Combined iron sucrose and protoporphyrin treatment protects against ischemic and toxin-mediated acute renal failure
2016; Elsevier BV; Volume: 90; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2016.01.022
ISSN1523-1755
AutoresRichard A. Zager, Ali C.M. Johnson, Kirsten B. Frostad,
Tópico(s)Neonatal Health and Biochemistry
ResumoTissue preconditioning, whereby various short-term stressors initiate organ resistance to subsequent injury, is well recognized. However, clinical preconditioning of the kidney for protection against acute kidney injury (AKI) has not been established. Here we tested whether a pro-oxidant agent, iron sucrose, combined with a protoporphyrin (Sn protoporphyrin), can induce preconditioning and protect against acute renal failure. Mice were pretreated with iron sucrose, protoporphyrin, cyanocobalamin, iron sucrose and protoporphyrin, or iron sucrose and cyanocobalamin. Eighteen hours later, ischemic, maleate, or glycerol models of AKI were induced, and its severity was assessed the following day (blood urea nitrogen, plasma creatinine concentrations; post-ischemic histology). Agent impact on cytoprotective gene expression (heme oxygenase 1, hepcidin, haptoglobin, hemopexin, α1-antitrypsin, α1-microglobulin, IL-10) was assessed as renal mRNA and protein levels. AKI-associated myocardial injury was gauged by plasma troponin I levels. Combination agent administration upregulated multiple cytoprotective genes and, unlike single agent administration, conferred marked protection against each tested model of acute renal failure. Heme oxygenase was shown to be a marked contributor to this cytoprotective effect. Preconditioning also blunted AKI-induced cardiac troponin release. Thus, iron sucrose and protoporphyrin administration can upregulate diverse cytoprotective genes and protect against acute renal failure. Associated cardiac protection implies potential relevance to both AKI and its associated adverse downstream effects. Tissue preconditioning, whereby various short-term stressors initiate organ resistance to subsequent injury, is well recognized. However, clinical preconditioning of the kidney for protection against acute kidney injury (AKI) has not been established. Here we tested whether a pro-oxidant agent, iron sucrose, combined with a protoporphyrin (Sn protoporphyrin), can induce preconditioning and protect against acute renal failure. Mice were pretreated with iron sucrose, protoporphyrin, cyanocobalamin, iron sucrose and protoporphyrin, or iron sucrose and cyanocobalamin. Eighteen hours later, ischemic, maleate, or glycerol models of AKI were induced, and its severity was assessed the following day (blood urea nitrogen, plasma creatinine concentrations; post-ischemic histology). Agent impact on cytoprotective gene expression (heme oxygenase 1, hepcidin, haptoglobin, hemopexin, α1-antitrypsin, α1-microglobulin, IL-10) was assessed as renal mRNA and protein levels. AKI-associated myocardial injury was gauged by plasma troponin I levels. Combination agent administration upregulated multiple cytoprotective genes and, unlike single agent administration, conferred marked protection against each tested model of acute renal failure. Heme oxygenase was shown to be a marked contributor to this cytoprotective effect. Preconditioning also blunted AKI-induced cardiac troponin release. Thus, iron sucrose and protoporphyrin administration can upregulate diverse cytoprotective genes and protect against acute renal failure. Associated cardiac protection implies potential relevance to both AKI and its associated adverse downstream effects. Despite great advancements in our understanding of acute kidney injury (AKI) pathogenesis, no effective prophylaxis for it currently exists. The need for such a therapy is underscored by a plethora of data indicating that AKI increases morbidity and mortality and can initiate the onset of progressive renal disease.1Ishani A. Xue J.L. Himmelfarb J. et al.Acute kidney injury increases risk of ESRD among elderly.J Am Soc Nephrol. 