Introduction

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 (NFκB) and activation of proinflammatory NFκB signaling pathway genes ₇ , induced apoptosis and activated apoptosis-related signal transduction genes ₈ and, modulated angiogenesis-related signal transduction ₉ . While these mediators are clearly involved in local tissue damage after ischemia/reperfusion (I/R), their role in remote organ injury has been less well defined. However, increasing evidence suggests that single organ ischemia can generate remote organ injury ₁₀ . Furthermore, Tnfα -dependent bilateral renal injury after unilateral renal ischemia/reperfusion (UR-I/R) has been demonstrated ₁₁ . Such signaling events may represent the earliest domino in the cascade of inflammatory events that culminate in contra lateral kidney after UR-I/R.

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 NF-κB signaling pathway is a beneficial strategy in I/R models ₁₃,₁₄ . Recently we demonstrated that fenoldopam a short-acting, parenteral vasodilator, attenuate a number of signaling pathways including NF-κB signaling pathway, apoptosis signaling pathway and, angiogenesis signaling pathway ₇,₈,₉ . It is thus appropriate to investigate the effect of fenoldopam on UR-I/R-modulated genetic alterations in the distant non-ischemic kidney. We utilized a rat model of surgical ischemic renal failure to test the hypothesis that (1) UR-I/R modulate the proinflammatory NFκB signaling and angiogenesis signaling in the non-ischemic distant kidney and, (2) fenoldopam attenuates UR-I/R-induced NFκB and angiogenic gene transcription in the distant kidney .

Materials and Methods

Animals and Instrumentation. All experiments were carried out in accordance with the guidelines laid down by the National Research Council ₁₅ and were approved by our institutional animal care and use committee. Twenty-four adult male Sprague-Dawley rats weighing 350-400 g each were acclimatized in standard animal quarters for at least 3 days prior to the study. Six rats were used for each group. In all animals, anesthesia was induced with an inhaled mixture of 3-5% isoflurane in oxygen. When the animal was sufficiently anesthetized (judged by loss of ear pinch reflex), a tracheotomy was performed, and the lungs were mechanically ventilated (Inspira ASV ventilator, Harvard Apparatus Inc., Holliston, MA, USA). Anesthesia was maintained using intraperitoneal urethane (50 mg. kg ^-1 ). The inspired oxygen tension (FiO2) was maintained at 35% throughout the experiment. The carotid artery and jugular vein were cannulated (PE-20, Harvard Apparatus) to facilitate monitoring of arterial blood pressure and infusion of fluids. Muscle relaxation to permit mechanical ventilation (volume controlled, time cycled with tidal volume of 2 ml/100 g body weight and respiratory rate of 30 breaths per minute and zero PEEP, Inspira, Harvard apparatus) was achieved with intravenous boluses of pancuronium bromide (20 µg 100g ^-1 ) as required. An intravenous infusion of 0.9% saline was administered to prevent dehydration during the experiment (1 ml 100g ^-1 hr ^-1 ). Core body temperature was maintained at 37°C by an automated rodent homeothermic blanket warming system (Harvard Apparatus).

Experimental protocol.

Microarray analysis

Results

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

Gene array analysis: Oligonucleotide microarray analysis was performed using group wise pooled RNA. With both NFκB signaling pathway and angiogenesis signaling pathway gene chips, we observed intervention specific gene transcriptional response (Fig. 1A and 1B).

Figure 1

Figure 1: heat map: Color representation of relative expression levels (percent GAPDH, a positive control) of genes encoding (A) proinflammatory NF?B signal transduction pathway genes and (B) angiogenesis signaling pathway genes in the distant kidney of rats that underwent sham operation or unilateral renal ischemia/reperfusion in the presence or absence of fenoldopam

NFκB signaling pathway

Expression of inflammatory molecules in distant kidney after UR-I/R: Of the 96 NFκB signaling pathway genes analyzed, UR-I/R induced 92 genes and suppressed 3 genes in the distant kidney. Expression of one gene, NFκB inhibitor-like 1 (NFκBIL-1) is not altered after UR-I/R. Eighty of the 92 upregulated genes including Rel/NF-κB/IκB family NF-κB1, NF-κB2, Nfκbib, Nfκbib, Nfκbie, nfκbil2, Rel, Rela, Relb; NF-κB responsive adhesion molecules, Icam1, Icam2, Icam5, Sele, Sell, Selpl, Ncam1,Vcam1; NFκB responsive cytokines, Csf1, Csf2 Csf3, Ifnβ, Ifnγ, Il2, Il6, Il12α, Il12β, Irf1, Scya2, Tnfα, Lta; NFκB responsive acute phase response proteins C3, Orm1, Orm2, Saa1, Srf; extracellular ligands, Il1α, Il1β, Tnfsf11; transmembrane receptors, Il1r1, Il2rα, Il2rγ, Ripk1, Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, Tlr9; adaptor proteins, Fadd, Myd88, Tnfaip3, Traf1, Traf2, Traf3, Traf4, Traf5, Traf6; signal transduction kinases, Chuk, Iκbkβ, Map3k1, Map3k14, Map3k2, Map3k5, Map3k7, Mapk14, Mapk9, Tab1, TBK1 and, transcription factors, Creb1,Elk1,Elk3, Max, Myc, Raf1 met stringent criteria for upregulation (at least a two-fold increase over control).

