Oxidant- induced preconditioning: A pharmacologic approach for triggering renal 'self defense'.

Acute kidney injury (AKI) is a common event, occurring in ~5% and ~35% of hospitalized and ICU patients, respectively. The development of AKI portends an increased risk of morbidity, mortality, prolonged hospitalization, and subsequent development of chronic kidney disease (CKD). Given these facts, a multitude of experimental studies have addressed potential methods for inducing AKI prevention in high-risk patients. However, successful clinical translation of promising experimental data has remained elusive. Over the past decade, our laboratory has focused on developing a method for safely triggering AKI protection by inducing "kidney preconditioning" in mice by the intravenous administration of a combination of Fe sucrose (FeS) + tin protoporphyrin (SnPP). These agents induce mild, but short lived, 'oxidant stress' which synergistically activate a number of kidney 'self-defense' pathways (e.g., Nrf2, ferritin, IL-10). Within 18-24 h of Fe/SnPP administration, marked protection against diverse forms of experimental toxic and ischemic AKI results. FeS/SnPP-mediated reductions in kidney injury can also indirectly decrease injury in other organs by mitigating the so called "organ cross talk" phenomenon. Given these promising experimental data, three phase 1b clinical trials were undertaken in healthy subjects and patients with stage 3 or 4 CKD. These studies demonstrated that FeS/SnPP were well tolerated and that they up-regulated the cytoprotective Nrf2, ferritin, and IL-10 pathways. Two subsequent phase 2 trials, conducted in patients undergoing 'on-pump' cardiovascular surgery or in patients hospitalized with COVID 19, confirmed FeS/SnPP safety. Furthermore, interim data analyses revealed statistically significant improvements in several clinical parameters. The goals of this review are to: (i) briefly discuss the historical background of renal "preconditioning"; (ii) present the experimental data that support the concept of FeS/SnPP- induced organ protection; and (iii) discuss the initial results of clinical trials that suggest the potential clinical utility of an 'oxidant preconditioning' strategy.

Early loss of glutathione -s- transferase (GST) activity during diverse forms of acute renal tubular injury.

Glutathione-S-transferases (GSTs) are a diverse group of phase II detoxification enzymes which primarily evoke tissue protection via glutathione conjugation to xenobiotics and reactive oxygen species. Given their cytoprotective properties, potential changes in GST expression during AKI has pathophysiologic relevance. Hence, we evaluated total GST activity, and the mRNA responses of nine cytosolic GST isotypes (GST alpha1, kappa1, mu1/5, omega1, pi1 sigma1, theta1, zeta1 mRNAs), in five diverse mouse models of AKI (glycerol, ischemia/reperfusion; maleate, cisplatin, endotoxemia). Excepting endotoxemia, each AKI model significantly reduced GST activity (~35%) during both the AKI "initiation" (0-4 h) and "maintenance" phases (18 or 72 h). During the AKI maintenance phase, increases in multiple GST mRNAs were observed. However, no improvement in GST activity resulted. Increased urinary GST excretion followed AKI induction. However, this could not explain the reduced renal GST activity given that it also fell in response to ex vivo renal ischemia (i.e., absent urinary excretion). GST alpha, a dominant proximal tubule GST isotype, manifested 5-10-fold protein increases following AKI, arguing against GST proteolysis as the reason for the GST activity declines. Free fatty acids (FFAs) and lysophospholipids, which markedly accumulate during AKI, are known to bind to, and suppress, GST activity. Supporting this concept, arachidonic acid addition to renal cortical protein extracts caused rapid GST activity reductions. Based on these results, we conclude that diverse forms of AKI significantly reduce GST activity. This occurs despite increased GST transcription/translation and independent of urinary GST excretion. Injury-induced generation of endogenous GST inhibitors, such as FFAs, appears to be a dominant cause.

Catalytic iron mediated renal stress responses during experimental cardiorenal syndrome 1 ("CRS-1").