2009; 20: 223-228Crossref PubMed Scopus (885) Google Scholar, 2Xue J.L. Daniels F. Star R.A. et al.Incidence and mortality of acute renal failure in Medicare beneficiaries, 1992 to 2001.J Am Soc Nephrol. 2006; 17: 1135-1142Crossref PubMed Scopus (621) Google Scholar, 3Liangos O. Wald R. O'Bell J.W. et al.Epidemiology and outcomes of acute renal failure in hospitalized patients: a national survey.Clin J Am Soc Nephrol. 2006; 1: 43-51Crossref PubMed Scopus (437) Google Scholar, 4Wald R. Quinn R.R. Luo J. et al.Chronic dialysis and death among survivors of acute kidney injury requiring dialysis.JAMA. 2009; 302: 1179-1185Crossref PubMed Scopus (555) Google Scholar, 5Goldberg A. Kogan E. Hammerman H. et al.The impact of transient and persistent acute kidney injury on long-term outcomes after acute myocardial infarction.Kidney Int. 2009; 76: 900-906Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 6Chertow G.M. Burdick E. Honour M. et al.Acute kidney injury, mortality, length of stay, and costs in hospitalized patients.J Am Soc Nephrol. 2005; 16: 3365-3370Crossref PubMed Scopus (2502) Google Scholar, 7Lo L.J. Go A.S. Chertow G.M. et al.Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease.Kidney Int. 2009; 76: 893-999Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar One potentially promising prophylactic approach could be the administration of agents that upregulate cytoprotective proteins in kidney (e.g., heme oxygenase 1 [HO-1], haptoglobin, interleukin-10 [IL-10]). Indeed, a plethora of experimental data support the concept that such redox-sensitive "stress proteins" can exert dramatic renal-protective effects (reviewed in Zager8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and Hill-Kapturczak et al.9Hill-Kapturczak N. Jarmi T. Agarwal A. Growth factors and heme oxygenase-1: perspectives in physiology and pathophysiology.Antioxid Redox Signal. 2007; 9: 2197-2207Crossref PubMed Scopus (15) Google Scholar). However, the most effective and safest way(s) of inducing these proteins in kidney remains to be defined. We recently reported that administration of a subtoxic dose of nitrited myoglobin, in combination with Sn protoporphyrin (SnPP), can safely and synergistically upregulate potent cytoprotective proteins (e.g., HO-1, haptoglobin, IL-10) in mouse kidney.8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Thus, by 18 hours after agent administration, striking protection against both ischemia–reperfusion (I/R) and toxic (maleate, glycerol) acute renal failure (ARF) was induced. Furthermore, these same cytoprotective genes were activated in extrarenal organs (most notably in liver). As a result, marked protection against ischemic and toxic hepatic injuries was expressed.8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar The rationale for the above pharmacologic approach was as follows: (i) heme proteins, such as myoglobin, are potent inducers of redox-sensitive cytoprotective genes;8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 9Hill-Kapturczak N. Jarmi T. Agarwal A. Growth factors and heme oxygenase-1: perspectives in physiology and pathophysiology.Antioxid Redox Signal. 2007; 9: 2197-2207Crossref PubMed Scopus (15) Google Scholar, 10Nath K.A. Balla G. Vercellotti G.M. et al.Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.J Clin Invest. 1992; 90: 267-270Crossref PubMed Scopus (601) Google Scholar, 11Agarwal A. Bolisetty S. Adaptive responses to tissue injury: role of heme oxygenase-1.Trans Am Clin Climatol Assoc. 2013; 124: 111-122PubMed Google Scholar (ii) nitrite binding to myoglobin Fe decreases its cytotoxic potential;8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 12Rassaf T. Totzeck M. Hendgen-Cotta U.B. et al.Circulating nitrite contributes to cardioprotection by remote ischemic preconditioning.Circ Res. 2014; 114: 1601-1610Crossref PubMed Scopus (266) Google Scholar, 13Totzeck M. Hendgen-Cotta U.B. Luedike P. Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation.Circulation. 2012; 126: 325-334Crossref PubMed Scopus (157) Google Scholar, 14Goyal A. Semwal B.C. Yadav H.N. Abrogated cardioprotective effect of ischemic preconditioning in ovariectomized rat heart.Hum Exp Toxicol. 11 August 2015; ([e-pub ahead of print])https://doi.org/10.1177/0960327115597980Crossref PubMed Scopus (15) Google Scholar, 15Hendgen-Cotta U.B. Merx M.W. Shiva S. et al.Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury.Proc Natl Acad Sci U S A. 2008; 105: 10256-10261Crossref PubMed Scopus (306) Google Scholar (iii) concomitant SnPP administration enhances nitrite–myoglobin signaling;8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and (iv) SnPP can independently upregulate HO-1 and haptoglobin expression, and synergize nitrited myoglobin–mediated cytoprotective gene induction.8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16Sardana M.K. Kappas A. Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.Proc Natl Acad Sci U S A. 1987; 84: 2464-2468Crossref PubMed Scopus (154) Google Scholar Despite the observed efficacy and safety of the above approach,8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar theoretical reservations might exist vis-à-vis the use of a potential nephrotoxin (myoglobin) as a renal cytoprotective agent. Furthermore, although SnPP has been safely administered to patients (e.g., to mitigate neonatal jaundice), no clinically available SnPP preparation exists. Thus, the present study was undertaken to address the following 2 questions: (i) can a widely used Fe-containing macromolecule, iron sucrose (FeS; Venofer), be substituted for nitrited myoglobin in the above prophylactic strategy? and (ii) can a readily available protoporphyrin (the vitamin B12 analogue cyanocobalamin [CCB]) be substituted for SnPP and used with FeS in a renal prophylaxis strategy? These 2 issues form the basis of this report. Blood urea nitrogen (BUN) and plasma creatinine (PCr) values for normal mice were 24 ± 2 and 0.31 ± 0.01 mg/dl. None of the test agents, used either alone or in combination, induced any significant changes in these BUN and PCr levels. Maleate injection into control mice caused severe AKI as denoted by marked BUN and PCr increases (Figure 1, left).17Kellerman P.S. Exogenous adenosine triphosphate (ATP) preserves proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion.J Clin Invest. 1993; 92: 1940-1949Crossref PubMed Scopus (35) Google Scholar, 18Zager R.A. Johnson A.C. Naito M. Bomsztyk K. Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death.Am J Physiol. 2008; 294: F187-F197Crossref PubMed Scopus (50) Google Scholar Neither SnPP alone nor FeS alone significantly reduced injury severity. However, combined FeS+SnPP conferred marked protection, denoted by ∼75% decreases in BUN and PCr concentrations. Within 18 hours of inducing I/R injury in control mice, approximately 4-fold elevations in BUN and PCr concentrations resulted (Figure 1, right panel). Pretreatment with FeS+SnPP conferred significant protection, lowering the PCr to near normal levels. SnPP alone, but not FeS alone, exerted a modest independent protective effect. Renal protection was also denoted by a marked diminution of histologic injury (tubule necrosis, cast formation; P < 0.01), as presented in Figure 2. Again, maleate injection induced severe AKI in control mice (Figure 3, left panel). Pretreatment with either FeS alone or CCB alone failed to alter AKI severity. Conversely, combination FeS+CCB induced marked renal protection, as denoted by dramatic BUN and PCr reductions. Severe renal failure resulted within 18 hours of glycerol injection into control mice (Figure 3, right panel). FeS+CCB conferred substantial protection, as denoted by marked reductions in both 18-hour BUN and PCr concentrations. FeS alone also exerted an independent protective effect, albeit not as great as that seen with combined FeS+CCB administration. Three days following FeS and CCB