Attenuating effect of fenoldopam on UR-I/R modulated NFκB signaling pathway genes in distant kidney: Microarray analysis of the fenoldopam UR-I/R group compared that of saline UR-I/R group showed that, using less stringent criteria, of the 92 genes upregulated by UR-I/R injury, 88 genes were returned to the baseline level or less with fenoldopam. Using stringent criteria (≥ twofold) we observed that 76 of 80 upregulated genes were attenuated by fenoldopam. These include Rel/NF-κB/IκB family, NF-κB1, NF-κB2, Nfκbib, Nfκbib, Nfκbie, nfκbil2, Relb (Fig 2A); NF-κB responsive adhesion molecules, Icam1, Icam2, Icam5, Sele, Sell, Selpl, Ncam1,Vcam1 (Fig 2B); NFκB responsive cytokines, Csf1, Csf2 Csf3, Ifnβ, Ifnγ, Il2, Il6, Il12α, Il12β, Irf1, Scya2, Tnfα, Lta (Fig 2C); NFκB responsive acute phase response proteins C3, Orm1, Orm2, Saa1 (Fig 2D); extracellular ligands, Il1α, Il1β, Tnfsf11 (Fig 2E); transmembrane receptors, Il1r1, Il2rα, Il2rγ, Ripk1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, Tlr9 (Fig. 2F); adaptor proteins, Fadd, Myd88, Tnfaip3, Traf1, Traf2, Traf3, Traf4, Traf5, Traf6 (Fig 2G); signal transduction kinases, Chuk, Iκbkβ, Map3k1, Map3k14, Map3k2, Map3k5, Map3k7, Mapk14, Mapk9, Tab1, TBK1 (Fig. 2H) and, transcription factors, Creb1,Elk1,Elk3, Max, Myc, Raf1 (Fig. 2I).

Figure 2

Figure 2: Effect of fenoldopam on unilateral renal ischemia/reperfusion induced (>= 2 fold proinflammatory NK-KB signaling pathway molecules in distant kidney

Angiogenesis signaling pathway

Effect of UR-I/R on angiogenesis signaling genes in distant kidney: Of the 96 angiogenesis-related genes analyzed, we observed increased expression of 77 genes and suppression of 19 genes after UR-I/R injury in distant kidney. Sixty eight of 77 UR-I/R-induced genes including specific promoters and inhibitors, Agpt, Agpt2, Ang; growth factors and receptors, Efna2 Ephb4, Fgf1, Fgf16, Fgf2, Fgf6, Fgf7, Fgfr3, Fgfr4, Pdgfa, Pdgfb, Pdgfra, Pdgfrb, Pf4, Tgfa, Tgfb1, Tgfb2, Tgfb3, Tgfbr1, Tgfbr2, Figf, Pgf, Vegf, Vegfb, Cd36, Ctgf, Edg1, Egf, Egfr, Igf1, Gro1, Tie1; cytokines and chemokines, Csf3, Ifnb, Ifng, Il10, Il12a, MDK, Nrp, Ptn, Rsn, Scya2, Sparc, Tnf;, adhesion molecules, Cdh5, Itga5, Itgav, Itgb3, Pecam, Tnc, Vcam1; matrix proteins, proteases and inhibitors, Adamts8, Col18a1, Fn1, Mmp2, Mmp9, Plau, Serpinb5, Serpine2, Thbs2, Thbs3, Thbs4; transcription factors, Erb2, Ets1, Idb1, Idb3, Madh1 and, other related genes Eng, F2, Msr1, Nos3, Ptgs1, Ptgs2, Spp1 met stringent criteria for upregulation. Similarly, 18 of the 19 downregulated genes including Adamts1, Chga, Efna5, Efna2, Fgf4, Fgfr1, Flk1, Flt1, Hgf, Hif1α, Ifna1, Serpine1, Tek, Tgfbr3, Thbs1, Timp, Timp2 and Vegfc were completely suppressed (≥ 2 fold) after UR-I/R in the distant kidney.

