Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells

Autophagy is a cellular process of bulk degradation of damaged organelles, protein aggregates and other macromolecules in the cytoplasm. It is thought to be a general response to stress contributing to cell death; alternatively it might act as a cytoprotective mechanism. Here we found that administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys. In cultured proximal tubular cells, the nephrotoxin caused autophagy in a dose- and time-dependent manner prior to apoptosis. Notably, autophagy occurred within hours of cisplatin administration but this was partially suppressed by the p53 inhibitor pifithrin-alpha, suggesting that p53 is involved in autophagic signaling. This cisplatin-induced autophagy was attenuated in renal cells stably transfected with Bcl-2, suggesting an anti-autophagic role for this well-known anti-apoptotic protein. Blockade of autophagy with pharmacological inhibitors (3-methyladenine or bafilomycin) or shRNA knockdown of the autophagic gene Beclin increased tubular cell apoptosis during cisplatin treatment. Our study has found that autophagy occurs in acute kidney injury and this may be an important protective mechanism for cell survival.     

Transient proximal tubular renal injury following Ecstasy ingestion

Multiple renal adverse effects have been anecdotally reported with the ingestion of 3,4-methylenedioxymethamphetamine (Ecstasy), a widely used recreational drug. These side effects include acute renal failure, necrotizing vasculitis, and hyponatremia, the mechanisms for which are unknown. We report a case of transient acute proximal tubular injury and hyponatremia associated with Ecstasy use. An 18-year-old woman presented with new onset seizures and polydipsia. Her initial laboratory evaluation revealed hyponatremia (Na 117 mEq/L), polyuria (urine output >400 mL/h for several hours), renal glycosuria (blood glucose 120 mg/dL, urine glucose >1,000 mg/dL), and solute diuresis (urine osmolality 552 mOsm/kg H2O). Urine electrolyte values reflected a low tubular reabsorption of phosphorus (TRP) of 68.1% (expected TRP >85% at serum P 2.3 mg/dL) with an appropriate transtubular potassium gradient of 3.0 (serum K 3.7 mEq/L). Her hyponatremia was slowly corrected. A repeat TRP after 48 h had normalized to 86.5%, and her glycosuria resolved. An extensive toxin screen was later reported positive for Ecstasy. To our knowledge, this is the first example of an acute and transient proximal tubular injury with Ecstasy ingestion. This complication may become more apparent with increasing use of this drug.     

Activation of complement by hydroxyl radical in thermal injury

Complement activation resulting from local burn injury of skin and other soft tissues can be linked to systemic complications, such as intravascular hemolysis, neutrophil activation, and acute lung injury. This study was designed to clarify the relationship between cutaneous thermal injury, oxygen radical formation, and complement activation in vivo. A model for "selective" venous sampling from the area of a partial-thickness cutaneous burn over 25% to 30% of the total body surface in the rat was developed. Interventions involving oxygen radical scavengers, antioxidant enzymes, xanthine oxidase inhibitors, an iron chelator, complement depletion, and neutrophil depletion were used to probe the nature of the oxygen products involved in complement activation. Plasma from the area of burn was examined for total hemolytic complement activity, content of C5a-related chemotactic peptide, and relationship of oxygen products to appearance of this peptide. Xanthine oxidase inhibitors, hydroxyl radical scavengers, and complement depletion diminished the generation of C5a activity at the burn site, whereas neutrophil depletion was without effect. These data suggest that C5a activity may be related to oxygen products from xanthine oxidase. The catalase sensitivity and iron dependency of C5a generation suggest that hydroxyl radical may be related to complement activation and C5a appearance. This is the first report to directly link oxygen radical generation and complement activation in vivo.     

Effect of hydroxyl radical scavenging on endotoxin-induced lung injury

The release of oxygen radicals, in particular the hydroxyl radical, from sequestered neutrophils produces acute lung injury after a number of insults. Our purpose was to determine whether hydroxyl radical, OH., is responsible for the lung injury from endotoxin characterized by (1) pulmonary leukostasis, (2) increased thromboxane production leading to pulmonary hypertension and hypoxia, and (3) increased protein permeability. This hypothesis was tested by infusion of a selective OH. scavenger, dimethyl thiourea (0.75 gm/kg), into unanesthetized sheep before endotoxin and comparison of the response to that seen with endotoxin alone. Pulmonary vascular integrity was measured by the use of lung lymph flow, QL, and lymph protein transport. Thromboxane A2 was measured as TxB2 and prostacyclin as 6-keto-PGF1 alpha. We found no difference in the degree of leukopenia and hypoxia after endotoxin or the levels of TxB2, 6-keto-PGF1 alpha, and pulmonary hypertension with dimethyl thiourea, compared with endotoxin alone. The permeability injury was also identical, with a twofold to threefold increase in protein-rich lymph seen in both groups. It appears that OH. does not play a major causative role in either phase of endotoxin lung injury.     