administration, significant protection against glycerol AKI was still observed (BUNs, 86 ± 32 vs. 154 ± 4 mg/dl, P < 0.05; PCr concentrations, 1.04 ± 0.43 vs. 1.80 ± 0.18, P < 0.03). However, it was less pronounced than the protection with the 1-day-post-FeS/CCB preconditioning, as shown in Figure 3. As shown in Figure 4, left panel, FeS and SnPP each caused dramatic increases in renal cortical HO-1 mRNA levels by 4 hours after injection. However, when administered together, no additive HO-1 mRNA increase was observed. By 18 hours after agent injection, HO-1 mRNA levels had returned to normal levels for all treatment groups. As shown in Figure 4, right panel, a correlate of the 4-hour mRNA increases were significant increases in HO-1 protein levels. These remained significantly elevated at the 18-hour time point, particularly with FeS alone or the FeS+SnPP combination. No additive effects of FeS and SnPP on HO-1 protein levels were observed. As shown in Figure 5, left panel, at 4 hours after administration, SnPP alone, FeS alone, and combined FeS+SnPP modestly raised renal cortical haptoglobin mRNA levels. At 18 hours after agent administration, a synergistic FeS/SnPP–mediated haptoglobin mRNA increase was observed (greater than with either agent alone; P < 0.01). At the 4-hour time point, only the combined therapy caused a significant rise in renal cortical haptoglobin levels (Figure 5, right panel). By 18 hours, all 3 treatments led to cortical haptoglobin increases, with the greatest elevations being observed with combined FeS+SnPP treatment. As shown in Figure 6 (left panel), each of the test agents alone and in combination caused significant increases in HO-1 mRNA levels at 4 hours. However, by 18 hours, all of the values reverted to normal (recapitulating the findings with FeS+SnPP). Corresponding with the 4-hour HO-1 mRNA increases were significant increases in HO-1 protein (Figure 6, right). Unlike the 18-hour HO-1 mRNA levels that reverted to normal at 18 hours, significant increases in HO-1 protein levels persisted to the 18-hour time point, particularly with FeS or FeS+CCB treatment. CCB alone and FeS alone each caused small but significant increases in haptoglobin mRNA at the 4-hour but not at the 18-hour time point (Figure 7, left). Conversely, combined FeS+CCB caused dramatic haptoglobin mRNA increases. By 18 hours after FeS or FeS+CCB injection, modest haptoglobin mRNA increases were still observed. FeS+CCB treatment evoked significant haptoglobin increases at both 4 hours and 18 hours after injection (Figure 7, right). These exceeded the values observed with single agent injection. To assess whether a potentially more broad-based increase in cytoprotective genes might result from Fe and protoporphyrin administration, protein levels for the above-noted cytoprotective genes were assessed 18 hours after FeS+SnPP injection. As shown in Figure 8, with the exception of α1-microglobulin, which showed a borderline increase, each of the remaining 4 proteins was significantly increased at the 18-hour time point (i.e., the point in time at which superimposed AKI was induced in the previous experiments). To assess relative individual contributions of FeS or SnPP in this induction, the individual and combined effects of these agents on cognate mRNAs were assessed. As shown in Table 1, combination agent administration raised the mRNAs for hepcidin, hemopexin, α1-antitrypsin, and α1-microglobulin at both the 4- and 18-hour time points, with these increases typically being greater than those seen with either FeS or SnPP alone. Surprisingly, IL-10 mRNA increases were not observed, despite a doubling of renal cortical IL-10 protein levels. This suggests that the increased IL-10 levels may have arisen from increased mRNA translation, a widely recognized determinant of IL-10 production.8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google ScholarTable 1mRNAs at 4 and 18 hours of FeS, SnPP, or Fes+SnPP treatmentControl