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.

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.

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.

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.

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 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.

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.

Renal cortical pyruvate depletion during AKI.

Pyruvate is a key intermediary in energy metabolism and can exert antioxidant and anti-inflammatory effects. However, the fate of pyruvate during AKI remains unknown. Here, we assessed renal cortical pyruvate and its major determinants (glycolysis, gluconeogenesis, pyruvate dehydrogenase [PDH], and H2O2 levels) in mice subjected to unilateral ischemia (15-60 minutes; 0-18 hours of vascular reflow) or glycerol-induced ARF. The fate of postischemic lactate, which can be converted back to pyruvate by lactate dehydrogenase, was also addressed. Ischemia and glycerol each induced persistent pyruvate depletion. During ischemia, decreasing pyruvate levels correlated with increasing lactate levels. During early reperfusion, pyruvate levels remained depressed, but lactate levels fell below control levels, likely as a result of rapid renal lactate efflux. During late reperfusion and glycerol-induced AKI, pyruvate depletion corresponded with increased gluconeogenesis (pyruvate consumption). This finding was underscored by observations that pyruvate injection increased renal cortical glucose content in AKI but not normal kidneys. AKI decreased PDH levels, potentially limiting pyruvate to acetyl CoA conversion. Notably, pyruvate therapy mitigated the severity of AKI. This renoprotection corresponded with increases in cytoprotective heme oxygenase 1 and IL-10 mRNAs, selective reductions in proinflammatory mRNAs (e.g., MCP-1 and TNF-α), and improved tissue ATP levels. Paradoxically, pyruvate increased cortical H2O2 levels. We conclude that AKI induces a profound and persistent depletion of renal cortical pyruvate, which may induce additional injury.

Acute hepatic ischemic-reperfusion injury induces a renal cortical "stress response," renal "cytoresistance," and an endotoxin hyperresponsive state.

Hepatic ischemic-reperfusion injury (HIRI) is considered a risk factor for clinical acute kidney injury (AKI). However, HIRI's impact on renal tubular cell homeostasis and subsequent injury responses remain ill-defined. To explore this issue, 30-45 min of partial HIRI was induced in CD-1 mice. Sham-operated or normal mice served as controls. Renal changes and superimposed injury responses (glycerol-induced AKI; endotoxemia) were assessed 2-18 h later. HIRI induced mild azotemia (blood urea nitrogen ∼45 mg/dl) in the absence of renal histologic injury or proteinuria, implying a "prerenal" state. However, marked renal cortical, and isolated proximal tubule, cytoprotective "stress protein" gene induction (neutrophil gelatinase-associated lipocalin, heme oxygenase-1, hemopexin, hepcidin), and increased Toll-like receptor 4 (TLR4) expression resulted (protein/mRNA levels). Ischemia caused release of hepatic heme-based proteins (e.g., cytochrome c) into the circulation. This corresponded with renal cortical oxidant stress (malondialdehyde increases). That hepatic derived factors can evoke redox-sensitive "stress protein" induction was implied by the following: peritoneal dialysate from HIRI mice, soluble hepatic extract, or exogenous cytochrome c each induced the above stress protein(s) either in vivo or in cultured tubule cells. Functional significance of HIRI-induced renal "preconditioning" was indicated by the following: 1) HIRI conferred virtually complete morphologic protection against glycerol-induced AKI (in the absence of hyperbilirubinemia) and 2) HIRI-induced TLR4 upregulation led to a renal endotoxin hyperresponsive state (excess TNF-α/MCP-1 gene induction). In conclusion, HIRI can evoke "renal preconditioning," likely due, in part, to hepatic release of pro-oxidant factors (e.g., cytochrome c) into the systemic circulation. The resulting renal changes can impact subsequent AKI susceptibility and TLR4 pathway-mediated stress.

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.

Progressive endothelin-1 gene activation initiates chronic/end-stage renal disease following experimental ischemic/reperfusion injury.

This study assessed whether endothelin-1 (ET-1) helps mediate postischemic acute kidney injury (AKI) progression to chronic kidney disease (CKD). The impact(s) of potent ETA or ETB receptor-specific antagonists (Atrasentan and BQ-788, respectively) on disease progression were assessed 24 h or 2 weeks following 30 min of unilateral ischemia in CD-1 mice. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant ETA, but not ETB, mRNA elevations. Extensive histone remodeling consistent with gene activation and increased RNA polymerase II (Pol II) binding occurred at the ET-1 gene. Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- and post-ischemia or just postischemic treatment with Atrasentan conferred dramatic protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. Nuclear KI-67 staining was not increased by Atrasentan, implying that increased tubule proliferation was not involved. Conversely, ETB blockade had no protective effect. Thus, our findings provide the first evidence that ET-1 operating through ETA can have a critical role in ischemic AKI progression to CKD. Blockade of ETA provided dramatic protection, indicating the functional significance of these results.