Beta-2-microglobulin in sickle cell anaemia. Evidence of increased tubular reabsorption

Sickle cell nephropathy is characterized by severe defects in renal medullary functions. In contrast, both proximal tubular secretion and reabsorption are found to be elevated. Since proximal tubular cells are involved in reabsorption and catabolism of beta 2-microglobulin (beta 2-m), we studied serum concentration and urinary excretion of this low molecular weight protein in sickle cell nephropathy. Serum beta 2-m concentration was higher in sickle cell anaemia (SCA) patients compared to control persons. beta 2-m excretion, however, was normal and beta 2-m clearance was not significantly decreased. Fractional beta 2-m excretion was significantly lower and therefore tubular reabsorption of beta 2-m was increased in the SCA patients. There was no correlation between serum beta 2-m concentration and the tubular reabsorption of beta 2-m and phosphate. These findings are further evidence of a particular function of the proximal tubule in sickle cell nephropathy.     

Evidence suggesting a role for hydroxyl radical in puromycin aminonucleoside-induced proteinuria

A single intravenous injection of puromycin aminonucleoside (PAN) results in marked proteinuria and glomerular morphological changes that are similar to minimal change disease in humans. We examined the effect of hydroxyl radical scavengers and an iron chelator on PAN-induced proteinuria. PAN in a dose of 5 mg/100 g body wt significantly increased urinary protein by day 5 (saline: 15 +/- 2, N = 24: PAN: 63 +/- 17, N = 23, P less than 0.001); the proteinuria rapidly increased thereafter, reaching 216 +/- 34, N = 23 by day 7. Concurrent administration of hydroxyl radical scavengers dimethylthiourea, (DMTU 500 mg/kg followed by 125 mg/kg i.p. twice a day) and sodium benzoate (BENZ, 150 mg/kg followed by 125 mg/kg i.p. twice a day) starting the evening before PAN injection markedly reduced proteinuria throughout the course of the study (urinary protein, mg/24 hours on day 7, mean +/- SEM: PAN: 229 +/- 45, N = 15; PAN + DMTU: 30 +/- 5, N = 18; PAN + BENZ: 80 +/- 18, N = 16. Because of the participation of iron in biological systems to generate hydroxyl radical, we also examined the effect of deferoxamine (DFO, 30 mg/day), an iron chelator, on the PAN-induced proteinuria. Concurrent administration of DFO was also protective. In a second series of experiments, DMTU and DFO (administered as described above and then for two additional days after the PAN) provided marked protection even when they were stopped prior to the onset of proteinuria. The protective effects of two hydroxyl radical scavengers and iron chelator implicate an important role for hydroxyl radical in PAN-induced nephrotic syndrome.     

Evidence of oxygen free radical damage in human otitis media.

Recent work with a guinea pig model of otitis media has demonstrated evidence of oxygen free radical damage to the middle ear mucosa. However, the relevance of an animal model to human disease is uncertain. Accordingly, the following pilot study was conducted to examine human middle ear fluid for lipid hydroperoxides as evidence of free radical damage. Thirty-five specimens of middle ear fluid from children with chronic otitis media were collected and described as mucoid (n = 19), purulent (n = 10), or serous (n = 6); specimens were weighed and analyzed for lipid hydroperoxide content. The results demonstrated the presence of lipid hydroperoxide in all 3 types of middle ear fluid. Additionally, there was a statistically significant elevation of total lipid hydroperoxide content in mucoid effusions compared with serous effusions, as well as a significant elevation of lipid hydroperoxide divided by weight of purulent effusions compared with serous effusions. This is the first study to document free radical damage in human otitis media.     

Glutathione depletion and increased apoptosis rate in human cystinotic proximal tubular cells

We have determined levels of glutathione (GSH), ATP, mitochondrial complex activity and apoptosis rate in proximal tubular cells (PTCs) exfoliated from urine in cystinotic (n=9) and control (n=9) children. Intracellular GSH was significantly depleted in cystinotic PTCs compared with controls (6.8 nmol GSH/mg protein vs 11.8 nmol GSH/mg protein; P<0.001), but there were no significant differences in mitochondrial complex activities or ATP levels under basal conditions. Cystinotic PTCs showed significantly increased apoptosis rate. After PTCs had been stressed by hypoxia, there was further depletion of GSH in cystinotic and control PTCs (2.4 nmol GSH/mg protein vs 7.2 nmol GSH/mg protein; P<0.001). Hypoxic stress led to increased complex I and complex IV activities in control but not in cystinotic PTCs. ATP levels were significantly reduced in cystinotic PTCs after hypoxic stress (12.2 nmol/mg protein vs 26.9 nmol/mg protein; P<0.001). GSH depletion occurs in this in vitro model of cystinotic PTCs, is exaggerated by hypoxic stress and may contribute to reduced ATP and failure to increase complex I/IV activities. Apoptotic rate is also increased, and these mechanisms may contribute to cellular dysfunction in cultured, human cystinotic PTCs.     