Effect of fenoldopam on UR-I/R modulated angiogenesis related genes in distant kidney: In general, administration of low dose fenoldopam (0.1µg. kg ^-1 . min ^-1 ) produced a blunting effect on the UR-I/R injury-modulated angiogenesis related gene expression. Forty eight of the 77 UR-I/R injury-induced genes including specific promoters and inhibitors, Agpt (Fig. 3A); growth factors and receptors, Cd36, Ctgf, Edg1, Egfr, Fgf16, Fgf2, Fgfr3, Fgfr4, Figf, Igf1, Pdgfa, Pdgfb, Pdgfra, Pdgfrb, Pf4, Pgf, Tgfb1, Tgfbr2, Tie1 (Fig. 3B); cytokines and chemokines, Csf3, Ifng, Il12a, MDK, Nrp, Tnf (Fig. 3C); adhesion molecules, Cdh5, Itgav, Pecam, Tnc, Vcam1 (Fig. 3A); matrix proteins, proteases and inhibitors, Adamts8, Col18a1, Fn1, Mmp2, Mmp9, Plau, Serpinb5, Thbs2 (Fig. 3D); transcription factors, Idb3, Madh1 (Fig. 3C) and, other related genes Eng, Msr1 (Fig. 3A) met stringent criteria for upregulation. Similarly, 15 of 19 UR-I/R-suppressed genes showed enhanced expression with fenoldopam. This blunting effect was significant (brought to baseline level) for 4 genes, Efnb2, Flk1, Timp and Vegfc (Fig. 4).

Figure 3

Figure 3: Effect of fenoldopam on unilateral renal ischemia/reperfusion induced (>= 2 fold angiogenesis-related genes in distant kidney)

Figure 4

Figure 4: Effect of fenoldopam on unilateral renal ischemia/reperfusion supressed (>=2 fold) angionesis-related genes in distant kidney

Discussion

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, NF-κB a redox-sensitive transcription factor has been implicated in the pathophysiology of a number of inflammatory disorders including renal I/R injury ₁₈,₁₉,₂₀ . While NFκB signaling is clearly a important mediator of local I/R injury, its role in remote organ damage after UR-I/R, is just beginning to be elucidated. In this study, we demonstrated that UR-I/R activated the expression of 80 proinflammatory NFκB signaling molecules in the non-ischemic distant kidney. The induction of Rel/NF-κB/IκB family genes ₁₈,₁₉,₂₁,₂₂,₂₃ after UR-I/R injury points to the general role of these genes in regulating the expression of cytokines, chemoattractant proteins, cell adhesion molecules, and other pro-inflammatory genes in the remote organ that subsequently lead to the cellular damage. Cytokines produced by renal tubular epithelial cells and infiltrated cells are critical factors in inflammatory processes of renal I/R injury. Induced expression of cytokines in remote organs ₁₀ including non-ischemic kidney ₁₁ after renal I/R injury has been well documented. These served as positive controls for the system. The induction of adhesion molecules after UR-I/R is in response to cytokines after I/R injury. Recently, Kelly demonstrated induced expression of adhesion molecule in remote organ after I/R ₁₀ . We also observed numerous UR-I/R injury-induced transmembrane receptors and adaptor proteins in the distant kidney. The induced Tlrs expression during renal inflammation was found to be mediated by both Ifn and TNF-α and is associated with a major increase of renal Tlr protein expression ₂₄ . Tlrs recruit an adapter protein, Myd88, to initiate a signaling pathway that is involved in the sequential activation of Iraks, Trafs, Map3k14, and the IκB kinase complex. Activated IKKs lead to NFκBiα phosphorylation and degradation, resulting in NF-κB activation ₂₅ . Li and his colleagues have previously suggested that the Tlr- mediated Myd88-dependent NF-κB signaling pathway may play a role in the inflammatory response to I/R injury ₂₅ . Consistently, in the present study, we observed the induction of all these molecules in the distant kidney which confirmed this concept of UR-I/R-injury induced cytokine mediated activation of NFκB signaling pathway.

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 NF-κB, apoptosis and, angiogenesis signaling pathway molecules ₇,₈,₉ . In the present study, fenoldopam significantly (≥2 fold) suppressed 76 of 80 UR-I/R induced NFκB signaling molecules in the distant kidney. The reduced expression of these UR-I/R injury-induced proinflammatory NF-κB family genes, cytokines, adaptor proteins and MAP kinases in distant kidney after fenoldopam demonstrate the inhibitory effect of fenoldopam on UR-I/R induced NF-κB signaling molecules and subsequent mediated inflammation. Several studies have demonstrated that direct inhibition NFκB prevents I/R injury-induced tissue damage ₂₀,₂₃ and other renal diseases ₁₃ . Cao and colleagues (2004) demonstrated that inhibition of I/R injury-induced NF-κB attenuated renal dysfunction, macrophage/monocyte infiltration and tubular damage in the cortex and outer medullary stripe ₂₀ . Consistently, we found that the administration of low-dose fenoldopam, an established renoprotectant vasodilator ₂₆,₂₇,₂₈ , blocked UR-I/R injury-induced inflammatory genes.

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 Efnb2 with fenoldopam administration observed in the present study suggests that it may be able to prevent UR-I/R-induced renal vasculature disruption in the distant kidney.

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.

Financial support

Correspondence to

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: naravind@ouhsc.edu Fax (405) 271-3820