Post-ischemic azotemia as a partial 'brake', slowing progressive kidney disease.

BACKGROUND: Recent experimental work suggests a paradox: although uremia evokes systemic toxicities, in the setting of AKI, it can induce intrarenal cytoprotective and anti-inflammatory effects. Whether these influences can attenuate post-ischemic kidney disease progression remains unknown. METHODS: To explore this possibility, male CD-1 mice were subjected to a 30-min unilateral (left) kidney ischemia model, previously shown to reduce renal mass by ∼50% over 2-3 weeks. Stepwise azotemia/acute uremia was superimposed by inducing different lengths of contralateral (right) kidney ischemia (0, 15, 18, 20 min). Subsequent loss of left renal mass (kidney weight) was assessed 2 weeks later and contrasted with the degree of initial azotemia 24-h BUN. RESULTS: A striking correlation between 24-h BUNs and 2-week left renal mass was observed (r, 0.77; P < 0.001). With 20 min of right kidney ischemia, left kidney size was completely preserved. This preservation did not result from increased tubular cell proliferation or decreased microvascular loss, as gauged by KI-67 and CD-34 immunohistochemistry, respectively. Rather, an early reduction in proximal tubule cell dropout (as judged by renal cortical N-acetyl-glucosaminidase content), with a subsequent preservation of tubule mass, was observed. CONCLUSIONS: In summary, these findings advance a novel concept: acute uremia can confer early post-ischemic cytoprotection resulting in a slowed progression of post-ischemic kidney disease.

Plasma and urinary heme oxygenase-1 in AKI.

AKI induces upregulation of heme oxygenase 1 (HO-1), which exerts cytoprotective effects and modulates the renal response to injury, suggesting that a biomarker of intrarenal HO-1 activity may be useful. Because HO-1 largely localizes to the endoplasmic reticulum and has no known secretory pathway, it is unclear whether plasma or urinary levels of HO-1 reflect intrarenal HO-1 expression. We measured plasma and urinary levels of HO-1 by ELISA during the induction and/or maintenance phases of four mouse models of AKI: ischemia/reperfusion, glycerol-induced rhabdomyolysis, cisplatin nephrotoxicity, and bilateral ureteral obstruction. In addition, we measured levels of HO-1 mRNA and protein in the renal cortex. Each AKI model increased renal HO-1 gene expression, which corresponded with release of HO-1 into plasma and urine by 4 hours. Over time, the magnitudes of plasma and urinary HO-1 paralleled renal cortical gene expression. AKI and the associated uremia did not seem to affect extrarenal HO-1 gene activity assessed in the liver, lung, and spleen. In iron-challenged, cultured proximal tubule cells, we observed a positive correlation between HO-1 mRNA level and HO-1 release. In humans, 10 patients with AKI demonstrated markedly higher levels of plasma and urine HO-1 levels than 10 critically ill patients without AKI or 20 patients with CKD or ESRD. In summary, these data suggest that plasma and urinary HO-1 levels may serve as biomarkers of AKI and intrarenal HO-1 gene activity.

Renal cortical hemopexin accumulation in response to acute kidney injury.

Hemopexin (Hpx) is a liver-generated acute phase reactant that binds and neutralizes prooxidant free heme. This study tested whether acute kidney injury (AKI) triggers renal Hpx accumulation, potentially impacting heme Fe-mediated tubular injury. Mice were subjected to glycerol, cisplatin, ischemia-reperfusion (I/R), or endotoxemic [lipopolysaccharide (LPS)] AKI. In each instance, 3- to 30-fold renal cortical and isolated proximal tubule segment (PTS) Hpx increases resulted. Although renal cortex and PTS showed variable Hpx mRNA increases, due, in part, to increased mRNA stability, mRNA levels did not correlate with renal Hpx protein accumulation. Conversely, AKI evoked three- to fourfold increases in hepatic Hpx gene induction, which corresponded with three- to fourfold plasma Hpx increases. Renal immunohistochemistry, and increased urinary Hpx excretion, indicated that circulating Hpx gains tubule luminal/urinary access, followed by proximal tubule endocytic uptake. Paradoxically, in cultured renal cells (HK-2, HEK-293), Fe depletion, and not free heme excess, increased Hpx mRNA. LPS acutely increased HK-2 cell Hpx mRNA. This finding, coupled with observations that LPS evoked ∼30-fold greater renal Hpx mRNA increases than any other AKI model, suggests that inflammation, not heme exposure, activates the renal Hpx gene. Each form of AKI evoked early increases in circulating free heme, which subsequently fell to subnormal levels as plasma Hpx rose. In addition, purified Hpx blunted free Fe-mediated HK-2 cell death. In sum, these data indicated that AKI-associated hepatic stress generates Hpx, which gains renal tubule access. Given its ability to bind free heme and mitigate free Fe toxicity, Hpx loading can potentially confer cytoprotective effects.