Cardiorenal syndrome I (CRS-1) denotes a state in which acute kidney injury occurs in the setting of acute heart failure (AHF). Isoproterenol (Iso) administration is widly used as an AHF model by transiently inducing extreme tachycardia, hypotension, and myocyte apoptosis and/or necrosis. To gain potential insights into renal manifestations of CRS-1, mice were subjected to the Iso-AHF model (50 mg Iso/kg), followed by renal functional and renal cortical assessments over 4 hours Iso induced acute azotemia (doubling of BUN, plasma creatinine) and significantly reduced renal plasma flow (prolonged plasma para-amino-hippurate clearance). Although no morphologic tubular injury was identified, marked increases in renal cortical 'stress markers' (NGAL, HO-1, IL-6, MCP-1 mRNAs) and oxidant stress (decreased glutathione, increased malondialdehyde) were observed. These changes were catalytic Fe dependent, given that the iron chelator desferrioxamine (DFO) significantly blunted, or completely reversed, these renal cortical abnormalities. Despite these acute changes, no lasting renal injury was observed (assessed over 3 days). To determine whether Iso directly impacts tubular cell integrity, cultured proximal tubule (HK-2) cells were exposed to Iso. Substantial Fe dependent cell injury (decreased MTT uptake), and Fe independent increases in HO-1/IL-6 mRNA expression were observed. We conclude that Iso-induced AHF is a useful reversible model of CRS-1. Despite its largely hemodynamic ('pre-renal') nature, Fe-mediated oxidative stress and pro-inflammatory reactions are induced. These arise, at least in part, from direct Iso- induced tubular cell toxicity, rather than simply being secondary to Iso-mediated hemodynamic events. Finally, Iso-triggered renal cytokine production can potentially contribute to 'organ cross talk' and a systemic pro-inflammatory state.

Iron sucrose ('RBT-3') activates the hepatic and renal HAMP1 gene, evoking renal hepcidin loading and resistance to cisplatin nephrotoxicity.

BACKGROUND: Iron sucrose (FeS) administration induces a state of renal preconditioning, protecting against selected forms of acute kidney injury (AKI). Recent evidence suggests that recombinant hepcidin also mitigates acute renal damage. Hence the goals of this study were to determine whether a new proprietary FeS formulation ('RBT-3') can acutely activate the hepcidin (HAMP1) gene in humans, raising plasma and renal hepcidin concentrations; assess whether the kidney participates in this posited RBT-3-hepcidin generation response; test whether RBT-3 can mitigate a clinically relevant AKI model (experimental cisplatin toxicity) and explore whether mechanisms in addition to hepcidin generation are operative in RBT-3's cytoprotective effects. METHODS: Healthy human volunteers (n = 9) and subjects with Stages 3-4 CKD (n = 9) received 120, 240 or 360 mg of RBT-3 (intravenously over 2 h). Plasma and urine samples were collected and assayed for hepcidin levels (0-72 h post-RBT-3 injection). In complementary mouse experiments, RBT-3 effects on hepatic versus renal hepcidin (HAMP1) messenger RNA (mRNA) and protein levels were compared. RBT-3's impact on the mouse Nrf2 pathway and on experimental cisplatin nephrotoxicity was assessed. Direct effects of exogenous hepcidin on in vivo and in vitro (HK-2 cells) cisplatin toxicity were also tested. RESULTS: RBT-3 induced rapid, dose-dependent and comparable plasma hepcidin increases in both healthy volunteers and chronic kidney disease subjects (∼15 times baseline within 24 h). Human kidney hepcidin exposure was confirmed by 4-fold urinary hepcidin increases. RBT-3 up-regulated mouse hepcidin mRNA, but much more so in kidney (>25 times) versus liver (∼2 times). RBT-3 also activated kidney Nrf2 [increased Nrf2 nuclear binding; increased Nrf2-responsive gene mRNAs: heme oxygenase-1, sulfiredoxin-1, glutamate-cysteine ligase catalytic subunit and NAD(P)H quinone dehydrogenase 1]. RBT-3 preconditioning (18 h time lapse) markedly attenuated experimental cisplatin nephrotoxicity (∼50% blood urea nitrogen/creatinine decrements), in part by reducing renal cisplatin uptake by 40%. Exogenous hepcidin (without RBT-3) treatment conferred protection against mild in vivo (but not in vitro) cisplatin toxicity. CONCLUSIONS: RBT-3 acutely and dramatically up-regulates cytoprotective hepcidin production, increasing renal hepcidin levels. However, additional cytoprotective mechanisms are activated by RBT-3 (e.g. Nrf2 activation; reduced cisplatin uptake). Thus RBT-3-induced preconditioning likely confers renal resistance to cisplatin via an interplay of multiple cytoprotective activities.

