Acute tubular injury causes dysregulation of cellular cholesterol transport proteins

Abstract

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.

Figure 1.

Free cholesterol (FC) and cholesteryl ester (CE) levels in renal cortex from control (C) mice and from mice 18 hours post-induction of glycerol mediated acute renal failure (Gly). Striking FC and CE levels were apparent following glycerol treatment, compared to controls (n = 7 for each determination).

Figure 2.

SR-B1 and ABCA-1 levels in renal cortical samples obtained from control (Cont) mice and from mice 18 hours post-induction of glycerol (Gly)-induced renal failure. As shown in the left panel, there was a marked reduction in SR-B1 in the glycerol-treated mice (n = 6), compared to controls (n = 5). As shown in the right panel, reductions in ABCA-1 bands were also observed (n = 12 for both the control and glycerol-treated group). SR-B1 appeared as a single dense band at 82 kd. In contrast, there was much weaker expression of ABCA-1, assuming a characteristic triplet appearance at ∼220 kd. Each of the three bands were diminished in the glycerol group. (Note the different y axis scales for ABCA-1 and SR-B1, reflecting much lesser apparent expression for the former versus the latter protein.)

Figure 3.

SR-B1 and ABCA-1 in HK-2 cell extracts obtained 4 hours following the addition of an Fe challenge. Paralleling the changes observed in renal cortex, significant reductions in each protein were observed in the Fe-challenged cells, compared to the controls (Cont) (n = 6 separate cell preparations for each of the 4 groups). As with the renal cortical samples, ABCA-1 expression was, in general, remarkably lower than SR-B1, reflecting the fact that low levels of ABCA-1 exist in non-steroidogenic tissues.

Figure 4.

SR-B1 and ABCA-1 in HK-2 cell extracts obtained 18 hours following the addition of an Fe challenge. The 18-hour results mimicked those seen at 4 hours, with significant reductions in SR-B1 and ABCA-1 (n = 6 per group).

Figure 5.

LDL-R protein expression in HK-2 cells 18 hours following the addition of an Fe challenge. LDL-R appears as a major (nonglycosylated) and a minor (glycosylated) band at 120 and 160 kd, respectively. There was greater expression of the dominant band in the Fe-treated cells versus the control group (P < 0.005; n = 5 per group).

Figure 6.

mRNA levels for ABCA-1, SR-B1, and LDL-R in HK-2 cells at either 4 or 18 hours post Fe-induced injury, as determined by multiplexing RT-PCR. The value for each mRNA (x) was factored by simultaneously obtained GAPDH values (x/GAPDH; y axis). Significant depressions in ABCA-1 and LDL-R mRNAs were apparent at both 4 and 18 hours post-Fe addition, compared to controls. In the case of SR-B1, a significant reduction was apparent in the Fe-challenged cells, but only at the 18-hour time point. (4-hour time points, n = 8 samples for each; 18-hour time points, n = 15 determinations for each).

Figure 7.

mRNA levels for ABCA-1, SR-B1, and LDL-R in control HK-2 cells and in HK-2 cells following 18 hours of HMG CoA reductase inhibition with mevastatin treatment (statin). The value of each mRNA (x) was factored by the simultaneously obtained GAPDH values (x/GAPDH). Statin treatment caused a marked reduction in ABCA-1 mRNA and a modest but highly significant reduction in SR-B1 mRNA. In contrast, LDL-R mRNA values manifested a highly significant increase in response to statin treatment. Each of these statin-mediated responses were judged to be biologically appropriate for a statin-induced cholesterol reduction state (n = 4 determinations for each depicted group).