Proximal tubule haptoglobin gene activation is an integral component of the acute kidney injury "stress response".

Haptoglobin (Hp) synthesis occurs almost exclusively in liver, and it is rapidly upregulated in response to stress. Because many of the pathways that initiate hepatic Hp synthesis are also operative during acute kidney injury (AKI), we tested whether AKI activates the renal cortical Hp gene. CD-1 mice were subjected to six diverse AKI models: ischemia-reperfusion, glycerol injection, cisplatin nephrotoxicity, myoglobinuria, endotoxemia, and bilateral ureteral obstruction. Renal cortical Hp gene induction was determined either 4-72 h or 1-3 wk later by measuring Hp mRNA and protein levels. Relative renal vs. hepatic Hp gene induction during endotoxemia was also assessed. Each form of AKI induced striking and sustained Hp mRNA increases, leading to ∼10- to 100-fold renal Hp protein elevations (ELISA; Western blot). Immunohistochemistry, and isolated proximal tubule assessments, indicated that the proximal tubule was the dominant (if not only) site of the renal Hp increases. Corresponding urinary and plasma Hp elevations were surrogate markers of this response. Endotoxemia evoked 25-fold greater Hp mRNA increases in kidney vs. liver, indicating marked renal Hp gene reactivity. Clinical relevance of these findings was suggested by observations that urine samples from 16 patients with established AKI had statistically higher (∼12×) urinary Hp levels than urine samples from either normal subjects or from 15 patients with chronic kidney disease. These AKI-associated urinary Hp increases mirrored those seen for urinary neutrophil gelatinase-associated lipoprotein, a well accepted AKI biomarker gene. In summary, these studies provide the first evidence that AKI evokes rapid, marked, and sustained induction of the proximal tubule Hp gene. Hp's known antioxidant, as well as its protean pro- and anti-inflammatory, actions imply potentially diverse effects on the evolution of acute tubular injury.

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.

Acute unilateral ischemic renal injury induces progressive renal inflammation, lipid accumulation, histone modification, and "end-stage" kidney disease.

There is an emerging concept in clinical nephrology that acute kidney injury (AKI) can initiate chronic kidney disease (CKD). However, potential mechanisms by which this may occur remain elusive. Hence, this study tested the hypotheses that 1) AKI triggers progressive activation of selected proinflammatory genes, 2) there is a relative failure of compensatory anti-inflammatory gene expression, 3) proinflammatory lipid accumulation occurs, 4) these changes correspond with "gene-activating" histone acetylation, and 5) in concert, progressive renal disease results. CD-1 mice were subjected to 30 min of unilateral renal ischemia. Assessments were made 1 day, 1 wk, or 3 wk later. Results were contrasted to those observed in uninjured contralateral kidneys or in kidneys from normal mice. Progressive renal injury occurred throughout the 3-wk postischemic period, as denoted by stepwise increases in neutrophil gelatinase-associated lipocalin gene induction and ongoing histologic damage. By 3 wk postischemia, progressive renal disease was observed (massive tubular dropout; 2/3rds reduction in renal weight). These changes corresponded with progressive increases in proinflammatory cytokine/chemokine gene expression (MCP-1, TNF-α, TGF-ß1), a relative failure of anti-inflammatory enzyme/cytokine (heme oxygenase-1; IL-10) upregulation, and progressive renal lipid (cholesterol/triglyceride) loading. Stepwise increases in collagen III mRNA and collagen deposition (Sirius red staining) indicated a progressive profibrotic response. Postischemic dexamethasone treatment significantly preserved renal mass, indicating functional significance of the observed proinflammatory state. Progressive gene-activating H3 acetylation was observed by ELISA, rising from 5% at baseline to 75% at 3 wk. This was confirmed by chromatin immunoprecipitation assay of target genes. In sum, these results provide experimental support for the clinical concept that AKI can trigger CKD, this is partially mediated by progressive postischemic inflammation, ongoing lipid accumulation results (potentially evoking "lipotoxicity"), and increasing histone acetylation at proinflammatory/profibrotic genes may contribute to this self-sustaining injury-promoting state.