The NRF2 stimulating agent, tin protoporphyrin, activates protective cytokine pathways in healthy human subjects and in patients with chronic kidney disease.

BACKGROUND: Tin protoporphyrin (SnPP), a heme oxygenase 1 (HO-1) inhibitor, triggers adaptive tissue responses that confer potent protection against acute renal- and extra-renal tissue injuries. This effect is mediated, in part, via SnPP-induced activation of the cytoprotective Nrf2 pathway. However, it remains unclear as to whether SnPP can also upregulate humoral cytokine defenses, either in healthy human subjects or in patients with CKD. If so, then systemically derived cytokines could contribute SnPP-induced tissue protection. METHODS: SnPP (90 mg IV) was administered over 2 hr to six healthy human volunteers (HVs) and 12 subjects with stage 3-4 CKD. Plasma samples were obtained from baseline upto 72 hr post injection. Two representative anti-inflammatory cytokines (IL-10, TGFß1), and a pro-inflammatory cytokine (TNF-α), were assayed. Because IL-6 has been shown to induce tissue preconditioning, its plasma concentrations were also assessed. In complementary mouse experiments, SnPP effects on renal, splenic, and hepatic IL-10, IL-6, TGFß1, and TNF-α production (as gauged by their mRNAs) were tested. Tissue HO-1 mRNA served as an Nrf2 activation marker. RESULTS: SnPP induced marked (~5-7x) increases in plasma IL-10 and IL-6 concentrations within 24-48 hr, and to equal degrees in HVs and CKD patients. SnPP modestly raised plasma TGFß1 without impacting plasma TNF-α levels. In mouse experiments, SnPP did not affect IL-6, IL-10, TNF-α, or TGFß1 mRNAs in kidney despite marked renal Nrf2 activation. Conversely, SnPP increased splenic IL-10 and hepatic IL-6/TGFß1 mRNA levels, suggesting these organs as sites of extra-renal cytokine generation. CONCLUSIONS: SnPP can trigger cytoprotective cytokine production, most likely in extra-renal tissues. With ready glomerular cytokine filtration, extra-renal/renal "organ cross talk" can result. Thus, humoral factors seemingly can contribute to SnPP's cytoprotective effects.

A Pharmacologic "Stress Test" for Assessing Select Antioxidant Defenses in Patients with CKD.

BACKGROUND AND OBJECTIVES: Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves. DESIGN: , setting, participants, & measurementsA total of 18 healthy volunteers and 24 participants with stage 3 CKD (n=12; eGFR 30-59 ml/min per 1.73 m2) or stage 4 CKD (n=12; eGFR 15-29 ml/min per 1.73 m2) were injected once with SnPP (9, 27, or 90 mg). Plasma and/or urinary antioxidant proteins were measured at baseline and for up to 4 days post-SnPP dosing. Kidney safety was gauged by serial measurements of BUN, creatinine, eGFR, albuminuria, and four urinary AKI biomarkers (kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, cystatin C, and N-acetyl glucosaminidase). RESULTS: Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (r=-0.85 to -0.95). All four proteins manifested statistically significant dose- and time-dependent elevations after SnPP injection. However, marked intersubject differences were observed. p21 responses to high-dose SnPP and HO-1 responses to low-dose SnPP were significantly suppressed in participants with CKD versus healthy volunteers. SnPP was well tolerated by all participants, and no evidence of nephrotoxicity was observed. CONCLUSIONS: SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.

