R A Zager1, A Johnson, K Anderson, S Wright. 1. University of Washington and Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA. dzager@fhcrc.org
Abstract
BACKGROUND: Cholesterol is a major constituent of plasma membranes, and recent evidence indicates that it is up-regulated during the maintenance phase of acute renal failure (ARF). However, cholesterol's fate and that of the cholesterol ester (CE) cycle [shuttling between free cholesterol (FC) and CEs] during the induction phase of ARF have not been well defined. The present studies sought to provide initial insights into these issues. METHODS: FC and CE were measured in mouse renal cortex after in vivo ischemia (15 and 45 minutes)/reperfusion (0 to 120 minutes) and glycerol-induced myoglobinuria (1 to 2 hours). FC/CE were also measured in (1) cultured human proximal tubule (HK-2) cells three hours after ATP depletion and in (2) isolated mouse proximal tubule segments (PTSs) subjected to plasma membrane damage (with cholesterol oxidase, sphingomyelinase, phospholipase A2, or cytoskeletal disruption with cytochalasin B). The impact of cholesterol synthesis inhibition (with mevastatin) and FC traffic blockade (with progesterone) on injury-evoked FC/CE changes was also assessed. RESULTS: In vivo ischemia caused approximately threefold to fourfold CE elevations, but not FC elevations, that persisted for at least two hours of reperfusion. Conversely, myoglobinuria had no effect. Isolated CE increments were observed in ATP-depleted HK-2 cells. Neither mevastatin nor progesterone blocked this CE accumulation. Plasma membrane injury induced with sphingomyelinase or cholesterol oxidase, but not with phospholipase A(2) or cytochalasin B, increased tubule CE content. High CE levels, induced with cholesterol oxidase, partially blocked hypoxic PTS attack. CONCLUSIONS: In vivo ischemia/reperfusion acutely increases renal cortical CE, but not FC, content, indicating perturbed CE/FC cycling. The available data suggest that this could stem from specific types of plasma membrane damage, which then increase FC flux via aberrant pathways to the endoplasmic reticulum, where CE formation occurs. That CE levels are known to inversely correlate with both renal and nonrenal cell injury suggests the potential relevance of these observations to the induction phase of ischemic ARF.
BACKGROUND:Cholesterol is a major constituent of plasma membranes, and recent evidence indicates that it is up-regulated during the maintenance phase of acute renal failure (ARF). However, cholesterol's fate and that of the cholesterol ester (CE) cycle [shuttling between free cholesterol (FC) and CEs] during the induction phase of ARF have not been well defined. The present studies sought to provide initial insights into these issues. METHODS:FC and CE were measured in mouse renal cortex after in vivo ischemia (15 and 45 minutes)/reperfusion (0 to 120 minutes) and glycerol-induced myoglobinuria (1 to 2 hours). FC/CE were also measured in (1) cultured human proximal tubule (HK-2) cells three hours after ATP depletion and in (2) isolated mouse proximal tubule segments (PTSs) subjected to plasma membrane damage (with cholesterol oxidase, sphingomyelinase, phospholipase A2, or cytoskeletal disruption with cytochalasin B). The impact of cholesterol synthesis inhibition (with mevastatin) and FC traffic blockade (with progesterone) on injury-evoked FC/CE changes was also assessed. RESULTS: In vivo ischemia caused approximately threefold to fourfold CE elevations, but not FC elevations, that persisted for at least two hours of reperfusion. Conversely, myoglobinuria had no effect. Isolated CE increments were observed in ATP-depleted HK-2 cells. Neither mevastatin nor progesterone blocked this CE accumulation. Plasma membrane injury induced with sphingomyelinase or cholesterol oxidase, but not with phospholipase A(2) or cytochalasin B, increased tubule CE content. High CE levels, induced with cholesterol oxidase, partially blocked hypoxic PTS attack. CONCLUSIONS: In vivo ischemia/reperfusion acutely increases renal cortical CE, but not FC, content, indicating perturbed CE/FC cycling. The available data suggest that this could stem from specific types of plasma membrane damage, which then increase FC flux via aberrant pathways to the endoplasmic reticulum, where CE formation occurs. That CE levels are known to inversely correlate with both renal and nonrenal cell injury suggests the potential relevance of these observations to the induction phase of ischemic ARF.
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