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. 2011 Jan;22(1):165-75.
doi: 10.1681/ASN.2010060641. Epub 2010 Nov 11.

MCP-1 gene activation marks acute kidney injury

Raj Munshi  1 , Ali Johnson, Edward D Siew, T Alp Ikizler, Lorraine B Ware, Mark M Wurfel, Jonathan Himmelfarb, Richard A Zager
Affiliations

MCP-1 gene activation marks acute kidney injury

Raj Munshi et al. J Am Soc Nephrol. 2011 Jan.

Abstract

Monocyte chemoattractant protein 1 (MCP-1) mediates acute ischemic and toxic kidney injury, but whether this can be used as a biomarker of acute kidney injury (AKI) is unknown. We obtained kidney and urine samples from mice with intrarenal (maleate), prerenal (endotoxemia), or postrenal (ureteral obstruction) injury. We also studied the independent effects of uremia without concomitant kidney injury by performing bilateral ureteral transection in mice. Additionally, we obtained urine samples from APACHE II-matched critically ill patients with or without advancing azotemia (n = 10 in each group). We assayed selected samples for MCP-1, MCP-1 mRNA, and for an activating histone mark (H3K4m3) at urinary fragments of the MCP-1 gene and contrasted the results with those obtained for neutrophil gelatinase-associated lipocalin (NGAL), a comparator "AKI biomarker" gene. Maleate increased urinary MCP-1 protein and mRNA more than the corresponding increases in NGAL. Endotoxemia and ureteral obstruction also increased NGAL and MCP-1 gene expression. Uremia, in the absence of renal injury, induced the NGAL gene, but not MCP-1, suggesting the possibility of better specificity of MCP-1 for AKI. Clinical assessments supported the utility of MCP-1 as a biomarker (e.g., nonoverlapping concentrations of urinary MCP-1 in patients with and without AKI). Elevated levels of urinary MCP-1 mRNA and levels of H3K4m3 at the MCP-1 gene supported MCP-1 gene activation in patients with renal injury. In conclusion, these data suggest that MCP-1 has potential as a biomarker of AKI and provide "proof of concept" that urinary histone assessments provide mechanistic insight among patients with kidney disease.

