Richard A. Zager

Acute Renal Failure Following Bone Marrow Transplantation: A Retrospective Study of 272 Patients

To assess the incidence, risk factors, and course of acute renal failure (ARF) following bone marrow transplantation (BMT), a retrospective analysis of 272 patients receiving transplants at the Fred Hutchinson Cancer Research Center during 1986 was undertaken. The patients were divided into three groups: group 1, hemodialysis requiring ARF; group 2, mild renal insufficiency (doubling of serum creatinine, Scr, but no dialysis); group 3, relatively normal post-BMT renal function (no doubling of Scr). Fifty-three percent of patients at least doubled their Scr (Groups 1 and 2), and 24% required dialysis. The degree of renal functional impairment had a dramatic impact on patient mortality rates (84%, 37%, and 17% in groups 1, 2, and 3, respectively). Jaundice (bilirubin greater than or equal to 2.0 mg/dL), weight gain (greater than or equal to 2.0 kg), amphotericin B use, and a pretransplant Scr greater than or equal to 0.7 mg/dL were independently associated with the subsequent development of dialysis-requiring ARF (P less than 0.001; relative risks, 3.0 to 7.7). Neither aminoglycoside/vancomycin/cyclosporine A use nor acute graft v host disease correlated with the development of ARF. A mismatched graft was a significant risk factor for ARF by univariate but not by multivariate analysis. Within 48 hours before doubling the Scr, 63% of group 1 patients had positive blood cultures and 39% developed hypotension. Of the 26 group 1 patients who had urine Na concentrations measured, 85% had values less than or equal to 40 mEq/L. Autopsy kidney specimens provided no clear explanation for ARF in the vast majority of patients in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)

assessment of Low-flow Sevoflurane and Isoflurane Effects on Renal Function Using Sensitive Markers of Tubular Toxicity

Background: Carbon dioxide absorbents degrade sevoflurane, particularly at low gas flow rates, to fluoromethyl‐2,2‐difluoro‐1‐(trifluoromethyl)vinyl ether (compound A). Compound A causes renal proximal tubular injury in rats but has had no effect on blood urea nitrogen (BUN) or creatinine concentrations in patients. This investigation compared the effects of low‐flow sevoflurane and isoflurane on renal tubular function in surgical patients using conventional (BUN and creatinine) and finer indices of renal injury, specifically those biomarkers sensitive for compound A toxicity in rats (glucosuria, proteinuria, and enzymuria [N‐acetyl‐beta‐D‐glucosaminidase (NAG) and alpha‐glutathione‐S‐transferase (alpha GST)]). Methods: Consenting patients with normal preoperative renal function at two institutions were randomized to receive sevoflurane (n = 36) or isoflurane (n = 37) in oxygen and air. Total gas flow was 1 l/min, opioid doses were minimized, and barium hydroxide lime was used to maximize anesthetic degradation. Inspiratory and expiratory compound A concentrations were quantified every 30–60 min. Blood and urine were obtained before and 24–72 h after anesthesia for laboratory evaluation. Results: Sevoflurane and isoflurane groups were similar with respect to age, weight, sex, American Society of Anesthesiologists status, anesthetic duration (3.7 or 3.9 h), and anesthetic exposure (3.6 or 3 minimum alveolar concentration [MAC]‐hour). Maximum inspired compound A concentration (mean +/‐ standard deviation) was 27 +/‐ 13 ppm (range, 10–67 ppm). Areas under the inspired and expired compound A concentration versus time curves (AUC) were 79 +/‐ 54‐ppm‐h (range, 10–223 ppm‐h) and 53 +/‐ 40 ppm‐h (range, 6–159 ppm‐h), respectively. There was no significant difference between anesthetic groups in postoperative serum creatinine or BUN, or urinary excretion of protein, glucose, NAG, proximal tubular alpha GST, or distal tubular pi GST. There was no significant correlation between compound A exposure (AUC) and protein, glucose, NAG, alpha GST, or pi GST excretion. Postoperative alanine and aspartate aminotransferase concentrations were not different between the anesthetic groups, and there were no significant correlations between compound A exposure and alanine or aspartate aminotransferase concentrations. Conclusions: The renal tubular and hepatic effects of low‐flow sevoflurane and isoflurane were similar as assessed using both conventional measures of hepatic and renal function and more sensitive biochemical markers of renal tubular cell necrosis. Moderate duration low‐flow sevoflurane anesthesia, during which compound A formation occurs, appears to be as safe as low‐flow isoflurane anesthesia.

