Effect Of Chelator BPCBG On The Decorporation Of Uranium In Rats And Its Transport Mechanism In Human Kidney Cells

Uranium (U) has been widely used in industrial production and military field as important nuclear fuel and nuclear weapons charge. Since the1991Gulf War, armor piercing shell and armored tank made of depleted uranium were used in war. More recently, after2011Japan’s fukushima nuclear plant accident occurred, threat of radionuclides internal contamination on human health attracts worldwide attention. Experimental studies demonstrated that kidney and skeleton are the major early and long-term uranium storage organs, respectively. Acute and chronic intakes of natural uranium resulted in renal damages and can even lead to renal failure. The long-term irradiation of bone from deposited isotopically enriched uranium can result in a consequent risk of osteosarcoma. Chelating therapy is the only known effective method to accelerate the excretion of uranium from the body and reduce the toxicity of uranium. At present, the clinical therapy proposed after internal contamination with uranium is still an infusion with sodium bicarbonate solution, but it is effective only when human body are slightly metabolic alkalosis. DTPA-CaNa3is used clinically as a drug of choice for the decoporation of actinides such as plutonium and americium, but it has a lower effectiveness for the decorporation of uranium. Therefore, scientists have been committed to looking for new high effective and low toxicity chelators for uranium.N,N’-1,2-ethanediylbis[N-[(2,3-dihydroxyphenyl)methyl]]-glycine (BPCBG) is a new chelator containing two catechol groups and two aminocarboxylic acid groups synthesized by our laboratory. The present study intends to investigate the dose-and time-effects of BPCBG on the decorporation of uranium and its protective effects for uranium-induced renal injury in rats, and further explore the transport mechanism of BPCBG in human renal cells and its relationship with protecting against uranium-induced renal cell injury with comparison to DTPA-CaNa3for the first time. Our data provide the inspiration for new effective uranium decorporating agents research and development. Part I Effect of BPCBG on the decorporation of uranium and protection against uranium-induced kidney injury in ratsObjective To explore the dose-and time-related effects of BPCBG on the decorporation of uranium and its protective effects for uranium-induced kidney injury in rats.Methods Sprague-Dawley (SD) male rats were randomly divided into4～7groups by body weight:normal control group, uranium exposure alone group, different doses of BPCBG group and DTPA-CaNa3group. Rats in chelating agents-treated groups were either injected intramuscularly (im) with60umol/kg,120μmol/kg and600μmol/kg of BPCBG or120μmol/kg and600μmol/kg of DTPA-CaNa3immediately after intraperitoneal injection of100μg uranyl acetate dihydrate to each rat, or injected with120μmol/kg of BPCBG0.5,2h before or0,0.5,1and2h after injection of uranium. Rats in the uranium exposure alone group were injected im with normal saline after intraperitoneal injection of uranyl acetate dihydrate, and the normal control group was merely injected with normal saline. The uranium content in urine, kidney and femurs were detected24h after chelator injections by ICP-MS method. Chelating agent were injected im at600μmol/kg BPCBG or1200μmol/kg DTPA-CaNa3immediately after ip injection of500μg uranyl acetate dihydrate to each rat. Histopathological changes in the kidney and serum creatinine and urea nitrogen were examined48h after chelator administration. The adult Kunming mice were randomly divided into three BPCBG administration groups with5male and5female mice in each group. The BPCBG dosage at2254mg/kg,1691mg/kg and1268mg/kg were chosed in the geometric progression with ratio0.75according to the preliminary experiment data. BPCBG was administered by a single intravenous injection to mice, and the symptoms, its severity, duration and survival in BPCBG-treated mice were observed for14days. The median lethal dose (LD50) value was calculated with Bliss method.Results Prompt injections of BPCBG resulted in37%-61%(P<0.05, P<0.001and P<0.001) increase in24h-urinary uranium excretion, and significantly decreased the levels of uranium in kidney and bone by59%-69%(P<0.01, P<0.001and P<0.001) and14%-58%(P<0.05, P<0.001and P<0.001) respectively in a dose-dependent manner. Advanced0.5h or delayed0.5and1h administrations of BPCBG were both effective