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Figures

Figure 1.
Figure 1.
Maleate induces azotemia and increases urinary and plasma NGAL and MCP-1 levels within 4 hours of injection. Renal injury 4 hours after maleate injection was assessed by BUN, NGAL, and MCP-1 concentrations. Significant azotemia was already apparent at 4 hours after maleate (MAL) injection, versus controls (C) with corresponding increases in plasma NGAL and MCP-1 concentrations. This injury was associated with massive increases in urinary NGAL and MCP-1 concentrations, indicating their extreme sensitivity as early AKI biomarkers. Plasma NGAL and MCP-1 protein levels were also markedly elevated, indicating increased synthesis and possibly a reduction in glomerular filtration.
Figure 2.
Figure 2.
Maleate increases NGAL and MCP-1 gene expression within 4 hours of injection. NGAL and MCP-1 mRNAs were assessed in renal cortex and in urine at 4 hours after maleate injection. By 4 hours after maleate injection, marked increases in renal cortical NGAL and MCP-1 mRNAs were apparent (indicating that the increase in urine NGAL and MCP-1 protein levels likely reflected, at least in part, renal production). There were corresponding threefold and ninefold increases in urinary NGAL and MCP-1 mRNAs, compared with control values. However, there was also a doubling of the mRNA for GAPDH, a housekeeping gene. The latter finding suggests that at least part of the urinary NGAL and MCP-1 mRNA increases reflected nonspecific tubular cell sloughing.
Figure 3.
Figure 3.
Endotoxemia, ureteral obstruction, and bilateral ureteral transection affect MCP-1 and NGAL gene expression. NGAL and MCP-1 gene expression were assessed in mouse renal cortex after induction of either a prerenal form of ARF (endotoxemia “LPS”), postrenal ARF (unilateral ureteral obstruction, UUO), or the induction of uremia in the presence of structurally normal kidneys (BUTx). Both LPS and UUO caused marked increases in renal cortical NGAL and MCP-1 mRNAs (assessed at 2 and 18 hours after LPS and UUO). Uremia (BUN 14 ± 14 mg/dl), as induced by BUTx, caused a massive increase in NGAL mRNA. Conversely, BUTx did not impact MCP-1 mRNA expression.
Figure 4.
Figure 4.
Clinical acute kidney injury increases plasma NGAL and MCP-1 levels. Serum creatinine, serum MCP-1, and serum NGAL values were assessed for individuals within the three test clinical groups. (C = normal individuals; AKI− = ICU patients without ARF; AKI+ = ICU patients with ARF. As expected, the serum creatinines were significantly elevated in the AKI+ group compared with the AKI− group (mean values of 3.8 versus 1.0 mg/dl, respectively). Serum MCP-1 values did not significantly differ between the three groups (although the trend was clearly toward increasing MCP-1 concentrations from controls → to AKI− → AKI+). No significant correlation was observed between serum MCP-1 and serum creatinine concentrations. However, in the case of NGAL, a significant correlation did exist between serum NGAL and serum creatinine (r = 0.5; P < 0.05) concentrations. However, no overt statistical difference between the means for the three groups was observed. Solid horizontal lines = mean values.
Figure 5.
Figure 5.
Clinical acute kidney injury increases urinary NGAL and MCP-1 protein levels. Urinary MCP-1 and NGAL concentrations were measured in the control, AKI−, and AKI+ groups. There was a complete separation (no overlap) of urine MCP-1 concentrations between the AKI− and AKI+ groups (<0.01). Conversely, significant overlap in urinary NGAL values for the AKI− and AKI+ groups was observed. Despite this overlap, the mean NAGL urine values for these two groups were statistically different (P < 0.03). The solid horizontal lines reflect mean values.
Figure 6.
Figure 6.
Clinical acute kidney injury increases urinary NGAL and MCP-1 protein levels, as assessed following log transformation and factoring by urine creatinine. Log base 10 conversions of urinary MCP-1 and NGAL concentrations for the three study groups were assessed. Because the absolute NGAL and MCP-1 concentrations did not have a Gaussian distribution, the values were converted to log base 10. The values were then factored by corresponding urine creatinine (Cr) values. As shown, using these corrected values, MCP-1 showed less overlap between the AKI+ and AKI− groups, versus substantial overlap with the converted NGAL values. It is noteworthy that even the AKI− patients showed a trend toward higher NGAL and MCP-1 values, compared with the normal control group, suggesting that even the AKI− group had sustained subclinical renal injury.
Figure 7.
Figure 7.
Clinical acute kidney injury increases urinary protein concentrations. Total urine protein concentrations as determined by the pyrogallol red-molybdate assay method were assessed with the results factored by the corresponding urine creatinine concentrations. As shown, there was no statistical difference between the control and AKI− patient groups. However, an average tenfold increase in urine protein concentrations were observed between the AKI− and AKI+ groups with only two AKI− patients showing overlap with the AKI+ values.
Figure 8.
Figure 8.
Clinical acute kidney injury increases urinary concentrations of NGAL, MCP-1, and BRG-1 mRNAs. Band densities for MCP-1, NGAL, and BRG-1 mRNAs were determined in urines obtained from the three clinical groups (controls, C; AKI− and AKI+). Statistically significant differences were observed for MCP-1 and BRG-1 mRNAs between the AKI− and AKI+ groups. Conversely, no significant differences for urinary NGAL mRNA levels were observed.
Figure 9.
Figure 9.
Clinical acute kidney injury can increase the urinary concentrations of GAPDH mRNA, actin mRNA, and 18s rRNA. Urinary mRNA levels were determined for two housekeeping genes (GAPDH and β-actin) and for 18s rRNA, used an RNA reference for nonspecific RNA sloughing). As can be seen, although there were no significant differences among the three groups, there was a trend toward increasing housekeeping RNA excretion into urine across the three patient groups (AKI+ > AKI− > controls, albeit NS). This suggests that at least some of the mRNA differences shown in Figure 8 may have reflected nonspecific RNA sloughing.
Figure 10.
Figure 10.
Detection of H3K4m3 in urine from patients with acute kidney injury. Amounts of H3K4m3 recovered in urine that were specifically associated with exon 1 of the MCP-1 and NGAL genes were assessed. The values were factored by simultaneously obtained amounts of H3K4m3 at exon 1 of β-actin. The AKI+ patients manifested highly significant increases in H3K4m3 at both the MCP-1 and the NGAL genes. However, even the AKI− negative patients showed a trend toward increased H3K4m3 at both genes, consistent with intermediate gene activation, compared with the AKI− and normal volunteer groups.
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