Parenterial iron sucrose-induced renal preconditioning: differential ferritin heavy and light chain expression in plasma, urine, and internal organs.

Experimental data suggest that iron sucrose (FeS) injection, used either alone or in combination with other prooxidants, can induce "renal preconditioning," in part by upregulating cytoprotective ferritin levels. However, the rapidity, degree, composition (heavy vs. light chain), and renal ferritin changes after FeS administration in humans remain to be defined. To address these issues, healthy human volunteers (n = 9) and patients with stage 3-4 chronic kidney disease(n = 9) were injected once with FeS (120, 240, or 360 mg). Plasma ferritin was measured from 0 to 8 days postinjection as an overall index of ferritin generation. Urinary ferritin served as a "biomarker" of renal ferritin production. FeS induced rapid (≤2 h), dose-dependent, plasma ferritin increases in all study participants, peaking at approximately three to five times baseline within 24-48 h. Significant urinary ferritin increases (~3 times), without dose-dependent increases in albuminuria, neutrophil gelatinase-associated lipocalin, or N-acetyl-ß-d-glucosaminidase excretion, were observed. Western blot analysis with ferritin heavy chain (Fhc)- and light chain (Flc)-specific antibodies demonstrated that FeS raised plasma Flc but not Fhc levels. Conversely, FeS increased both Fhc and Flc in urine. To assess sites of FeS-induced ferritin generation, organs from FeS-treated mice were probed for Fhc, Flc, and their mRNAs. FeS predominantly raised hepatic Flc. Conversely, marked Fhc and Flc elevations developed in the kidney and spleen. No cardiopulmonary ferritin increases occurred. Ferritin mRNAs remained unchanged throughout, implying posttranscriptional ferritin production. We conclude that FeS induces rapid, dramatic, and differential Fhc and Flc upregulation in organs. Renal Fhc and Flc increases, in the absence of nephrotoxicity, suggest potential FeS utility as a clinical renal "preconditioning" agent.

Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.

The cyclin kinase inhibitor p21 is acutely upregulated during acute kidney injury (AKI) and exerts cytoprotective effects. A proposed mechanism is oxidant stress-induced activation of p53, the dominant p21 transcription factor. Glycerol-induced rhabdomyolysis induces profound renal oxidant stress. Hence, we studied this AKI model to determine whether p53 activation corresponds with p21 gene induction and/or whether alternative mechanism(s) might be involved. CD-1 mice were subjected to glycerol-induced AKI. After 4 or 18 h, plasma, urinary, and renal cortical p21 protein and mRNA levels were assessed. Renal p53 activation was gauged by measurement of both total and activated (Ser15-phosphorylated) p53 and p53 mRNA levels. Glycerol evoked acute, progressive increases in renal cortical p21 mRNA and protein levels. Corresponding plasma (~25-fold) and urinary (~75-fold) p21 elevations were also observed. Renal cortical ratio of total to phosphorylated (Ser15) p53 rose three- to fourfold. However, the p53 inhibitor pifithrin-α failed to block glycerol-induced p21 gene induction, suggesting that an alternative p21 activator might also be at play. To this end, it was established that glycerol-induced AKI 1) dramatically increased plasma (~5-fold) and urinary (~75-fold) cortisol levels, 2) the glucocorticoid receptor antagonist mifepristone blocked glycerol-induced p21 mRNA and protein accumulation, and 3) dexamethasone or cortisol injections markedly increased p21 protein and mRNA in both normal and glycerol-treated mice, although no discernible p53 protein or mRNA increases were observed. We conclude that AKI-induced "systemic stress" markedly increases plasma and urinary cortisol, which can then activate renal p21 gene expression, at least in part, via a glucocorticoid receptor-dependent signaling pathway. Discernible renal cortical p53 increases are not required for this dexamethasone-mediated p21 response.