Bioflavonoids attenuate renal proximal tubular cell injury during cold preservation in Euro-Collins and University of Wisconsin solutions

BACKGROUND: Cold ischemia and reperfusion during kidney transplantation are associated with release of free oxygen radicals and damage of renal tubular cells. Bioflavonoids may diminish cold storage-induced injury due to antioxidant and iron chelating activities. This study was designed to delineate the renoprotective mechanisms of bioflavonoids and to define the structural features conferring cytoprotection from cold injury. METHODS: LLC-PK1 cells were preincubated for three hours with bioflavonoids and cold stored in University of Wisconsin (UW)- or Euro-Collins (EC)-solution for 20 hours. After rewarming, cell viability was assessed by the lactate dehydrogenase (LDH) release, MTT-test, and amino acid transport activity. Lipid peroxidation was assessed from the generation of thiobarbituric acid-reactive substances. RESULTS: Twenty-hours of cold storage of LLC-PK1 cells resulted in a substantial loss of cell integrity that was more pronounced in the EC (LDH release, 93.6 +/- 1.6%) than the UW solution (67.2 +/- 6.9%; P < 0.0001). Pretreatment with quercetin significantly enhanced cell survival (LDH release, 5.4 +/- 2.7% for UW and 8.4 +/- 4.2% for EC) in a concentration dependent manner. Structure-activity studies revealed similar renoprotection for kaempferol, luteolin and fisetin, unlike myricetin, morin, apigenin, naringenin, catechin, silibinin and rutin. Lipid peroxidation was reduced (UW alone, 2.7 +/- 1.2 vs. UW+quercetin 0.5 +/- 0.2 nmol/mg protein, P < 0.01), and l-threonine uptake completely sustained by pretreatment with quercetin, kaempferol, luteolin, and fisetin. However, renoprotection by fisetin was rapidly lost during rewarming. Protective properties of bioflavonoids were governed by the number and arrangement of hydroxyl substitutes, electron-delocalization, sterical planarity, and lipophilicity of the basic diphenylpyran skeleton. CONCLUSION: Cold storage-induced renal tubular cell injury is ameliorated by bioflavonoids. Renoprotective effects of bioflavonoids are defined by structure, suggesting that flavonoids are incorporated into membrane lipid bilayers and interfere with membrane lipid peroxidation.     

IL-1 alpha stimulated TNF alpha production by cultured human proximal tubular epithelial cells.

Tumor necrosis factor alpha (TNF alpha) production by proximal tubuli was studied by immunoperoxidase staining of 20 renal biopsies from transplant patients. A positive staining for TNF alpha on proximal tubuli was seen in nine out of 15 patients with interstitial infiltrate, five without clinical significant rejection and four with moderate to severe interstitial rejection. TNF alpha was only expressed on tubuli within areas of interstitial infiltrate. Expression of TNF alpha in the mononuclear cell infiltrate was seen only in three patients with interstitial rejection. Absence of TNF alpha could be seen in biopsies with no renal abnormalities. To obtain more information on the regulation of TNF alpha production, proximal tubular epithelial cells (PTEC) cultures were established and assessed for production of TNF alpha. Heterogenicity in production of TNF alpha was found in 14 tested PTEC lines cultured under serum free conditions. The presence of IL-1 alpha in the cultures induced a time- and dose-dependent enhancement of TNF alpha production by PTEC. Enhanced production of TNF alpha was not seen after stimulation with other cytokines such as IL-2 or IFN gamma. Inhibition studies with cycloheximide indicated de novo synthesis of TNF alpha. Western blot analysis of supernatants of unstimulated and IL-1 alpha stimulated PTEC indicated a 17 kd product, a size similar to that of recombinant TNF alpha. Northern blot analysis revealed the presence of a 2.0 kb hybridization signal in total RNA of PTEC cultures and up regulation upon treatment of PTEC with 1 ng/ml of IL-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against increased glomerular pressure initiating diabetic nephropathy