Combined mannitol and deferoxamine therapy for myohemoglobinuric renal injury and oxidant tubular stress. Mechanistic and therapeutic implications.

Mannitol (M) and deferoxamine (DFO) can each protect against myohemoglobinuric acute renal failure (MH-ARF). This study assessed M-DFO interactions during MH-ARF to help discern mechanisms of renal injury, and to define whether M + DFO exerts additive or synergistic antioxidant/cytoprotective effects. Rats subjected to the glycerol model of MH-ARF were treated with (a) M; (b) DFO; (c) M + DFO; or (d) no protective agents. Relative degrees of protection (24-h plasma urea/creatinine concentrations) were M + DFO greater than M greater than DFO greater than or equal to no therapy. To assess whether catalytic Fe is generated during MH-ARF, the bleomycin assay was applied to plasma/urine samples obtained 0-2 h post-glycerol injection. Although striking plasma and urinary increments were noted, excess renal hydroxyl radical (.OH) production was not apparent (gauged by the salicylate trap method). M increased catalytic Fe excretion (four times), whereas DFO eliminated its urinary (but not plasma) activity. To determine direct M/DFO effects on proximal tubular cell oxidant injury, isolated rat proximal tubular segments (PTS) were incubated with toxic dosages of FeSO4 or H2O2. Despite inducing cell injury (lactic dehydrogenase release), Fe caused no .OH production. DFO conferred dose-dependent cytoprotection, correlating with increased, not decreased, .OH generation. Although M scavenged this .OH excess, it had no additive or independent, protective effect. H2O2 cytotoxicity correlated with increased catalytic Fe (but not .OH) generation. The fact that DFO (but not .OH scavengers [M and dimethylthiourea]) blocked H2O2 toxicity implied Fe-dependent, .OH-independent cell killing. In conclusion, (a) striking catalytic Fe generation occurs during MH-ARF, but augmented intrarenal .OH production may not develop; (b) DFO can block Fe toxicity despite a prooxidant effect; (c) H2O2 PTS toxicity is Fe, but possibly not .OH, dependent; and (d) M does not mitigate oxidant PTS injury, either in the presence or absence of DFO, suggesting that its additive benefit with DFO in vivo occurs via a diuretic, not antioxidant effect.

Effects of inorganic iron and myoglobin on in vitro proximal tubular lipid peroxidation and cytotoxicity.

Recent in vivo studies suggest that heme Fe causes proximal tubular lipid peroxidation and cytotoxicity, thereby contributing to the pathogenesis of myoglobinuric (Mgb) acute renal failure. Because hydroxyl radical (.OH) scavengers [dimethylthiourea (DMTU), benzoate, mannitol] can mitigate this injury, it is postulated that .OH is a mediator of Mgb-induced renal damage. The present study has tested these hypotheses using an isolated rat proximal tubular segment (PTS) system. An equal mixture of Fe2+/Fe3+ (4 mM total), when added to PTS, caused marked cytotoxicity [as defined by lactate dehydrogenase (LDH) release] and lipid peroxidation [assessed by malondialdehyde (MDA) increments]. Fe2+ or Fe3+ alone each induced massive MDA elevations, but only Fe2+ caused cytotoxicity. Although both DMTU and benzoate decreased LDH release during the Fe2+/Fe3+ challenge, mannitol and GSH did not, despite equivalent reductions in .OH (gauged by the salicylate trap method). GSH and catalase (but not DMTU, benzoate, or mannitol) decreased MDA concentrations, suggesting the Fe-driven lipid peroxidation was more H2O2 than .OH dependent. Deferoxamine totally blocked Fe-induced LDH release, even under conditions in which it caused an apparent increase in .OH generation. Mgb paradoxically protected against Fe-mediated PTS injury, an effect largely reproduced by albumin. In conclusion, these data suggest that: (a) Fe can cause PTS lipid peroxidation and cytotoxicity by a non-.OH-dependent mechanism; (b) Fe-mediated cytotoxicity and lipid peroxidation are not necessarily linked; and (c) Mgb paradoxically protects PTS against Fe-mediated injury, suggesting that: (i) Mgb Fe may require liberation from its porphyrin ring before exerting toxicity; and (ii) the protein residue may blunt the resulting injury.

