Protection From Unilateral Renal Ischemia/Reperfusion Induced Injury In The Non-Ischemic Distant Kidney With Fenoldopam
N Aravindan, S Aravindan, A Shaw
angiogenesis, distant kidney, ischemiareperfusion, microarray, nfκb signaling pathway, remote organ injury
N Aravindan, S Aravindan, A Shaw. Protection From Unilateral Renal Ischemia/Reperfusion Induced Injury In The Non-Ischemic Distant Kidney With Fenoldopam. The Internet Journal of Emergency and Intensive Care Medicine. 2006 Volume 10 Number 1.
Ischemic acute renal failure (ARF) remains a formidable clinical problem in perioperative and critically ill patients, that is associated with high morbidity and mortality 1,2 . The pathophysiology of ARF is complex and multifactorial and includes persistent intra-renal vasoconstriction, hypoxic injury to microvascular endothelial cells and tubular epithelial cells and leukocyte-mediated cytotoxicity 1,2,3 . Pathophysiologic mechanisms of ARF include intracellular damage with ATP depletion 4 , and intracellular Ca 2+ accumulation 5 . Cellular activation leads to reactive oxygen species generation, cytokine generation, neutrophil sequestration/activation, phospholipase activation, and membrane lipid alterations 6 . Recently, we demonstrated that a period of unilateral ischemia followed by reperfusion induced the DNA binding activity of the transcription factor Nuclear factor kappa B (
Although several experimental ameliorative strategies have been tested in I/R induced ARF, there is still a remarkable lack of definitive evidence supporting specific prophylactic therapies in any setting 12 . Increasing evidence suggests that inhibition of inflammatory pathways, such as inhibition of redox-sensitive
Materials and Methods
After instrumentation, and once the animal had reached a stable baseline, the randomization envelope was opened and the animal was assigned to one of the following four groups. Study infusions were begun immediately thereafter.
At the end of each experiment, the animals were euthanized by inhalation of 5% isoflurane and rapid exsanguination via the arterial line. At that time right kidneys were removed from the animal and snap frozen.
Total cellular RNA from distant non-ischemic kidney tissues was isolated by phenol/chloroform extraction 17 . Gene array expression analysis was performed as described earlier 7 . In brief, single-stranded cDNA was synthesized from 2 µg of the group-wise pooled total RNA extract using MMLV reverse transcriptase and labeled with biotin-16-dUTP (Roche Applied Science, Branchburg, NJ, USA). Each membrane was prehybridized with hybridization solution supplemented with 100 µg ml -1 of heat-inactivated salmon sperm DNA (Invitrogen Corporation, Carlsbad, CA, USA). The cDNA probes were denatured and then hybridized with nylon-based arrays spotted with cDNA fragments from 96 genes. We used
When comparing each gene's signal intensity between arrays, we used a twofold or more increase in the experimental group compared with the control group to represent “stringent” criteria for upregulation and an increase of less than twofold to represent “less stringent” criteria. Classifying gene regulation criteria in this manner can provide an index of the reliability of the gene microarray data . Also, we recently validated 7,9 the quality of these microarray expression levels using Reverse transcriptase-polymerase chain reaction (RT-PCR).
All 24 rats survived for at least the 6 hours after the reperfusion period (i.e. there was no operative mortality, and all animals required euthanasia in order to obtain kidney samples). In six cases (25%) an additional dose of urethane was required
NFκB signaling pathway
Angiogenesis signaling pathway
This study constitutes the initial demonstration of that UR-I/R induces proinflammatory NFκB- and angiogenesis-signaling in the non-ischemic distant kidney. We have previously shown that increased NFκB-DNA binding activity in response to renal I/R injury induces multiple pro-inflammatory molecules and mediate local cellular injury 7 . Consistently,
A number of earlier studies demonstrated the protective effect of fenoldopam, a selective dopamine1-receptor (D1) agonist, in renal I/R injury, establishing the therapeutic potential of fenoldopam in this setting 26,27,28 . The role of fenoldopam in multiple aspects of renal protection suggests that a complex interplay of transcriptional events underpin the renoprotective effect of fenoldopam in experimental I/R injury. Recently we demonstrated that fenoldopam attenuate I/R injury modulated
In addition to the ischemic kidney induced cytokine mediated injury, UR-I/R will significantly increase the functional burden in the non-ischemic kidney. An important consequence of I/R is stimulation of angiogenesis, which in turn leads to vascular discontinuity, proliferation and migration of endothelial cells, and structural reorganization of the new vasculature 29 . A complex interaction of biochemical and physiological processes, including imbalances in levels of angiogenic regulators, occurs during I/R. In the present study, we found that UR-I/R significantly (≥2 fold) induced 68 angiogenesis related genes and downregulated another 18 in the distant kidney. Administration of fenoldopam attenuated 43 of UR-I/R injury-induced and 15 of UR-I/R-suppressed genes. Fenoldopam associated attenuation in the UR-I/R modulated angiogenesis related genes might be highly beneficial in preventing the UR-I/R induced injury in distant kidney. For example, the ephrins, play a vital role in angiogenesis including primary network remodeling and tissue vascularization by the generation of new capillaries from existing vessels 30 and, hence the enhanced expression of
While it is tempting to speculate that these fenoldopam-mediated alterations in UR-I/R-induced gene expression might be beneficial to patients (if indeed these findings could be reproduced in the clinical setting), association is by no means proof of causation and we do not claim any such extrapolation here. Nevertheless, our data do provide an interesting platform on which to form future hypotheses about the role of vasodilator drugs in renal I/R injury. In the clinical setting it is rarely possible to administer an agent before an ischemic insult occurs to the kidney. The situation of descending thoracic aortic aneurysm (TAA) repair is just such a scenario however, and the high incidence of renal failure after this procedure shows that this is a serious, and presently unmet, medical need. Although it can never completely replicate the overall procedural insult, we feel that our model at least reproduces the temporal aspects of the renal ischemia induced by this operation, and the reason we gave the drug before the insult was because this would certainly be possible in the actual setting of TAA repair.
In conclusion, in the present study using pathway focused microarrays, we demonstrate that (a) UR-I/R significantly modulated the expression of both proinflammatory NFκB signaling pathway genes and angiogenesis related genes in the non-ischemic distant kidney and, (b) administration of fenoldopam significantly attenuates UR-I/R-modulated genes. These data provide further evidence that UR-I/R causes inflammatory injury to the contralateral kidney and possibly to other remote organs. The degree of injury to the distant kidney may be insufficient to cause detectable functional impairment; nevertheless, it may make the distant kidney susceptible to further injury during states of additional physiological stress.
This work was supported by departmental funds and institutional research grant #1-8779701
Natarajan Aravindan OUPB 1430, Department of Radiation Oncology, The University of Oklahoma Health Science Center 825 N.E. 10 th , Oklahoma City, OK 73104, USA Phone: 405-271-3825 email: firstname.lastname@example.org Fax (405) 271-3820