mRNA4 h FeS4 h SnPP4 h FeS+SnPPHepcidin0.10 ± 0.020.86 ± 0.540.23 ± 0.091.1 ± 0.53bHemopexin0.1 ± 0.020.41/0.20.56/0.310.73 ± 0.32cα1-Microglobulin0.40 ± 0.080.76/0.131.03/0.421.3 ± 0.30aα1-Antitrypsin0.95 ± 0.081.96/0.662.15/0.902.62 ± 0.93bIL-100.58 ± 0.090.55 ± 0.290.29 ± 0.070.34 ± 0.06 NSControl mRNA18 h FeS18 h SnPP18 h FeS+SnPPHepcidin0.10 ± 0.030.12 ± 0.050.20 ± 0.091.47 ± 0.12cHemopexin0.11 ± 0.020.45/0.250.26/0.161.01 ± 0.14aα1-Microglobulin0.41 ± 0.090.61/0.100.83/.0271.1 ± 0.21aα1-Antitrypsin0.91 ± 0.082.36/1.12.73/1.53.86 ± 0.61cIL-100.59 ± 0.090.35 ± 0.090.65 ± 0.180.40 ± 0.06 NSEither 4 or 18 hours after administration of FeS, SnPP, or FeS+SnPP, the above-noted mRNAs in renal cortex were measured (see text). Excepting IL-10 mRNA, all of the other mRNAs were significantly elevated at the 4- and 18-hour time points with combination, but not single agent, treatments. In general, the combination therapy induced greater increases than either agent alone.FeS, iron sucrose; IL-10, interleukin-10; NS, not significant; SnPP, Sn protoporphyrin.Values versus control: aP < 0.05; bP < 0.01; cP < 0.001. Open table in a new tab Either 4 or 18 hours after administration of FeS, SnPP, or FeS+SnPP, the above-noted mRNAs in renal cortex were measured (see text). Excepting IL-10 mRNA, all of the other mRNAs were significantly elevated at the 4- and 18-hour time points with combination, but not single agent, treatments. In general, the combination therapy induced greater increases than either agent alone. FeS, iron sucrose; IL-10, interleukin-10; NS, not significant; SnPP, Sn protoporphyrin. Values versus control: aP < 0.05; bP < 0.01; cP < 0.001. By 18 hours after induction of maleate and IRI-induced AKI in control mice, approximately 6- to 8-fold increases in plasma troponin I levels were observed (Figure 9). Combined FeS+SnPP pretreatment markedly blunted these troponin increases. This finding was particularly pronounced in the maleate model, where >90% reductions in troponin levels were observed. Maleate injection into non-conditioned (control) mice caused severe AKI, as depicted in Figure 10. Preconditioning with FeS+SnPP conferred marked protection (Figure 10). When a second dose of SnPP was administered into preconditioned mice at the time of maleate injection (to inhibit pre-formed HO-1; see Materials and Methods), a significant diminution of renal protection resulted (Figure 10). However, some degree of protection persisted, given that both BUN and PCr concentrations remained lower in the preconditioned mice that had received a second SnPP dose versus untreated maleate controls. Thus, these experiments indicate that increased HO-1 activity is mechanistically important to the induction of the cytoresistant state. Confirming that HO-1 was indeed active at 18 hours after FeS and SnPP administration, renal cortical and plasma HO-1 enzymatic activity was determined. (Notably, plasma HO-1 levels tightly correlate with intrarenal HO-1 levels.19Zager R.A. Johnson A.C. Becker K. Plasma and urinary heme oxygenase-1 in AKI.J Am Soc Nephrol. 2012; 23: 1048-1057Crossref PubMed Scopus (91) Google Scholar) Two- to threefold increases in HO-1 enzyme activity were observed (plasma: 30 ± 8.9 vs. 8 ± 0.9, P < 0.01; tissue: 42 ± 5 vs. 20 ± 1.5, P < 0.01 [μg HO-1 activity per ml plasma or μg per mg tissue protein]). FeS has become a mainstay in the treatment of anemia of chronic kidney disease. Although rare episodes of anaphylactoid reactions have occurred following its i.v. administration, its widespread and repetitive use attests to its overall safety in patients with renal and nonrenal diseases. As with other Fe-containing macromolecules, such as heme proteins, FeS has the capacity to induce transient oxidative stress, which can then upregulate the expression of diverse cytoprotective proteins (e.g., HO-1, haptoglobin, hemopexin, ferritin).20Zager R.A. Johnson A.C. Hanson S.Y. Wasse H. Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury.Am J Kidney Dis. 2002; 40: 90-103Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 21Johnson A.C. Becker K. Zager R.A. Parenteral iron formulations differentially affect MCP-1, HO-1, and NGAL gene expression and renal responses to injury.Am J Physiol Renal Physiol. 2010; 299: F426-F435Crossref PubMed Scopus (42) Google Scholar, 22Zager R.A. Parenteral iron compounds: potent oxidants but mainstays of anemia management in chronic renal disease.Clin J Am Soc Nephrol. 2006; : S24-S31Crossref PubMed Scopus (41) Google Scholar Given this property, we previously demonstrated that by 18 hours after FeS administration in mice, partial renal resistance to the glycerol ARF model emerges.21Johnson A.C. Becker K. Zager R.A. Parenteral iron formulations differentially affect MCP-1, HO-1, and NGAL gene expression and renal responses to injury.Am J Physiol Renal Physiol. 2010; 299: F426-F435Crossref PubMed Scopus (42) Google Scholar However, this protection was relatively weak, and its ability to protect against other forms of AKI was not assessed. We recently demonstrated that administration of another Fe-containing macromolecule (nitrited myoglobin), in combination with SnPP, induces striking and broad-ranging protection against diverse forms of AKI as well as acute liver disease (I/R, hepatotoxicity).8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Thus, we have now tested whether i.v. FeS with or without SnPP, or a clinically available protoporphyrin (the vitamin B12 analogue CCB), can also upregulate renal cytoprotective proteins, and thus induce a broad-based renal-protected state. As shown in Figure 1, when administered alone, FeS had an inconstant effect on AKI: although it decreased the severity of glycerol-induced AKI, it had no discernible impact on I/R-induced ARF. In contrast, when given alone, SnPP conferred partial protection against I/R injury, but it could not mitigate maleate-induced ARF. However, when FeS and SnPP were administered together, dramatic protection against both AKI models resulted. To ascertain whether CCB could be substituted for SnPP, we administered it either alone or in combination with FeS. Eighteen hours later, protection against both glycerol- and maleate-induced injuries was assessed. Despite the fact that CCB had no independent influence, it increased FeS-mediated protection in both models of ARF (Figure 2). Finally, the fact that significant protection was also observed when the glycerol challenge was imposed 72 hours after FeS and CCB administration indicates that the induced preconditioning response was not simply a momentary effect. HO-1 is widely considered to be a highly potent renal-protective protein.8Zager R.A. Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.Transl Res. 2015; 166: 485-501Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 9Hill-Kapturczak N. Jarmi T. Agarwal A. Growth factors and heme oxygenase-1: perspectives in physiology and pathophysiology.Antioxid Redox Signal. 2007; 9: 2197-2207Crossref PubMed Scopus (15) Google Scholar, 10Nath K.A. Balla G. Vercellotti G.M. et al.Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.J Clin Invest. 1992; 90: 267-270Crossref PubMed Scopus (601) Google Scholar, 11Agarwal A. Bolisetty S. Adaptive responses to tissue injury: role of heme oxygenase-1.Trans Am Clin Climatol Assoc. 2013; 124: 111-122PubMed Google Scholar Therefore, to ascertain the impact of our test agents on HO-1 expression, its mRNA and protein levels were determined 4 and 18 hours after agent administration. As shown in Figure 4, Figure 6, FeS, SnPP, and CCB each upregulated HO-1 mRNA at 4 hours, and by 18 hours, the time of cytoresistance, marked increases in HO-1 protein levels were observed. Given that combination therapies were more effective in producing broad-based protection against AKI than any single agent, we predicted that combination therapies would produce higher renal cortical HO-1 protein levels than any single agent alone. However, in no