Thresholds for cellular disruption and activation of the stress response in renal epithelia.

Renal ischemia causes a rapid fall in cellular ATP, increased intracellular calcium (Ca(i)), and dissociation of Na(+)-K(+)-ATPase from the cytoskeleton along with initiation of a stress response. We examined changes in Ca(i), Na(+)-K(+)-ATPase detergent solubility, and activation of heat-shock transcription factor (HSF) in relation to graded reduction of ATP in LLC-PK(1) cells to determine whether initiation of the stress response was related to any one of these perturbations alone. Ca(i) increased first at 75% of control ATP. Triton X-100 solubility of Na(+)-K(+)-ATPase increased below 70% control ATP. Reducing cellular ATP below 50% control consistently activated HSF. Stepped decrements in cellular ATP below the respective thresholds caused incremental increases in Ca(i), Na(+)-K(+)-ATPase solubility, and HSF activation. ATP depletion activated both HSF1 and HSF2. Proteasome inhibition caused activation of HSF1 and HSF2 in a pattern similar to ATP depletion. Lactate dehydrogenase release remained at control levels irrespective of the degree of ATP depletion. Progressive accumulation of nonnative proteins may be the critical signal for the adaptive induction of the stress response in renal epithelia.

Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion.

Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 microM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

Heat shock proteins in renal injury and recovery.

Heat shock proteins, or stress proteins, are molecular chaperones responsible for protein processing and protection against cellular injury through the prevention of inappropriate peptide interactions. The distribution of individual stress proteins varies between regions of the kidney and within subcellular compartments both in normal and pathological conditions. Novel molecular chaperones have been identified in renal medullary cells which are unique, among mammalian cells, in routinely facing osmotic stress. Heat shock proteins can participate in renal injury as antigenic targets, but their primary role is beneficial, and these proteins may function by interacting with the cytoskeleton to protect against and assist recovery from cellular 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.

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.

Severe hypertension without urinary abnormalities in a patient with Henoch-Schönlein purpura.

Hypertension as a complication of Henoch-Schönlein purpura (HSP) is almost uniformly accompanied by evidence of renal involvement, either decreased renal function or urinary abnormalities. We report a 4.5-year-old male with HSP who developed severe hypertension without other manifestations of glomerulonephritis, including no decline in renal function and no development of urinary abnormalities. Extensive evaluation for other identifiable causes for his hypertension was not productive. His hypertension resolved with the resolution of his HSP. This case demonstrates that patients with HSP may on occasion develop severe hypertension without other evidence of nephritis. An extensive evaluation for other causes of severe hypertension may be deferred in this setting until well after all other manifestations of HSP have resolved.

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.

Expression and molecular regulation of Na(+)-K(+)-ATPase after renal ischemia.

Renal ischemia causes redistribution of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) to the apical membrane of proximal tubules. We determined the time course of regeneration of Na(+)-K(+)-ATPase polarity and sought evidence of increased enzyme production during recovery as a means to restore polarity. Anesthetized rats underwent 45 min renal ischemia and reflow of 15 min, 2 h, 6 h, and 24 h. Immunofluorescent and electron microscopy showed loss of strict basolateral localization of Na(+)-K(+)-ATPase at 15 min reflow with repolarization by 24 h in sublethally injured cells. Both alpha 1- and beta-subunits were only in microsomal fractions at all reflow intervals. Immunodetectable levels of both subunits declined to 60-70% of control by 24 h reflow. Levels of mRNA for each subunit declined in parallel through 24 h to 55% of control. Overall transcription was profoundly depressed through 6 h but had recovered to near control by 24 h. Specific transcription of alpha 1- and beta-subunit mRNA was markedly decreased after ischemia and only partially recovered by 24 h. These results suggest that recycling of misplaced units rather than new Na(+)-K(+)-ATPase production is the means by which renal epithelia initially repolarize after ischemic injury.

Altered renovascular resistance after spontaneous recovery from hemolytic uremic syndrome.

Twenty-three patients were evaluated from 1-15 (mean 6) years after recovering from an episode of diarrhea-associated associated childhood hemolytic uremic syndrome (DA-HUS). All patients had received only conservative treatment; none had been given experimental, anti-coagulant, or immunological therapies. Follow-up studies included morphologic and duplex Doppler sonograms. Doppler sonography was used to determine the resistive index, a measure of renovascular resistance. Histories and physical examinations revealed no abnormalities. Results of laboratory studies, which included calculated glomerular filtration rates, were all within normal limits, except for one patient with minor urinary abnormalities. Renal sonograms showed no significant abnormalities of kidney length or parenchymal appearance. However, Doppler sonographic examinations revealed that the DA-HUS patients demonstrated less of a decrease in renovascular resistance with age than did the control group (p < 0.0002). After recovery, patients treated exclusively with conservative management during an acute episode of DA-HUS appeared to have an excellent long-term prognosis. Comparison of our results with those from other studies in which investigational therapies have been used during the acute phase of DA-HUS suggests that latent toxicities which cause long term sequelae may not have been appreciated previously. The clinical significance of the altered renal vascular resistance remains to be delineated.

ddAVP does not stimulate acute changes in levels of medullary trimethylamines in humans.