Renal ischemia-reperfusion injury upregulates histone-modifying enzyme systems and alters histone expression at proinflammatory/profibrotic genes

Ischemic renal injury can produce chronic renal inflammation and fibrosis. This study tested whether ischemia-reperfusion (I/R) activates histone-modifying enzyme systems and alters histone expression at selected proinflammatory/profibrotic genes. CD-1 mice were subjected to 30 min of unilateral I/R. Contralateral kidneys served as controls. At 1, 3, or 7 days of reflow, bilateral nephrectomy was performed. Renal cortices were probed for monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta1 (TGF-beta1), and collagen III mRNAs and cytokine levels. RNA polymerase II (Pol II) binding, which initiates transcription, was quantified at exon 1 of the MCP-1, TGF-beta1, collagen III genes (chromatin immunoprecipitation assay). Two representative gene-activating histone modifications [histone 3 lysine 4 (H3K4) trimethylation (m3) (H3K4m3); histone 2 variant H2A.Z] were sought. Degrees of binding of two relevant histone-modifying enzymes (Set1, BRG1) to target genes were assessed. Renal cortical Set1, BRG1, and H2A.Z mRNAs were measured. Finally, the potential utility of urinary mRNA concentrations as noninvasive markers of these in vivo processes was tested. I/R caused progressive increases in Pol II binding to MCP-1, TGF-beta1, and collagen III genes. Parallel increases in cognate mRNAs also were expressed. Progressive increases in renal cortical Set1, BRG1, H2A.Z mRNAs, and increased Set1/BRG1 binding to target genes occurred. These changes corresponded with: 1) progressive elevations of H3K4m3 and H2A.Z at each test gene; 2) increases in renal cortical TGF-beta1/MCP-1 cytokines; and 3) renal collagen deposition (assessed by histomorphology). Postischemic increases in urinary TGF-beta1, MCP-1, Set1, and BRG1 mRNAs were also observed. We conclude that: 1) I/R upregulates histone-modifying enzyme systems, 2) histone modifications at proinflammatory/profibrotic genes can result, and 3) urinary mRNA assessments may have utility for noninvasive monitoring of these in vivo events.

BRG1 Increases Transcription of Proinflammatory Genes in Renal Ischemia

Acute kidney injury stimulates renal production of inflammatory mediators, including TNF-alpha and monocyte chemoattractant protein 1 (MCP-1). These responses reflect, in part, injury-induced transcription of proinflammatory genes by proximal tubule cells. Because of the compact structure of chromatin, a series of events at specified loci remodel chromatin to provide access for transcription factors and RNA polymerase II (Pol II). Here, we examined the role of Brahma-related gene-1 (BRG1), a chromatin remodeling enzyme, in the transcription of TNF-alpha and MCP-1 in response to renal ischemia. Two hours after renal ischemic injury in mice, renal TNF-alpha and MCP-1 mRNA increased and remained elevated for at least 1 wk. Matrix chromatin immunoprecipitation assays revealed sustained increases in Pol II at these genes, suggesting that the elevated mRNA levels were, at least in part, transcriptionally mediated. The profile of BGR1 binding to the genes encoding TNF-alpha and MCP-1 resembled Pol II recruitment. Knockdown of BRG1 by small interfering RNA blocked an ATP depletion-induced increase in TNF-alpha and MCP-1 transcription in a human proximal tubule cell line; this effect was associated with decreased recruitment of BRG1 and Pol II to these genes. In conclusion, BRG1 promotes increased transcription of TNF-alpha and MCP-1 by the proximal tubule in response to renal ischemia.

Acute Tubular Injury Causes Dysregulation of Cellular Cholesterol Transport Proteins

Acute renal injury causes accumulation of free and esterified cholesterol (FC, CE) in proximal tubules, mediated, at least in part, by increased cholesterol synthesis. Normally, this would trigger compensatory mechanisms such as increased efflux and decreased influx to limit or reverse the cholesterol overload state. This study sought to determine the integrity of these compensatory pathways following acute renal damage. Rhabdomyolysis-induced acute renal failure was induced in mice by glycerol injection. Normal mice served as controls. After 18 hours, BUN levels and renal cortical FC/CE content were determined. Expression of ABCA-1 and SR-B1 (cholesterol efflux proteins) were assessed by Western blot. Renal cortical LDL receptor (LDL-R; a cholesterol importer) regulation was gauged by quantifying its mRNA. To obtain proximal tubule cell-specific data, the impact of oxidant (Fe) stress on cultured HK-2 cell LDL-R, SR-B1, and ABCA-1 proteins and their mRNAs (versus controls) was assessed. Glycerol evoked marked azotemia and striking FC/CE increments (44%, 384%, respectively). Paradoxically, renal cortical SR-B1 and ABCA-1 protein reductions and LDL-R mRNA increments resulted. Fe-induced injury suppressed HK-2 cell SR-B1, ABCA-1, and their mRNAs. LDL-R protein rose with the in vitro Fe challenge. Renal tubular cell injury causes dysregulation of SR-B1, ABCA-1, and LDL-R protein expression, changes which should contribute to a cholesterol overload state. Reductions in HK-2 cell SR-B1 and ABCA-1 mRNAs and increases in renal cortical LDL-R mRNA imply that this dysregulation reflects, at least in part, altered genomic/transcriptional events.

