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. 2011 Dec;301(6):F1334-45.
doi: 10.1152/ajprenal.00431.2011. Epub 2011 Sep 14.

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

Richard A Zager  1 , Ali C M Johnson, Kirsten Becker
Affiliations

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

Richard A Zager et al. Am J Physiol Renal Physiol. 2011 Dec.

Abstract

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-β1), 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.

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Figures

Fig. 1.
Fig. 1.
Kidney weights following unilateral ischemic injury. Following left renal ischemia, the right kidney slowly increased in weight, reaching statistical significance by the 3-wk time point (P < 0.01 vs. baseline kidney values; consistent with compensatory hypertrophy). The postischemic kidney showed an initial increase in weight at 1 day postischemia (due to edema), but then progressively decreased weight, such that by 3 wk, a 2/3rds renal weight reduction was apparent (right: consistent with progressivep fibrosis/atrophy). 1d, 1 day postischemia; 1, 3 wk, weeks postischemia.
Fig. 2.
Fig. 2.
Renal histologic hematoxylin and eosin (H&E) sections from normal kidneys, 1 day postischemia and 3 wk postischemia. A: normal kidney section demonstrating histology of renal cortex and part of outer medulla. B: normal kidney outer medulla. C: outer medulla at 1 day postischemia. Cast formation and tubular necrosis are seen at the cortical/outer medullary junction but with preservation of most of the cortical tubular segments. D: outer medullary stripe at 1 day postischema. Widespread tubular necrosis and cast formation are apparent. E: renal cortex at 3 wk postischemia. A marked cellular interstitial infiltrate with massive tubular dropout and periglomerular fibrosis are apparent. F: outer medulla at 3 wk postischemia. As with the cortex, there is a marked cellular interstitial infiltrate, tubular dropout, and debris/casts in lumina of remaining tubules.
Fig. 3.
Fig. 3.
Sirius red staining for collagen. Kidney sections were obtained at 3 wk postischemia (A) and examined under polarized light. The postischemic kidney demonstrates interstitial collagen deposition, as evidenced by the deposition of yellow and red staining strands laced between tubules. This gave a “chicken wire” appearance. In addition, extensive tubular dilatation and marked amounts of tubular cell debris within tubular lumina were apparent. In contrast, a normal kidney section (B) showed only a minimal amount of collagen between the tubules. As a “positive internal control,” the presence of collagen in the adventitia of a medium-sized blood vessel is apparent.
Fig. 4.
Fig. 4.
Neutrophil gelatinase-associated lipocalin (NGAL) expression postischemia. Renal cortical NGAL mRNA levels (left) progressively increased postischemia, compared with values observed in contralateral kidneys (all contralateral kidney values depicted as a single group for ease of presentation). This was associated with a marked increase in renal cortical NGAL protein levels (middle), as assessed at the 3-wk time point. CL, contralateral kidney values (which did not significantly differ from values observed in normal kidneys). Right: corresponding with the increase in renal cortical NGAL protein was a corresponding increase in plasma NGAL concentrations, compared with control plasma.
Fig. 5.
Fig. 5.
Renal cortical TNF-α, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor (TGF)-β1 mRNAs following unilateral renal ischemia. By 1 day postischemia, significant increases in all 3 mRNAs were apparent, compared with values in the nonischemic CL kidneys. Further increases in each were observed at the 1-wk time point. TGF-β1 mRNA levels continued to increase throughout the course of the experiment. The contralateral kidney values did not significantly differ over time, and these results are presented as a single group. P values were derived by comparing postischemic vs. contralateral kidney values at each time point.
Fig. 6.
Fig. 6.
MCP-1 and TGF-β1 protein levels in renal cortex and plasma after unilateral ischemia. Renal cortical MCP-1 and TGF-β1 protein levels were measured at 3 wk in the postischemic and CL kidneys, and dramatic increases in both protein levels were observed (left). There were associated increases in plasma MCP-1 and TGF-β1 levels (compared to values in normal mouse plasma), suggesting renal efflux of these cytokines into the systemic circulation.
Fig. 7.
Fig. 7.
Collagen III and HMG CoA reductase mRNA levels postischemia. These 2 mRNAs were measured in postischemic and CL kidneys at 1 day, 1 wk, and 3 wk postischemia. Progressive increases in both mRNA levels were observed over time. The CL kidney results did not significantly differ over time, and these results are presented as a single group. P values were derived by comparing postischemic vs. contralateral kidney values at each time point.
Fig. 8.
Fig. 8.
Free cholesterol, esterified cholesterol, and total triglyceride levels in renal cortex postischemia. By 1 wk postischemia, an ∼35% increase in free cholesterol levels were observed (left), compared with values observed in the nonischemic CL kidneys. These elevated levels remained relatively stable thereafter. Conversely, cholesterol ester levels continued to increase over time, such that by 3 wk an ∼20-fold increase was observed, compared with the values seen in CL kidneys (middle). Triglyceride levels were significantly elevated at 1 day postischemia, further increases were seen by 1 wk, and they remained elevated at the 3-wk time point. The CL kidney results did not differ significantly over time for any of these measurements, and, thus, the CL kidney results are presented as a single group. P values were derived by comparing postischemic vs. CL kidney results at each time point.
Fig. 9.
Fig. 9.
Heme oxygenase 1 (HO-1) mRNA and IL-10 mRNA levels postischemia. Both mRNAs were significantly elevated by 1 day postischemia. HO-1 mRNA remained elevated over the course of the experiments, albeit at just 50% of the 1-day value. Conversely, IL-10 mRNA levels manifested further increases from 1 day vs. 1 wk postischemia. The CL kidney results did not differ over time, and these results are presented as a single group. P values were derived from comparing ischemic vs. contralateral kidney values at each time point.
Fig. 10.
Fig. 10.
Renal cortical HO-1 and IL-10 protein levels postischemia. Both HO-1 and IL-10 protein levels were markedly elevated at 1 day postischemia. At 1 and 3 wk, the HO-1 levels remained elevated, albeit at just 50% of the 1-day values. Thus, HO-1 protein expression almost exactly paralleled its cognate mRNA. In contrast, IL-10 levels manifested a drastic fall at 1 wk postischemia, and by 3 wk postischemia, the values were significantly less than those observed in normal or CL kidneys. The CL kidney results did not significantly differ over time, and these results are presented as a single group. P values were derived from ischemic vs. CL kidneys at each time point.
Fig. 11.
Fig. 11.
Percent histone 3 (H3) acetylation in normal kidneys, in postischemic kidneys, and in CL controls. Nonacetylated and acetylated H3 values were measured by independent ELISAs, and the relative % acetylation was calculated. No increase in acetylation was observed in the CL (contralat) kidneys, compared with values in normal (control) kidneys. Conversely, the postischemic kidneys manifested a dramatic increase in % H3 acetylation at 1 day postischemia and by 3 wk postischemia, ∼75% of total H3 was in an acetylated form.
Fig. 12.
Fig. 12.
Effect of dexamethasone (Dex) treatment on postischemic-reperfusion (I/R) cytokine expression and renal mass reduction, as assessed by renal weight. Cytokine mRNAs were measured at 3-day postunilateral ischemic injury with and without Dex treatment. Renal weights at 3 wk postischemia with and without Dex treatment were also assessed. Dex lowered postischemic cytokine expression and conferred partial protection against renal injury, as assessed by a relative preservation of kidney weight at the 3-wk time point. I/R, ischemia-reperfusion.
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