Studies were carried out to determine whether exaggerated glomerular hydraulic pressure (PG) initiates the development of glomerular pathology and proteinuria in insulin-dependent diabetic rats. Normotensive (WKY) and hypertensive rats (SHR) were made diabetic by streptozotocin injection. One group of SHR diabetic rats was treated with antihypertensive drugs to reduce blood pressure. One week after onset of diabetes, micropuncture determinations of PG, measured by stopped-flow technique, revealed that PG was higher in WKY diabetics than in non-diabetic WKY controls, and that PG was even higher in SHR diabetics (P less than 0.05). Similarly prepared groups of animals were followed for six months, approximately one fifth to one third of the expected life span of these rats. Tail systolic blood-pressure measurements documented continuous severe systolic-hypertension in SHR diabetics, normal pressure in the WKY diabetics and hypotension in the SHR diabetics treated continuously with antihypertensive drugs. Urinary protein excretion, measured monthly, was statistically the same in all groups, with no evidence of a progressive rise in the SHR diabetics. PG measured in two rats from each group after four months of diabetes was similar to values found after one week of diabetes. Semiquantitative histologic scoring of glomerular mesangial expansion after six months of diabetes failed to demonstrate any significant difference between the normotensive WKY diabetics and the hypertensive SHR diabetics. These observations suggest that elevated PG does not in itself initiate glomerular pathology or proteinuria in diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactic acidosis during sepsis is related to increased pyruvate production, not deficits in tissue oxygen availability.

OBJECTIVE. The purpose of this study was to quantitate the derangements in intermediary carbohydrate metabolism and oxygen use in severely septic patients in comparison with healthy volunteers. SUMMARY BACKGROUND DATA. It commonly has been assumed that the development of lactic acidosis during sepsis results from a deficit in tissue oxygen availability. Dichloroacetate (DCA), which is known to increase pyruvate oxidation but only when tissue oxygen is available, provides a means to assess the role of hypoxia in lactate production. METHODS. Stable isotope tracer methodology and indirect calorimetry was used to determine the rates of intermediary carbohydrate metabolism and oxygen use in five severely septic patients with lactic acidosis and six healthy volunteers before and after administration of DCA. RESULTS. Oxygen consumption and the rates of glucose and pyruvate production and oxidation were substantially greater (p < 0.05) in the septic patient compared with healthy volunteers. Administration of DCA resulted in a further increase in oxygen consumption and the percentage of glucose and pyruvate directed toward oxidation. Dichloroacetate also decreased glucose and pyruvate production, with a corresponding decrease in plasma lactate concentration. CONCLUSIONS. These findings clearly indicate that the accumulation of lactate during sepsis is not the result of limitations in tissue oxygenation, but is a sequelae to the markedly increased rate of pyruvate production. Furthermore, the substantially higher rate of pyruvate oxidation in the septic patients refutes the notion of a sepsis-induced impairment in pyruvate dehydrogenase activity.     

Increased oxygen radical and eicosanoid formation in immune-mediated mesangial cell injury.

To evaluate whether monocytes/macrophages derived from glomeruli could be a source of increased eicosanoid and free oxygen radical formation in glomerular disease, monocytes/macrophage (M/M) were isolated from nephritic glomeruli and their in vitro generation of eicosanoids and superoxides were measured. Glomerular immune injury was induced by i.v. injection of a rabbit-anti-rat thymocyte antiserum (ATS). Kidneys were removed two, five, and 24 hours, and three and eight days after ATS. Adhesive glomerular macrophages were obtained by isolation of glomeruli, enzymatic digestion and incubation of the single cell suspensions in culture dishes. O2-production was evaluated by superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C; PGE2 and TxB2 release was assessed by direct RIA. Glomerular macrophage infiltration was maximal 24 hours after intravenous antibody (35.9 +/- 5.1 M/M per glomerulus). In vitro production of superoxide was significantly enhanced (P less than 0.001) five hours after ATS administration (51.6 +/- 4.4 nmol O2/10(6) MM/hr), when compared with M/M from controls (30.4 +/- 2.0 nmol O2/10(6) MM/hr). TxB2 formation of glomerular M/M was increased (P less than 0.001) two hours and five hours after ATS administration (1056 +/- 75 and 1182 +/- 112 pg TxB2/10(6) MM/hr) compared with controls (390 +/- 34 pg TxB2/10(6) MM/hr). PGE2 synthesis, however, was decreased (P less than 0.01) at five hours after ATS (629 +/- 43 pg PGE2/10(6) MM/hr) compared with controls (950 +/- 125 pg PGE2/10(6) MM/hr). Furthermore, there was release of leukotriene B4 (LTB4) in monocytes of nephritic glomeruli five hours after ATS administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack.

BACKGROUND: Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. METHODS: Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. RESULTS: Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. CONCLUSIONS: (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.