1H nuclear magnetic resonance has been used to determine the effect of acute iv administration of the arginine vasopressin analog 1-(3-mercaptopropionic acid)-8-D-arginine vasopressin monoacetate (ddAVP; 2 micrograms) on renal medullary trimethylamine (TMA) levels in human volunteers. In subjects deprived of food and water for 15 h, urine osmolality (Uosm) was 889 +/- 47 mosmol/kg and had not changed significantly 3 h after ddAVP administration. Medullary TMA did not change significantly over 3 h after ddAVP. In a second group of subjects who were well hydrated, acute ddAVP infusion increased Uosm from 203 +/- 63 to 421 +/- 47 mosmol/kg in 3 h (P < 0.05). However, medullary TMA did not change significantly over this time period. These results indicate that ddAVP, and presumably arginine vasopressin, do not acutely influence medullary TMA levels, and they support the view that results previously reported for animal and isolated cell systems are also applicable to human physiology.

Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia.

Parathyroid hormone-related protein (PTHrP) is widely expressed in normal adult and fetal tissues, where it acts in an autocrine/paracrine fashion, stimulates growth and differentiation, and shares early response gene characteristics. Since recovery from renal injury is associated with release of local growth factors, we examined the expression and localization of PTHrP in normal and ischemic adult rat kidney. Male Sprague-Dawley rats underwent complete bilateral renal artery occlusion for 45 min, followed by reperfusion for 15 min, and 2, 6, 24, 48, and 72 h. Renal PTHrP mRNA levels, when compared with sham-operated animals, increased twofold after ischemia, and peaked within 6 h after reperfusion. PTH receptor, beta-actin, and cyclophilin mRNA levels all decreased after ischemia. PTHrP immunohistochemical staining intensity increased in proximal tubular cells after ischemia, changing its location from diffusely cytoplasmic to subapical by 24 h after reperfusion. In addition, PTHrP localized to glomerular epithelial cells (visceral and parietal), but not to mesangial cells. PTHrP and PTH stimulated proliferation two- to threefold in cultured mesangial cells. We conclude that PTHrP mRNA and protein production are upregulated after acute renal ischemic injury, that PTHrP is present in glomerulus and in both proximal and distal tubular cells, and that PTHrP stimulates DNA synthesis in mesangial cells. The precise functions of PTHrP in normal and injured kidney remain to be defined.

Abdominal symptoms as presentation of hypertensive crisis.

OBJECTIVE: To describe a series of patients in hypertensive crisis who presented primarily with abdominal symptoms. DESIGN: Patient reports. SETTING: Referral center after initial presentation in the general community. PARTICIPANTS: Three children, aged 10 months to 4 years, in hypertensive crisis who presented with abdominal symptoms that promptly resolved and did not recur with blood pressure control. CONCLUSIONS: Blood pressure should be measured in all children undergoing physical examination; particular attention should be paid to the blood pressure of patients with unexplained abdominal symptoms before extensive diagnostic testing is pursued.

Induction and intracellular localization of HSP-72 after renal ischemia.

To determine whether heat shock proteins (HSPs) might be active in cellular recovery following transient ischemia, we examined rat kidneys for 70-kDa HSP (HSP-70) mRNA expression, protein elaboration, and intracellular localization after 45 min of renal ischemia and reflow of 15 min, 2, 6, and 24 h. Inducible HSP-70 mRNA is present at 15 min of reperfusion, peaks between 2 and 6 h, and falls by 24 h. Inducible 72-kDa HSP (HSP-72) protein accumulates progressively through 24 h and is found in both soluble and microsomal fractions following ischemia. Within proximal tubules, immunofluorescent localization of HSP-72 is restricted to the apical domain at 15 min, is dispersed through the cytoplasm in a vesicular pattern at 2 and 6 h, and has migrated away from the apical domain at 24 h. A portion of the vesicular HSP-72 is associated with lysosomes; no intranuclear HSP-72 is detected. The course of mRNA induction, protein elaboration, and HSP-72 localization coincides with previously described changes in proximal tubule morphology and polarity following sublethal ischemic injury. HSP-72 may be instrumental in cellular remodeling and restitution of epithelial polarity during recovery from ischemic renal injury.

Renal insufficiency after bone marrow transplantation in children.

Between 1975 and 1988, 92 pediatric patients have undergone bone marrow transplantation (BMT) at our institution for malignant or immune deficiency disease. We evaluated in a retrospective fashion 64 of these patients who survived beyond the first 60 days post-BMT. The clinical course was divided into: less than 60 days post-BMT (early) and greater than 60 days post-BMT (late). The presence or absence of renal insufficiency was noted as well as all known potential factors predisposing to insufficiency. Step-wise regression analysis was then performed to determine which of the factors were most significantly associated with renal dysfunction during the two periods. The follow-up period was 2 months to 11 years (mean 17.5 months). The mean age of the patients was 7.6 years (1 month-18 years). Fifty percent of the patients had renal insufficiency during the early period and 28% of the patients had insufficiency after the initial 60 days. Three major predictors of renal insufficiency were discovered. Cyclosporin A or amphotericin B early or late post-BMT was independently predictive of developing insufficiency during the same period. Conditioning with total body irradiation was a predictor for insufficiency in both periods. Early insufficiency was not predictive of late insufficiency. Hypertension was present in 31% of patients during the early period and in 16% during the late period. Hypertension was strongly associated with cyclosporin use and renal insufficiency. Renal insufficiency is a frequent sequela in children following BMT and likely results from a combination of radiation injury and drug toxicity.