Trimetazidine Protects from Mercury-Induced Kidney Injury.

INTRODUCTION: The presence of mercury in the environment is a worldwide concern. Inorganic mercury is present in industrial materials, is employed in medical devices, is widely used in batteries, is a component of fluorescent light bulbs, and it has been associated with human poisoning in gold mining areas. The nephrotoxicity induced by inorganic mercury is a relevant health problem mainly in developing countries. The primary mechanism of mercury toxicity is oxidative stress. Trimetazidine (TMZ) is an anti-ischemic drug, which inhibits cellular oxidative stress, eliminates oxygen-free radicals, and improves lipid metabolism. The aim of this study was to evaluate whether the administration of TMZ protects against mercuric chloride (HgCl2) kidney damage. METHODS: Adult male Wistar rats received only HgCl2 (4 mg/kg bw, sc) (Hg group, n = 5) or TMZ (3 mg/kg bw, ip) 30 min before HgCl2 administration (4 mg/kg bw, sc) (TMZHg group, n = 7). Simultaneously, a control group of rats (n = 4) was studied. After 4 days of HgCl2 injection, urinary flow, urea and creatinine (Cr) plasma levels, Cr clearance, urinary glucose, and sodium-dicarboxylate cotransporter 1 (NaDC1) in urine were determined. Lipid peroxidation (MDA) and glutathione (GSH) levels were measured in kidney homogenates. RESULTS: Rats only treated with HgCl2 showed an increase in urea and Cr plasma levels, urinary flow, fractional excretion of water, glucosuria, and NaDC1 urinary excretion as compared with the control group and a decrease in Cr clearance. TMZHg group showed a decrease in urea and Cr plasma levels, urinary flow, fractional excretion of water, glucosuria, NaDC1 urinary excretion, and an increase in Cr clearance when compared to the Hg group. Moreover, MDA and GSH levels observed in Hg groups were decreased and increased, respectively, by TMZ pretreatment. CONCLUSION: TMZ exerted a renoprotective action against HgCl2-induced renal injury, which might be mediated by the reduction of oxidative stress. Considering the absence of toxicity of TMZ, its clinical application against oxidative damage due to HgCl2-induced renal injury should be considered. The fact that TMZ is commercially available should simplify and accelerate the translation of the present data "from bench to bedside." In this context, TMZ become an interesting new example of drug repurposing.

Renal expression and urinary excretion of Na+/dicarboxylate cotransporter 1 (NaDC1) in obstructive nephropathy: a candidate biomarker for this pathology.

Obstructive nephropathy is characterized by alterations in renal function that depends on the degree and type of obstruction. To increase the knowledge about the physiopathological mechanisms involved in the renal damage associated with bilateral ureteral obstruction (BUO), we studied the renal expression and function (as urinary citrate excretion) of sodium-dependent dicarboxylate cotransporter (NaDC1) in rats. In addition, we evaluated the urinary excretion of NaDC1 as a candidate biomarker for this pathology. Male Wistar rats underwent bilateral ureteral obstruction for 1 (BUO1), 2 (BUO2), 5 (BUO5), and 24 (BUO24) h or sham operation. After 24 h of ureteral releasing, traditional parameters of renal function and citrate levels were determined, and NaDC1 levels were evaluated in total renal homogenates, apical plasma membranes, and urine by electrophoresis and Western blotting. Traditional parameters of renal function were only modified in BUO5 and BUO24. The renal expression of NaDC1 was decreased in BUO5 and BUO24, with a concomitant increase in urinary excretion of citrate. Moreover, the urinary excretion of NaDC1 increased after short times of ureteral obstruction (BUO1 and BUO2) and was positively correlated with the time elapsed after obstruction. The results obtained from the renal expression of NaDC1 could explain an adaptive mechanism to prevent the formation of kidney stones by increasing the levels of citrate, a calcium chelator. The urinary excretion of NaDC1 could be postulated as an early biomarker of obstructive nephropathy that also gives information about the duration of the obstruction.

Renal Expression and Urinary Excretion of Na-K-2Cl Cotransporter in Obstructive Nephropathy.