Renal cortical albumin gene induction and urinary albumin excretion in response to acute kidney injury.

This study evaluated the potential utility of albuminuria as a "biomarker" of acute kidney injury (AKI) and tested whether AKI induces renal expression of the normally silent albumin gene. Urine albumin concentrations were measured in mice with five different AKI models (maleate, ischemia-reperfusion, rhabdomyolysis, endotoxemia, ureteral obstruction). Albumin gene induction in renal cortex, and in antimycin A-injured cultured proximal tubular cells, was assessed (mRNA levels; RNA polymerase II binding to the albumin gene). Albumin's clinical performance as an AKI biomarker was also tested (29 APACHE II-matched intensive care unit patients with and without AKI). Results were contrasted to those obtained for neutrophil gelatinase-associated lipocalin (NGAL), an established "AKI biomarker" gene. The experimental and clinical assessments indicated albumin's equivalence to NGAL as an AKI biomarker (greater specificity in experimental AKI; slightly better receiver-operating curve in humans). Furthermore, experimental AKI markedly induced the albumin gene (mRNA/RNA polymerase II binding increases; comparable to those seen for NGAL). Albumin gene activation in patients with AKI was suggested by fivefold increases in RNA polymerase II binding to urinary fragments of the albumin gene (vs. AKI controls). Experimental AKI also increased renal cortical mRNA levels for α-fetoprotein (albumin's embryonic equivalent). A correlate in patients was increased urinary α-fetoprotein excretion. We conclude that AKI can unmask, in the kidney, the normally silent renal albumin and α-fetoprotein genes. In addition, the urinary protein data independently indicate that albuminuria, and perhaps α-fetoprotein, have substantial utility as biomarkers of acute tubular injury.

Progressive histone alterations and proinflammatory gene activation: consequences of heme protein/iron-mediated proximal tubule injury.

Rhabdomyolysis (Fe)-induced acute renal failure (ARF) causes renal inflammation, and, with repetitive insults, progressive renal failure can result. To gain insights into these phenomena, we assessed the impact of a single episode of glycerol-induced rhabdomyolysis on proinflammatory/profibrotic [TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta1 (TGF-beta1)] gene expression and the time course of these changes. CD-1 mice were studied 1-7 days after glycerol injection. Normal mice served as controls. RNA polymerase II (Pol II) binding to the TNF-alpha, MCP-1, and TGF-beta1 genes, "gene-activating" histone modifications [histone 3 lysine 4 (H3K4) trimethylation (H3K4m3) and histone 2 variant H2A.Z], and cognate mRNA levels were assessed. Results were contrasted to changes in anti-inflammatory heme oxygenase-1 (HO-1). Glycerol produced severe ARF (blood urea nitrogen approximately 150-180 mg/dl) followed by marked improvement by day 7 (blood urea nitrogen approximately 40 mg/dl). Early increases in TNF-alpha, MCP-1, and TGF-beta1 mRNAs, Pol II gene binding, and H3K4m3/H2A.Z levels were observed. These progressed with time, despite resolution of azotemia. Comparable early HO-1 changes were observed. However, HO-1 mRNA normalized by day 7, and progressive Pol II binding/histone alterations did not occur. Fe-mediated injury to cultured proximal tubule (HK-2) cells recapitulated these in vivo results. Hence, this in vitro model was used for mechanistic assessments. On the basis of these studies, it was determined that 1) the H3K4m3/H2A.Z increases are early events (i.e., they precede mRNA increases), 2) subsequent mRNA elevations reflect transcription, not mRNA stabilization (actinomycin D assessments), and 3) increased transcription, per se, helps sustain elevated H2A.Z levels. We conclude that 1) Fe/glycerol-induced tubular injury causes sustained proinflammatory gene activation, 2) decreasing HO-1 expression, as reflected by mRNA levels, may facilitate this proinflammatory state, and 3) gene-activating histone modifications are early injury events and progressively increase at selected proinflammatory genes. Thus they may help sustain a proinflammatory state, despite resolving ARF.