Renal Cortical Lactate Dehydrogenase: A Useful, Accurate, Quantitative Marker of In Vivo Tubular Injury and Acute Renal Failure

Studies of experimental acute kidney injury (AKI) are critically dependent on having precise methods for assessing the extent of tubular cell death. However, the most widely used techniques either provide indirect assessments (e.g., BUN, creatinine), suffer from the need for semi-quantitative grading (renal histology), or reflect the status of residual viable, not the number of lost, renal tubular cells (e.g., NGAL content). Lactate dehydrogenase (LDH) release is a highly reliable test for assessing degrees of in vitro cell death. However, its utility as an in vivo AKI marker has not been defined. Towards this end, CD-1 mice were subjected to graded renal ischemia (0, 15, 22, 30, 40, or 60 min) or to nephrotoxic (glycerol; maleate) AKI. Sham operated mice, or mice with AKI in the absence of acute tubular necrosis (ureteral obstruction; endotoxemia), served as negative controls. Renal cortical LDH or NGAL levels were assayed 2 or 24 hrs later. Ischemic, glycerol, and maleate-induced AKI were each associated with striking, steep, inverse correlations (r, −0.89) between renal injury severity and renal LDH content. With severe AKI, >65% LDH declines were observed. Corresponding prompt plasma and urinary LDH increases were observed. These observations, coupled with the maintenance of normal cortical LDH mRNA levels, indicated the renal LDH efflux, not decreased LDH synthesis, caused the falling cortical LDH levels. Renal LDH content was well maintained with sham surgery, ureteral obstruction or endotoxemic AKI. In contrast to LDH, renal cortical NGAL levels did not correlate with AKI severity. In sum, the above results indicate that renal cortical LDH assay is a highly accurate quantitative technique for gauging the extent of experimental acute ischemic and toxic renal injury. That it avoids the limitations of more traditional AKI markers implies great potential utility in experimental studies that require precise quantitation of tubule cell death.

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.

Rapid Renal Alpha-1 Antitrypsin Gene Induction in Experimental and Clinical Acute Kidney Injury

Alpha-1-antitrypsin (AAT) is a hepatic stress protein with protease inhibitor activity. Recent evidence indicates that ischemic or toxic injury can evoke selective changes within kidney that resemble a hepatic phenotype. Hence, we tested the following: i) Does acute kidney injury (AKI) up-regulate the normally renal silent AAT gene? ii) Does rapid urinary AAT excretion result? And iii) Can AATs anti-protease/anti-neutrophil elastase (NE) activity protect injured proximal tubule cells? CD-1 mice were subjected to ischemic or nephrotoxic (glycerol, maleate, cisplatin) AKI. Renal functional and biochemical assessments were made 4–72 hrs later. Rapidly following injury, 5–10 fold renal cortical and isolated proximal tubule AAT mRNA and protein increases occurred. These were paralleled by rapid (>100 fold) increases in urinary AAT excretion. AKI also induced marked increases in renal cortical/isolated proximal tubule NE mRNA. However, sharp NE protein levels declines resulted, which strikingly correlated (r, −0.94) with rising AAT protein levels (reflecting NE complexing by AAT/destruction). NE addition to HK-2 cells evoked ∼95% cell death. AAT completely blocked this NE toxicity, as well as Fe induced oxidant HK-2 cell attack. Translational relevance of experimental AAT gene induction was indicated by ∼100–1000 fold urinary AAT increases in 22 AKI patients (matching urine NGAL increases). We conclude: i) AKI rapidly up-regulates the renal cortical/proximal tubule AAT gene; ii) NE gene induction also results; iii) AAT can confer cytoprotection, potentially by blocking/reducing cytotoxic NE accumulation; and iv) marked increases in urinary AAT excretion in AKI patients implies clinical relevance of the AKI- AAT induction pathway.