Renal damage due to urinary tract obstruction accounts for up to 30% of acute kidney injury in paediatrics and adults. Bilateral ureteral obstruction (BUO) is associated with polyuria and reduced urinary concentrating capacity. We investigated the renal handling of water and electrolytes together with the renal expression and the urinary excretion of the Na-K-Cl cotransporter (NKCC2) after 1 (BUO-1), 2 (BUO-2), and 7 (BUO-7) days of release of BUO. Immunoblotting and immunohistochemical studies showed that NKCC2 expression was upregulated in apical membranes both from BUO-2 and from BUO-7 rats. The apical membrane expression, where NKCC2 is functional, may be sufficient to normalize water, potassium, sodium, and osmolytes tubular handling. NKCC2 abundance in homogenates and mRNA levels of NKCC2 was significantly decreased in almost all groups suggesting a decrease in the synthesis of the transporter. Urinary excretion of NKCC2 was increased in BUO-7 groups. These data suggest that the upregulation in the expression of NKCC2 in apical membranes during the postobstructive phase of BUO could contribute to improving the excretion of sodium and consequently also the excretion of potassium, osmolytes, and water. Moreover, the increase in urinary excretion of NKCC2 in BUO-7 group could be a potential additional biomarker of renal function recovery.

Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti).

The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl(-), and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti. Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A(1); its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl(-)-dependent uptake of (86)Rb(+) is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 µM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 µM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium.

Oat5 and NaDC1 protein abundance in kidney and urine after renal ischemic reperfusion injury.

The aim of this study was to evaluate the abundance of the organic anion transporter 5 (Oat5) and the sodium-dicarboxylate cotransporter 1 (NaDC1) in kidney and urine after renal ischemic reperfusion injury. Renal injury was induced in male Wistar rats by occlusion of both renal pedicles for 0 (Group Sham), 5 (Group I5R60), or 60 (Group I60R60) min. The studies were performed after 60 min of reperfusion. The expression of Oat5 and NaDC1 was evaluated by IHC and Western blotting. Oat5 and NaDC1 abundance and alkaline phosphatase activity (AP) were assayed in urine. A decreased expression in renal homogenates and apical membranes and an increase in urinary excretion of Oat5 and NaDC1 were observed in I60R60 rats, as well as alterations of other widely used parameters for renal dysfunction and injury (plasma creatinine, urinary AP activity, kidney weight, histological lesions). In contrast, in the I5R60 group, only an increase in urinary excretion of Oat5 and mild histopathological damage was detected. This is the first study on Oat5 and NaDC1 detection in urine. These results suggest that urinary excretion of Oat5 might be an early indicator of renal dysfunction, which is useful for detection of even minor alterations in renal structural and functional integrity.

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Regulation of the renal thiazide-sensitive Na-Cl cotransporter, blood pressure, and natriuresis in obese Zucker rats treated with rosiglitazone.

Previously, we showed an increase in protein abundance of the renal thiazide-sensitive Na-Cl cotransporter (NCC) in young, prediabetic, obese Zucker rats relative to lean age mates (Bickel CA, Verbalis JF, Knepper MA, and Ecelbarger CA. Am J Physiol Renal Physiol 281: F639-F648, 2001). To test whether this increase correlated with increased thiazide sensitivity (NCC activity) and blood pressure, and could be modified by insulin-sensitizing agents, we treated lean and obese Zucker rats (9 wk old) with either a control diet or this diet supplemented with 3 mg/kg body wt rosiglitazone (RGZ), a peroxisomal proliferator-activated receptor subtype gamma agonist and potent insulin-sensitizing agent, for 12 wk (n = 9/group). The rise in blood pressure, measured continuously by radiotelemetry, was significantly blunted in the RGZ-treated obese rats. Similarly, blood glucose and urinary albumin were markedly decreased in these rats. RGZ-treated rats whether lean or obese excreted a NaCl load faster but excreted less sodium in response to hydrochlorothiazide, applied as a novel in vivo measure of NCC activity. Obese rats had increased renal protein abundance and urinary excretion of NCC; however, this was not significantly reduced by RGZ (densitometry in cortex homogenate - %lean control): 100 +/- 9, 93 +/- 4, 124 +/- 9, and 141 +/- 14 for lean control, lean RGZ, obese control, and obese RGZ, respectively. Subcellular localization, as evaluated by confocal microscopy and immunoblotting following differential centrifugation, of NCC was not different between rat groups. Overall, RGZ reduced blood pressure and thiazide sensitivity; however, the mechanism(s) did not seem to involve a decrease in NCC protein abundance or cellular location. Decreased NCC activity may have contributed to the maintenance of normotension in RGZ-treated obese rats.

Identification and proteomic profiling of exosomes in human urine.

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented "cytoplasmic-side inward," consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.