Gunilla Thulin

Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat.

Renal ischemia results in both a profound fall in cellular ATP and a rapid induction of the 70 kD heat-shock protein family, HSP-70. The present studies examined the relationship between cellular ATP and induction of the stress response in renal cortex. Cellular ATP, continuously monitored by in vivo 31P-NMR spectroscopy, was reduced and maintained at specific, stable levels in renal cortex by partial aortic occlusion for 45 min. Activation of heat-shock transcription factor (HSF) was detected by gel retardation assay and transcription was confirmed by Northern analysis. Activation of HSF was not present, and HSP-70 mRNA induction did not occur when ATP levels were maintained above 60% preocclusion (control) levels. Reduction in cortical ATP levels to 35-50% preocclusion values resulted in HSF activation and low-level expression of inducible HSP-70 mRNA. Cellular ATP of 20-25% control values resulted in a greater level of HSF activation and subsequent HSP-70 mRNA elaboration. HSF was activated at the end of 15 min of total occlusion. The studies indicate that a 50% reduction in cellular ATP in the renal cortex must occur before the stress response is detectable, that reduction of ATP below 25% control levels produces a more vigorous response, and that reperfusion is not required for initiation of a heat-shock response in the kidney. Cellular ATP, or the metabolic consequences associated with ATP depletion, may be a threshold factor for initiation of a stress response in the kidney.

Hsp27 Associates with Actin and Limits Injury in Energy Depleted Renal Epithelia

The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine phalloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.

Functional Activation of Heat Shock Factor and Hypoxia-Inducible Factor in the Kidney

Renal ischemia is the result of a complex series of events, including decreases in oxygen supply (hypoxia) and the availability of cellular energy (ATP depletion). In this study, the functional activation of two stress-responsive transcription factors, i.e., heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1), in the kidney was assessed. When rats were subjected to 45 min of renal ischemia, electrophoretic mobility shift assays of kidney nuclear extracts revealed rapid activation of both HIF-1 and HSF. Western blot analyses further demonstrated that this activation resulted in increased expression of the HSF and HIF-1 target genes heat shock protein-72 and heme oxygenase-1, respectively. Whether hypoxia or ATP depletion alone could produce similar activation patterns in vitro was then investigated. Renal epithelial LLC-PK(1) cells were subjected to either ATP depletion (0.1 microM antimycin A and glucose deprivation) or hypoxia (1% O(2)). After ATP depletion, HSF was rapidly activated (within 30 min), whereas HIF-1 was unaffected. In contrast, hypoxia led to the activation of HIF-1 but not HSF. Hypoxic activation of HIF-1 was observed within 30 min and persisted for 4 h, whereas no HSF activation was detected even with prolonged periods of hypoxia. HIF-1 was transcriptionally active in LLC-PK(1) cells, as demonstrated by luciferase reporter gene assays using the vascular endothelial growth factor promoter or a synthetic promoter construct containing three hypoxia-inducible elements. Interestingly, intracellular ATP levels were not affected by hypoxia but were significantly reduced by ATP depletion. These findings suggest that HIF-1 is activated specifically by decreased O(2) concentrations and not by reduced ATP levels alone. In contrast, HSF is activated primarily by metabolic stresses associated with ATP depletion and not by isolated O(2) deprivation. In vivo, the two transcription factors are simultaneously activated during renal ischemia, which might account for observed differences between in vivo and in vitro epithelial cell injury and repair. Selective modulation of either pathway might therefore be of potential interest for modification of the response of the kidney to ischemia, as well as the processes involved in recovery from ischemia.

Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia

The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.

Preactivation of AMPK by metformin may ameliorate the epithelial cell damage caused by renal ischemia

Alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion. AMP-activated protein kinase (AMPK), a cellular energy sensor, is rapidly activated in response to renal ischemia, and we demonstrate that its activity is upregulated by energy depletion in Madin-Darby canine kidney (MDCK) cells. We hypothesized that AMPK activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion. MDCK cells were ATP depleted through a 1-h incubation with antimycin A and 2-deoxyglucose. Immunofluoresence localization demonstrated that this regimen induces mislocalization of the Na-K-ATPase from its normal residence at the basolateral plasma membrane to intracellular vesicular compartments. When cells were pretreated with the AMPK activator metformin before energy depletion, basolateral localization of Na-K-ATPase was preserved. In MDCK cells in which AMPK expression was stably knocked down with short hairpin RNA, preactivation of AMPK with metformin did not prevent Na-K-ATPase redistribution in response to energy depletion. In vivo studies demonstrate that metformin activated renal AMPK and that treatment with metformin before renal ischemia preserved cellular integrity, preserved Na-K-ATPase localization, and led to reduced levels of neutrophil gelatinase-associated lipocalin, a biomarker of tubular injury. Thus AMPK may play a role in preserving the functional integrity of epithelial plasma membrane domains in the face of energy depletion. Furthermore, pretreatment with an AMPK activator before ischemia may attenuate the severity of renal tubular injury in the context of acute kidney injury.

