Changes in free and esterified cholesterol: hallmarks of acute renal tubular injury and acquired cytoresistance

Abstract

Acute tubular cell injury is accompanied by plasma membrane phospholipid breakdown. Although cholesterol is a dominant membrane lipid which interdigitates with, and impacts, phospholipid homeostasis, its fate during the induction and recovery phases of acute renal failure (ARF) has remained ill defined. The present study was performed to ascertain whether altered cholesterol expression is a hallmark of evolving tubular damage. Using gas chromatographic analysis, free cholesterol (FC) and esterified cholesterol (CE) were quantified in: 1) isolated mouse proximal tubule segments (PTS) after 30 minutes of hypoxic or oxidant (ferrous ammonium sulfate) injury; 2) cultured proximal tubule (HK-2) cells after 4 or 18 hours of either ATP depletion/Ca(2+) ionophore- or ferrous ammonium sulfate-mediated injury; and 3) in renal cortex 18 hours after induction of glycerol-induced myoglobinuric ARF, a time corresponding to the so-called "acquired cytoresistance" state (ie, resistance to further renal damage). Hypoxic and oxidant injury each induced approximately 33% decrements in CE (but not FC) levels in PTS, corresponding with lethal cell injury ( approximately 50 to 60% LDH release). When comparable CE declines were induced in normal PTS by exogenous cholesterol esterase treatment, proportionate lethal cell injury resulted. During models of slowly evolving HK-2 cell injury, progressive CE increments occurred: these were first noted at 4 hours, and reached approximately 600% by 18 hours. In vivo myoglobinuric ARF produced comparable renal cortical CE (and to a lesser extent FC) increments. Renal CE accumulation strikingly correlated with the severity of ARF (eg, blood urea nitrogen versus CE; r, 0.84). Mevastatin blocked cholesterol accumulation in injured HK-2 cells, indicating de novo synthesis was responsible. Acute tubule injury first lowers, then raises, tubule cholesterol content. Based on previous observations that cholesterol has cytoprotectant properties, the present findings have potential relevance for both the induction and maintenance phases of ARF.

Figure 1.

Cholesterol (Chol) esters (left) and FC (right) in isolated mouse proximal tubular segments after 30 minutes of control incubation or 30 minutes of hypoxic/re-oxygenation (H/R) injury (15 minutes of hypoxia plus 15 minutes of re-oxygenation). Cell injury induced a significant reduction in CE levels (P < 0.01), without any discernible change in FC content (right).

Figure 2.

CE and FC in isolated mouse proximal tubular segments after 30 minutes of control incubation or 30 minutes of iron-mediated oxidative stress (Fe), induced by 25 μmol/L FeHQ. These results mimicked those produced by hypoxia/re-oxygenation, as depicted in Figure 1 ▶ : ie, cell injury induced an ∼38% reduction in CE levels without causing a discernible change in FC content.

Figure 3.

Influence of CEase treatment on mouse isolated proximal tubule CE and FC content. After a 30-minute incubation with CEase, an ∼50% reduction in CEs was observed. This corresponded with 64 ± 2% LDH release. Only slight and nonsignificant elevations in FC resulted from CEase treatment (as discussed in Results).

Figure 4.

HK-2 cell cholesterol/CE levels after 4 hours of acute cell injury. Left: CE results produced by either the Ca²⁺ ionophore/antimycin/deoxyglucose (CAD)- or the Fe-mediated challenge are depicted. Right: FC results are presented. Both CAD and Fe caused significant elevations in CE levels. This result appeared more substantial with the CAD versus the Fe challenge (indicating that it is not an ATP-dependent process). The CAD- mediated CE elevations occurred without any change in FC content. Conversely, in the case of Fe, a modest decrement in FC was also observed, consistent with oxidation of FC to an undetected oxidative by-product (eg, cholestenone).

Figure 5.

HK-2 cell cholesterol/CE results after 18 hours of Fe-mediated oxidative injury. An approximate fivefold increase in CEs resulted after the 18-hour iron challenge (left). Conversely, an ∼20% reduction in FC levels was noted (right). There was a corresponding 65% LDH release after this treatment, suggesting that despite the reductions in absolute FC levels, the amount of FC per residual viable cell was actually increased.

Figure 6.

Impact of de novo cholesterol synthesis on injury-mediated HK-2 cell cholesterol accumulation. HK-2 cells were incubated with Fe either in the presence of 10 μmol/L of mevastatin or mevastatin vehicle. As depicted, mevastatin (meva) prevented the Fe-mediated increase in CEs/FC, preserving essentially normal concentrations (as determined in above described experiments).

Figure 7.

CE and FC levels in renal cortex 18 hours after glycerol-induced myohemoglobinuria. Left: Glycerol-mediated myohemoglobinuria induced striking increases in CE levels. This was associated with a slight (∼11%), but statistically significant (P < 0.005), increase in FC content (right).

Figure 8.

Relationship between the severity of glycerol-mediated renal injury (BUN) and the extent of CE elevations in renal cortex 18 hours after glycerol-induced myohemoglobinuria. When the CE values (which form the composite data presented in Figure 7 ▶ ), are contrasted with the BUN levels at 18 hours after glycerol injection, a highly significant direct relationship (r, 0.84; P < 0.005) is apparent.