Distinct patterns of transcriptional and epigenetic alterations characterize acute and chronic kidney injury.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are considered early and late phases of a pathologic continuum of interconnected disease states. Although changes in gene expression patterns have recently been elucidated for the transition of AKI to CKD, the epigenetic regulation of key kidney injury related genes remains poorly understood. We used multiplex RT-qPCR, ChIP-qPCR and integrative analysis to compare transcriptional and epigenetic changes at renal disease-associated genes across mouse AKI and CKD models. These studies showed that: (i) there are subsets of genes with distinct transcriptional and epigenetically profiles shared by AKI and CKD but also subsets that are specific to either the early or late stages of renal injury; (ii) differences in expression of a small number of genes is sufficient to distinguish AKI from CKD; (iii) transcription plays a key role in the upregulation of both AKI and CKD genes while post-transcriptional regulation appears to play a more significant role in decreased expression of both AKI and CKD genes; and (iv) subsets of transcriptionally upregulated genes share epigenetic similarities while downregulated genes do not. Collectively, our study suggests that identified common transcriptional and epigenetic profiles of kidney injury loci could be exploited for therapeutic targeting in AKI and CKD.

Plasma and urinary p21: potential biomarkers of AKI and renal aging.

p21 is upregulated in renal tubules in response to acute kidney injury ( AKI). and localizes in the nucleus, where it induces cell cycle arrest (CCA). These events can mitigate early injury but can also facilitate the onset of the degenerative cell senescence/"aging" process. Hence, we asked the following: 1) can AKI-induced p21 upregulation be gauged by plasma and/or urinary p21 assay; 2) might p21 serve as an AKI/CCA biomarker; and 3) does p21 accumulate during normal renal aging, and might plasma p21 reflect this process? Mice were subjected to either ischemia-reperfusion (I/R) or nephotoxic (maleate) AKI. Renal cortical p21 expression (protein, mRNA) was assessed 2-18 h later and contrasted with plasma/urine p21 concentrations (ELISA). p21 mRNA/protein levels were also measured in aging mice (2, 12, 24 mo). AKI induced marked, progressive, increases in renal cortical p21 mRNA and protein levels. These changes were marked by acute (within 2-4 h) and profound increases (up to 200×) in both plasma and urine p21 concentrations. Renal I/R also activated p21 gene expression in extrarenal organs (heart, brain), consistent with so-called "organ cross talk". p21 efflux from damaged cells was confirmed with studies of hypoxia-injured, isolated proximal tubules. Aging was associated with progressive renal cortical p21 expression, which correlated ( r, 0.83) with rising plasma p21 concentrations. We concluded that 1) during AKI, renal p21 increases can be gauged by either plasma or urine p21 assay, serving as potentially useful AKI/CCA biomarkers; 2) AKI can activate p21 in extrarenal organs; and 3) plasma p21 levels may provide an index of the renal/systemic aging process.

Mechanisms Underlying Increased TIMP2 and IGFBP7 Urinary Excretion in Experimental AKI.