ATP releases HSP-72 from protein aggregates after renal ischemia.

The pattern of 72-kDa heat-shock protein (HSP-72) induction after renal ischemia suggests a role in restoring cell structure. HSP-72 activity in the repair and release from denatured and aggregated proteins requires ATP. Protein aggregates were purified from normal and ischemic rat renal cortex. The addition of ATP to cortical homogenates reduced HSP-72, Na+-K+-ATPase, and actin in aggregates subsequently isolated, suggesting that their interaction is ATP dependent. Altering ATP hydrolysis in the purified aggregates, however, had different effects. ATP released HSP-72 during reflow and preserved Na+-K+-ATPase association with aggregates at 2 h but had no effect in controls or at 6 h reflow and caused no change in actin. These results indicate that HSP-72 complexes with aggregated cellular proteins in an ATP-dependent manner and suggests that enhancing HSP-72 function after ischemic renal injury assists refolding and stabilization of Na+-K+-ATPase or aggregated elements of the cytoskeleton, allowing reassembly into a more organized state.

Beneficial effect of thyroxin in the treatment of ischemic acute renal failure.

To evaluate the effect of thyroxin (T4) on recovery from ischemic acute renal failure, rats were treated with T4 (10 or 20 μg/100 g body wt.) or normal saline (NS) either immediately prior to, immediately after or 24 h after 45 min of renal ischemia. Animals given T4 prior to ischemia had no significant increase in Inulin clearance (Cin) (377±40 μl/min per 100 g body wt.) as compared with saline-treated ischemic controls (306±54). In contrast, animals treated immediately after ischemia with either dose of T4 demonstrated significantly better kidney function (Cin 515±59 μl/min per 100 g body wt., Uosm 842±88 mosmol/kg, FENa 0.52%±0.12% and Cin 543±71, Uosm 939±103, FENa 0.48±0.12, for 10 and 20 μg/100 g body wt., respectively). Moreover, the improvement in renal function was sustained and Cin was significantly better at day 3 (748±70) and day 7 (990±75) compared with saline controls (560±30 and 732±45, respectively). Animals which received T4 24 h after ischemia showed significantly higher Cin when compared with ischemic controls. To assess the impact of T4 on recovery of renal ATP,31P-NMR was used. T4-treated rats demonstrated 90%±5% recovery of renal ATP by 120 min of reflow, whereas NS animals had only 64%±1%. In addition, cellular morphology was better preserved in T4 animals. These data indicate that animals treated postischemically with T4 showed accelerated and sustained recovery from acute renal failure. This beneficial effect appears to be related to cellular mechanisms which are essential for the restoration of sublethally injured cells.

Vitamin E Affects Lung Biochemical and Morphologic Response to Hyperoxia in the Newborn Rabbit

Summary: The effects of parenteral vitamin E treatment on aspects of the pulmonary biochemical and morphologic response to 100% oxygen were studied in newborn rabbits manifesting chemical evidence of vitamin E deficiency. Pups treated with 2 mg/100 g body weight increased serum vitamin E levels from 0.39 to 2.17 mg/dl by 72 hr and lung tissue vitamin E content from 3.52 to 17 mg/mg wet weight of lung. In vitro lipid peroxidation in lung homoginates of animals in 100% oxygen for 72 hr was inhibited by approximately 80% in animals receiving 100% oxygen plus vitamin E. Hyperoxia-induced increases in the pulmonary antioxidant enzymes, super-oxide dismutase, glutathione peroxidase, and glutathione reductase were diminished by vitamin E administration. Lungs from vitamin E-treated animals did not show the early lung epithelial injury seen in animals exposed to 100% oxygen but not treated with vitamin E. Mophometric analysis of lungs of animals in room air for 72 hr showed 81.6% of lung to be normal as compared with 43.3% normal lung in the group maintained in 100% oxygen for 72 hr. In the group treated with oxygen plus vitamin E., the lungs were similar to room air controls (82.6% normal). This study thus provides further evidence for a direct antioxident affect of vitamin E in lung.Speculation: These findings provide further evidence of antioxidant protection of lung by vitamin E and suggest that the antioxidant effect of vitamin E may involve an inactivation of the probable stimulus for antioxidant enzyme induction, oxygen-free radicals.