case was this observed. This suggests 2 possibilities: first, that FeS, SnPP, and CCB signal through a shared pathway, thereby obviating additive effects; and second, that HO-1 upregulation is not a complete explanation for the broad-based renal protection that followed combination (FeS+SnPP; FeS+CCB) therapy. Had it been the only mediator of protection, then additive HO-1 protein increases should have matched the greater renal protection that followed combination versus single agent administration. To further explore the theory that multiple protective pathways are activated by therapy with FeS and protoporphyrin, we tested whether an upregulation of other redox-sensitive cytoprotective genes, in addition to HO-1, were induced. To first explore this possibility, haptoglobin gene expression was assessed. Indeed, FeS, SnPP, and CCB each upregulated haptoglobin mRNA and protein levels, and in general, this effect was greater with combination versus single agent therapy. We next questioned whether 5 additional cytoprotective genes, α1-antitrypsin,23Zager R.A. Johnson A.C. Frostad K.B. Rapid renal alpha-1 antitrypsin gene induction in experimental and clinical acute kidney injury.PLoS One. 2014; 9: e9838Crossref Scopus (23) Google Scholar hemopexin,24Zager R.A. Johnson A.C. Becker K. Renal cortical hemopexin accumulation in response to acute kidney injury.Am J Physiol. 2012; 303: F1460-F1472Crossref PubMed Scopus (32) Google Scholar α1-microglobulin,25Åkerström B. Gram M. A1M, an extravascular tissue cleaning and housekeeping protein.Free Radic Biol Med. 2014; 74: 274-282Crossref PubMed Scopus (55) Google Scholar hepcidin,26Scindia Y. Dey P. Thirunagari A. et al.Hepcidin mitigates renal ischemia-reperfusion injury by modulating systemic iron homeostasis.J Am Soc Nephrol. 2015; 26: 2800-2814Crossref PubMed Scopus (95) Google Scholar and IL-10, might also have been induced by FeS+SnPP. As shown in Figure 8, with the exception of α1-microglobulin, increased protein levels for each were observed. Given these findings, it seems quite plausible that combination FeS and protoporphyrin administration can upregulate diverse cytoprotective pathways, which could then act in concert to induce the full expression of the renal cytoresistant state. Clearly, the above test proteins do not represent a complete list of potentially induced cytoprotective pathways. For example, SnPP has been reported to exert antiapoptotic effects,27Blumenthal S.B. Kiemer A.K. Tiegs G. et al.Metalloporphyrins inactivate caspase-3 and -8.FASEB J. 2005; 19: 1272-1279Crossref PubMed Scopus (29) Google Scholar and these have been previously implicated in conferring protection against postischemic ARF.28Kaizu T. Tamaki T. Tanaka M. et al.Preconditioning with tin-protoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney.Kidney Int. 2003; 63: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar HO-1 generates cytoprotective molecules—for instance, bilirubin, biliverdin, and carbon monoxide—via its enzymatic cleavage of heme. However, HO-1 may also exert renal-protective effects that are independent of its enzymatic activity.28Kaizu T. Tamaki T. Tanaka M. et al.Preconditioning with tin-protoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney.Kidney Int. 2003; 63: 1393-1403Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Thus, we needed to explore whether HO-1 activity was, indeed, involved in the observed cytoprotected state. First, we observed a 2- to 3-fold increase in HO-1 enzymatic activity at 18 hours after FeS and SnPP treatment, confirming active HO-1 generation (i.e., vs. production of a "dead" enzyme). Second, we utilized a functional way29Issan Y. Kornowski R. Aravot D. et al.Heme oxygenase-1 induction improves cardiac function following myocardial ischemia by reducing oxidative stress.PLoS One. 2014; 9: e92246Crossref PubMed Scopus (59) Google Scholar of determining HO-1's enzymatic participation
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