BACKGROUND: Recent clinical data support the utility/superiority of a new AKI biomarker ("NephroCheck"), the arithmetic product of urinary TIMP × IGFBP7 concentrations. However, the pathophysiologic basis for its utility remains ill defined. METHODS: To clarify this issue, CD-1 mice were subjected to either nephrotoxic (glycerol, maleate) or ischemic AKI. Urinary TIMP2/IGFBP7 concentrations were determined at 4 and 18 hours postinjury and compared with urinary albumin levels. Gene transcription was assessed by measuring renal cortical and/or medullary TIMP2/IGFBP7 mRNAs (4 and 18 hours after AKI induction). For comparison, the mRNAs of three renal "stress" biomarkers (NGAL, heme oxygenase 1, and p21) were assessed. Renal cortical TIMP2/IGFBP7 protein was gauged by ELISA. Proximal tubule-specific TIMP2/IGFBP7 was assessed by immunohistochemistry. RESULTS: Each AKI model induced prompt (4 hours) and marked urinary TIMP2/IGFBP7 increases without an increase in renal cortical concentrations. Furthermore, TIMP2/IGFBP7 mRNAs remained at normal levels. Endotoxemia also failed to increase TIMP2/IGFBP7 mRNAs. In contrast, each AKI model provoked massive NGAL, HO-1, and p21 mRNA increases, confirming that a renal "stress response" had occurred. Urinary albumin rose up to 100-fold and strongly correlated (r=0.87-0.91) with urinary TIMP2/IGFBP7 concentrations. Immunohistochemistry showed progressive TIMP2/IGFBP7 losses from injured proximal tubule cells. Competitive inhibition of endocytic protein reabsorption in normal mice tripled urinary TIMP2/IGFBP7 levels, confirming this pathway's role in determining urinary excretion. CONCLUSIONS: AKI-induced urinary TIMP2/IGFBP7 elevations are not due to stress-induced gene transcription. Rather, increased filtration, decreased tubule reabsorption, and proximal tubule cell TIMP2/IGFBP7 urinary leakage seem to be the most likely mechanisms.

Mechanisms and consequences of oxidant-induced renal preconditioning: an Nrf2-dependent, P21-independent, anti-senescence pathway.

Background: P21, a cyclin kinase inhibitor, is upregulated by renal 'ischemic preconditioning' (IPC), and induces a 'cytoresistant' state. However, P21-induced cell cycle inhibition can also contribute to cellular senescence, a potential adverse renal event. Hence, this study assessed whether: (i) IPC-induced P21 upregulation is associated with subsequent renal senescence; and (ii) preconditioning can be established 'independent' of P21 induction and avoid a post-ischemic senescent state? Methods: CD-1 mice were subjected to either IPC (5-15 min) or to a recently proposed 'oxidant-induced preconditioning' (OIP) strategy (tin protoporphyrin-induced heme oxygenase inhibition +/- parental iron administration). P21 induction [messenger RNA (mRNA)/protein], cell proliferation (KI-67, phosphohistone H3 nuclear staining), kidney senescence (P16ink4a; P19Arf mRNAs; senescence-associated beta-galactosidase levels) and resistance to ischemic acute kidney injury were assessed. Results: IPC induced dramatic (10-25×) and persistent P21 activation and 'downstream' tubular senescence. Conversely, OIP did not upregulate P21, it increased, rather than decreased, cell proliferation markers, and it avoided a senescence state. OIP markedly suppressed ischemia-induced P21 up-regulation, it inhibited the development of post-ischemic senescence and it conferred near-complete protection against ischemic acute renal failure (ARF). To assess OIP's impact on a non-P21-dependent cytoprotective pathway, its ability to activate Nrf2, the so-called 'master regulator' of endogenous cell defenses, was assessed. Within 4 h, OIP activated each of three canonical Nrf2-regulated genes (NQO1, SRXN1, GCLC; 3- to 5-fold mRNA increases). Conversely, this gene activation pathway was absent in Nrf2-/- mice, confirming Nrf2 specificity. Nrf2-/- mice also did not develop significant OIP-mediated protection against ischemic ARF. Conclusions: OIP (i) activates the cytoprotective Nrf2, but not the P21, pathway; (ii) suppresses post-ischemic P21 induction and renal senescence; and (iii) confers marked protection against ischemic ARF. In sum, these findings suggest that OIP may be a clinically feasible approach for safely activating the Nrf2 pathway, and thereby confer protection against clinical renal injury.

Alpha 1 Microglobulin: A Potentially Paradoxical Anti-Oxidant Agent.

