
- Borgnia, M, Nielson, S, Engel, A, Agre, P: Cellular and molecular biology of the aquaporin water channels. Annu Rev Biochem 1999 68: 425–458, | Article | PubMed | ISI | ChemPort |
- King, LS, Nielsen, S, Agre, P: Pespiratory aquaporins in lung inflammation: the night is young. Am J Respir Cell Mol Biol 2000 22: 8–10, | PubMed | ISI | ChemPort |
- Verkman, AS, Matthay, MA, Song, Y: Aquaporin water channels and lung physiology. Am J Physiol Lung Cell Mol Physiol 2000 278: L867–L879, | PubMed | ISI | ChemPort |
- Towne, JE, Harrod, KS, Krane, CM, Menon, AG: Decreased expression of aquaporin (AQP)1 and AQP5 in mouse lung after acute viral infection. Am J Respir Cell Mol Biol 2000 22: 34–44, | PubMed | ISI | ChemPort |
- Song, Y, Fukuda, N, Bai, C, et al: Role of aquaporins is alveolar fluid clearance in neonatal and adult lung, and in oedema formation following acute lung injury: Studies in transgenic aquaporin nullmice. J Physiol 2000 525 (Pt 3):771–779, | Article | PubMed | ISI | ChemPort |
- King, LS, Nielsen, S, Agre, P: Aquaporins and the respiratory system: Advice for a lung investigator. J Clin Invest 2000 105: 15–16, | PubMed | ISI | ChemPort |
- Hales, CA, Du, HK, Volokhov, A, et al: Aquaporin channels may modulate ventilator-induced lung injury. Respir Physiol 2001 124: 159–166, | Article | PubMed | ISI | ChemPort |
- Fu, Z, Costello, ML, Tsukimoto, K, et al: High lung volume increases stress failure in pulmonary capillaries. J Appl Physiol 1992 73: 123–133, | PubMed | ISI | ChemPort |
- Mead, J, Takishima, T, Leith, D: Stress distribution in lungs: A model of pulmonary elasticity. J Appl Physiol 1970 28: 596–608, | PubMed | ISI | ChemPort |
- Smith, JC, Stamenovic, D: Surface forces in lungs. I. Alveolar surface tension-lung volume relationships. J Appl Physiol 1986 60: 1341–1350, | PubMed | ISI | ChemPort |
- Hida, W, Hildebrandt, J: Alveolar surface tension, lung inflation, and hydration affect interstitial pressure. J Appl Physiol 1984 57: 262–270, | PubMed | ISI | ChemPort |
- Albert, RD: Prone ventilation. Clin Chest Med 2000 21: 511–517, | Article | PubMed | ISI | ChemPort |
- Hotchkiss, JR, Blanch, L, Naviera, A, et al: Relative roles of vascular and airspace pressures in ventilator induced lung injury. Crit Care Med 2001 29: 1593–1598, | PubMed | ISI |
- Dreyfuss, D, Saumon, G: Ventilator-induced lung injury: Lessons from experimental studies. Am J Respir Crit Care Med 1998 157: 294–323, | PubMed | ISI | ChemPort |
- Amato, MB, Barbas, CS, Medeiros, DM, et al: Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 1998 338: 347–354, | Article | PubMed | ISI | ChemPort |
- The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000 342: 1301–1308, | Article | PubMed | ISI
- Tremblay, L, Valenza, F, Ribeiro, SP, et al: Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997 99: 944–952, | PubMed | ISI | ChemPort |
- Dematte, JE, Sznajder, JJ: Mechanisms of pulmonary edema clearance: From basic research to clinical implication. Intensive Care Med 2000 26: 477–480, | Article | PubMed | ISI | ChemPort |
- Matthay, MA, Folkesson, HG, Verkman, AS: Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am J Physiol 1996 270: L487–L503, | PubMed | ISI | ChemPort |
- Icard, P, Saumon, G: Alveolar sodium and liquid transport in mice. Am J Physiol 1999 277: L1232–L1238, | PubMed | ISI | ChemPort |
- Saldias, F, Lecuona, E, Friedman, E, et al: Modulation of lung liquid clearance by isoproterenol in rat lungs. Am J Physiol 1998 274: L694–L701, | PubMed | ISI | ChemPort |
- Saldias, FJ, Comellas, A, Ridge, KM, et al: Isoproterenol improves ability of lung to clear edema in rats exposed to hyperoxia. J Appl Physiol 1999 86: 30–35,
- Saldias, FJ, Lecuona, E, Comellas, AP, et al: Beta adrenergic stimulation restores rat lung ability to clear edema in ventilator-associated lung injury. Am J Respir Crit Care Med 2000 162: 282–287, | PubMed | ISI | ChemPort |
- Barnard, ML, Ridge, KM, Saldias, F, et al: Stimulation of dopamine 1 receptor increases lung edema clearance. Am J Respir Crit Care Med. 1999 160: 982–986, | PubMed | ISI | ChemPort |
Acknowledgments
H.R. is supported by NIH DK54770 & NKF Clinical Scientist Award. J.R.H. is a Scientist Development Grantee of the American Heart Association. M.S. is supported by DK 52281 and DK 54430, a Merit Review Grant, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic Incorporated. The authors thank Gwen Treadwell for assistance with the manuscript preparation.