Role of heat stress response in the tolerance of immature renal tubules to anoxia

The stress response was studied in suspensions of tubules from immature (IT) and mature (MT) rats after noninjury, heat, oxygen, and anoxia. Under all conditions, IT exhibited more exuberant activation of heat shock transcription factor (HSF) than MT. Characterization of activated HSF in immature cortex revealed HSF1. Also, 2 h after each condition, heat shock protein-72 (HSP-72) mRNA was twofold in IT. As the metabolic response to 45 min of anoxia, 20-min reoxygenation was assessed by measuring O2 consumption (O2C). Basal O2C was manipulated with ouabain, nystatin, and carbonylcyanide p-chloromethyoxyphenylhydrazone (CCCP). Basal O2C in IT were one-half the value of MT. After anoxia, basal O2C was reduced by a greater degree in MT. Ouabain reduced O2C to half the basal value in both noninjured and anoxic groups. Basal O2C was significantly stimulated by nystatin but not to the same level following anoxia in MT and IT. Basal O2C was also stimulated by CCCP, but after anoxia, CCCP O2C was significantly less in MT with no decrease in IT, suggesting mitochondria are better preserved in IT. Also, O2C devoted to nontransport activity was better maintained in IT.The stress response was studied in suspensions of tubules from immature (IT) and mature (MT) rats after noninjury, heat, oxygen, and anoxia. Under all conditions, IT exhibited more exuberant activation of heat shock transcription factor (HSF) than MT. Characterization of activated HSF in immature cortex revealed HSF1. Also, 2 h after each condition, heat shock protein-72 (HSP-72) mRNA was twofold in IT. As the metabolic response to 45 min of anoxia, 20-min reoxygenation was assessed by measuring O2 consumption (O2C). Basal O2C was manipulated with ouabain, nystatin, and carbonylcyanide p-chloromethyoxyphenylhydrazone (CCCP). Basal O2C in IT were one-half the value of MT. After anoxia, basal O2C was reduced by a greater degree in MT. Ouabain reduced O2C to half the basal value in both noninjured and anoxic groups. Basal O2C was significantly stimulated by nystatin but not to the same level following anoxia in MT and IT. Basal O2C was also stimulated by CCCP, but after anoxia, CCCP O2C was significantly less in MT with no decrease in IT, suggesting mitochondria are better preserved in IT. Also, O2C devoted to nontransport activity was better maintained in IT.

Differential Inhibition of HSP72 and HSP25 Produces Profound Impairment of Cellular Integrity

To test a putative cause and effect relationship between heat-shock protein (HSP) expression and response to renal cell injury, HSP72 and HSP25 were differentially inhibited in LLC-PK1 cells by means of transcription factor decoy and short interference RNA (siRNA). Cellular injury was assessed by solubilization of NaK ATPase (S-NaK). An exonuclease-resistant, ethylene glycol-bridged, circular oligonucleotide decoy for heat-shock transcription factor (HSF)-1, based on the sequence of the porcine heat-shock element, was constructed and validated. It was found that under all experimental conditions, cells had comparable ATP levels; that decoy of unligated or scrambled sequence was ineffective; that HSP72 mRNA and HSP72/HSP25 proteins were significantly reduced in decoy-treated cells; and that the dampened response to HSF activation in decoy-treated, injured cells was accompanied by a substantially amplified loss of cellular integrity (S-NaK was 85% compared with baseline levels). Specific inhibition of HSP72 that used siRNA directed against an inducible porcine HSP72 gene resulted in complete ablation of injury-induced HSP72. Isolated inhibition of HSP72 was also associated with marked NaK ATPase detachment from the cytoskeleton (S-NaK was 135% compared with baseline levels). These studies suggest that an HSF-1 decoy effectively dampens the HSP72/HSP25 response to injury in renal cells; that HSP72 siRNA ablates injury-induced induction of HSP72; and that dampening of the HSP72/HSP25 response and ablation of the HSP72 response are both associated with impaired restitution of cellular polarity.