Alpha 1 microglobulin is a low molecular weight heme binding antioxidant protein with interesting, and potentially important, clinical applications. However, much remains to be learned about its in vivo effects. This invited review raises a number of physiologic issues regarding this compound as it pertains to clinical use.

Tin protoporphyrin activates the oxidant-dependent NRF2-cytoprotective pathway and mitigates acute kidney injury.

Tin protoporphyrin (SnPP), a heme oxygenase (HO) inhibitor, can paradoxically protect against diverse forms of acute kidney injury (AKI). This study sought potential underlying mechanisms. CD-1 mice received intravenous SnPP, followed 4-18 hours later by a variety of renal biochemical, histologic, and genomic assessments. Renal resistance to ischemic-reperfusion injury (IRI) was also sought. SnPP was rapidly taken up by kidney and was confined to proximal tubules. Transient suppression of renal heme synthesis (decreased δ aminolevulinic acid synthase expression), a 2.5-fold increase in "catalytic" Fe levels and oxidant stress resulted (decreased glutathione; increased malondialdehyde, and protein carbonyl content). Nrf2 nuclear translocation (∼2x Nrf2 increase; detected by enzyme-linked immunosorbent assay, Western blotting), with corresponding activation of ∼20 Nrf2-sensitive genes (RNA-Seq) were observed. By 18 hours after SnPP injection, marked protection against IRI emerged. This represented "preconditioning", not a direct SnPP effect, given that SnPP administered at the time of IRI exerted no protective effect. The importance of transient oxidant stress in SnPP "preconditioning" was exemplified by the following: (1) oxidant stress induced by a different mechanism (myoglobin injection) recapitulated SnPP's protective action; (2) GSH treatment blunted SnPP's protective influence; (3) SnPP raised cytoprotective heavy chain ferritin (Fhc), a response enhanced by exogenous Fe injection; and (4) SnCl2, a ∼35- to 50-fold HO-1 inducer (not inhibitor) evoked neither oxidant stress nor mitigated IRI (seemingly excluding HO-1 activity in SnPP's protective effect). SnPP specifically accumulates within proximal tubule cells; transient "catalytic" Fe overload and oxidative stress result; Nrf2-cytoprotective pathways are upregulated; and these changes help protect against ischemic AKI.

An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant.

α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought "proof of concept" in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) "catalytic" iron, 4) H2O2/Fenton reagents, 5) a Ca(2+) ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.

Combined iron sucrose and protoporphyrin treatment protects against ischemic and toxin-mediated acute renal failure.

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.

Marked protection against acute renal and hepatic injury after nitrited myoglobin + tin protoporphyrin administration.

The phenomenon known as renal "ischemic preconditioning," whereby an initial ischemic insult induces resistance against subsequent kidney damage, has been well established in the experimental literature. However, a clinically applicable way to safely recapitulate this state has not been defined. We hypothesized that a unique combination of agents (nitrited myoglobin [N-Mgb] + tin protoporphyrin [SnPP]) can achieve these ends safely and synergistically, increasing cytoprotective proteins (eg, heme oxygenase 1 [HO-1], interleukin 10 [IL-10], and haptoglobin) in kidney cells. To test this hypothesis, CD-1 mice received 1 mg of N-Mgb and 1 µmol of SnPP, either alone or in combination. Renal cortical HO-1, haptoglobin, and IL-10 gene expressions (messenger RNA [mRNA], protein levels) were determined 4 and 18 hours later. Cytoresistance to 3 forms of acute kidney injury (AKI; glycerol-induced rhabdomyolysis, maleate nephrotoxicity, and postischemic AKI progression to chronic kidney disease [CKD]) was assessed. To ascertain whether cytoresistance might emerge in extrarenal organs, hepatic HO-1, IL-10, and haptoglobin levels were also measured, and resistance to 25 minutes of hepatic ischemia-reperfusion injury and hepatotoxicity (intraperitoneal glycerol injection) was sought. N-Mgb + SnPP induced additive or synergistic increases in renal HO-1, haptoglobin, and IL-10 mRNA and protein levels (up to 20-fold) without inducing any apparent renal or extrarenal damage. After 18 hours of post-treatment, marked or complete protection against glycerol-induced AKI, maleate-induced AKI, and postischemic AKI progression to CKD had emerged. Combined N-Mgb + SnPP was more protective than either agent alone (assessed in glycerol model). N-Mgb + SnPP also upregulated cytoprotective pathways in liver and induced marked protection against both hepatic ischemia-reperfusion and toxic liver damage. In conclusion, we posit that "preconditioning" with combined administration of N-Mgb + SnPP represents a promising approach for protecting against diverse forms of renal and nonrenal (hepatic) forms of tissue damage.

Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes.

Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). As epigenetic processes control gene expression in a cell- and environment-defined manner, understanding the epigenetic pathways that regulate genes altered by AKI may open vital new insights into the complexities of disease pathogenesis and identify possible therapeutic targets. Here we used matrix chromatin immunoprecipitation and integrative analysis to study 20 key permissive and repressive epigenetic histone marks at transcriptionally induced Tnf, Ngal, Kim-1, and Icam-1 genes in mouse models of AKI; unilateral renal ischemia/reperfusion, lipopolysaccharide (LPS), and their synergistically injurious combination. Results revealed unexpected heterogeneity of transcriptional and epigenetic responses. Tnf and Ngal were transcriptionally upregulated in response to both treatments individually, and to combination treatment. Kim-1 was induced by ischemia/reperfusion and Icam-1 by LPS only. Epigenetic alterations at these genes exhibited distinct time-dependent changes that shared some similarities, such as reduction in repressive histone modifications, and also had major ischemia/reperfusion versus endotoxin differences. Thus, diversity of changes at AKI genes in response to different insults indicates involvement of several epigenetic pathways. This could be exploited pharmacologically through rational-drug design to alter the course and improve clinical outcomes of this syndrome.

Progression from acute kidney injury to chronic kidney disease: clinical and experimental insights and queries.

There is an increasing number of clinical studies suggesting that acute kidney injury (AKI) can be complicated by the onset of progressive renal disease. Indeed, given the frequency of AKI in hospitalized patients, it could potentially be a leading cause of, or contributor to, end-stage renal disease. Insights into the natural history of AKI and potential mechanisms for disease progression can be gleaned from experimental studies. Although such studies underscore the principle that AKI can 'heal with defects', whether ongoing renal disease develops remains a subject of debate. Indeed, in the aftermath of AKI, a variety of secondary renal protective pathways are activated, which may retard or prevent severe chronic kidney disease. Furthermore, the onset of acute uremia per se may exert surprisingly potent renal protective effects. The purpose of this brief report is to review some of the clinical and experimental data that deal with these complex issues.

Renal cortical pyruvate as a potentially critical mediator of acute kidney injury.

Pyruvate is a key intermediary in both aerobic and anaerobic energy metabolisms. In addition, a burgeoning body of experimental literature indicates that it can also dramatically impact oxidant, proinflammatory, and cytoprotective pathways. In sum, these actions can confer protection against diverse forms of tissue damage. However, the fate of pyruvate during the evolution of acute kidney injury (AKI) has remained ill defined. Recent experimental studies have indicated that following either ischemic or nephrotoxic renal injury, marked and sustained pyruvate depletion results. While multiple potential mechanisms for this pyruvate loss may be involved, experimental data suggest that a loss of lactate (a dominant pyruvate precursor) and enhanced gluconeogenesis (i.e. pyruvate utilization) are involved. The importance of pyruvate depletion for AKI pathogenesis is underscored by observations that pyruvate therapy can attenuate diverse forms of experimental AKI. This protection may stem from reductions in tissue inflammation, improved anti-inflammatory defenses, and an enhanced cellular energy metabolism. The pieces of information that give rise to these conclusions are discussed in this brief report.