in24h-urinary uranium excretion (For advanced0.5h or delayed0.5h:P<0.001, P<0.01), decreasing accumulation of kidney uranium (P<0.001, P<0.001and P<0.001) and accumulation of bone uranium (P<0.001, P<0.001and P<0.05), but the efficacy decreased with the interval time between uranium and BPCBG injection. BPCBG injection obviously reduced the severity of uranium-induced histological alterations in the kidney, which was in parallel with the amelioration noted in serum indicators, serum creatinine and urea nitrogen, of uranium nephrotoxicity. Although DTPA-CaNa3at120μmol/kg and600μmol/kg markedly reduced uranium retention in kidney (P<0.05and P<0.01), its efficacy in uranium removal was significantly lower than that of BPCBG, and it showed no protective effects against uranium-induced nephrotoxicity. The mice intravenous injection LD50value for BPCBG was1748.7mg/kg.Conclusions BPCBG can effectively decorporate uranium from rats and protect against uranium-induced kidney injury in rats. The preliminary acute toxicity test in mice showed that BPCBG has a low toxicity.Part Ⅱ Transport mechanism of chelator BPCBG in human renal cells and its protective effects on uranium-induced renal cell damageObjective To explore the uptake and secretion pathways of catechol chelator BPCBG containing two aminocarboxylic acid groups in renal tubular cells and the protective effects of intracellular BPCBG on the uranium-induced human renal cell damage with comparison to aminoalkyl chelator DTPA-CaNa3containing five aminocarboxylic acid groups.Methods The uptake experiments using human embryonic kidney (HEK293) cells stably transfected with human organic anion transporters1or3(hOATl-HEK293cells and hOAT3-HEK293cells) were performed to assess the inhibitory effects of50μM,100μM and1000μM BPCBG on the uptake of fluorescent substrates,6-carboxyfluorescence (6-CF) and5-carboxyfluorescence (5-CF), mediated by OAT1and OAT3respectively, as compared with probenecid, a well-known OAT inhibitor. The HEK293cell lines with stable human OAT4gene silencing and vector were established through the lentivirus-mediated short hairpin RNA (shRNA) method and their hOAT4protein expression was detected by western blot assay. To explore the secretion pathway of BPCBG from the HEK293,the uptake of5-CF mediated by hOAT3and hOAT4were detected after hOAT4knockdown cells and vector cells were pretreated with10μM BPCBG for24h. The concentration-and time-related effects of BPCBG on inhibiting the hOAT3/hOAT4mediated uptake of5-CF were observed to further confirm the uptake of BPCBG into HEK293cells. The hOAT3/hOAT4mediated uptake of5-CF was assayed in HEK293cells exposed to1.4mM uranyl acetate dihydrate for48h. To investigate the relationship of the transport of BPCBG into HEK293cells with its protective effect on the uranium-induced cell damage, HEK293cells were divided into the following groups with and without probenecid treatment:(1) untreated control,(2) uranium exposure alone control,(3) chelating agents treatment group. Cells in all groups were either treated with PBS or1.4mM uranyl acetate dehydrate for24h first followed by treatments with fresh culture medium with or without different concentration of chelating agents for another24h, micronucleus formation and apoptosis were detected by the citokinesis block method and flow cytometry technology, respectively. DTPA-CaNa3was used as chelating agent treatment control.Results BPCBG treatment at50μM,100μM and1000μM could significantly inhibit the intake of6-CF by hOAT1(P<0.001, P<0.001and P<0.001) and5-CF by hOAT3(P<0.05, P<0.001and P<0.001) in the hOAT1-HEK293cells and hOAT3-HEK293cells, and the inhibition effect was enhanced with the increase of drug concentration and was similar to effects of probenecid at the concentration of1000μM, indicating that BPCBG was the substrate of hOATl and hOAT3and possibly mediated by hOAT1and hOAT3to enter into cells. Althought DTPA-CaNa3could also markedly inhibit uptake of5-CF, the effect was weaker than that of BPCBG at the chelator concentration (For1000μM, P<0.05), and it didn’t inhibit the uptake of6-CF, suggesting that the hOAT3-mediated uptake of DTPA-CaNa3was much lower than that of BPCBG, and it may be not mediated by hOAT1to enter cells. In HEK293cells, BPCBG at50μM,100μM could significantly inhibited the hOAT3/hOAT4-mediated uptake of5-CF (P<0.001and P<0.001) just as the effect of probenecid, and its inhibitory effect was enhanced with the pre-treatment time of BPCBG increases. Moreover, inhibitory effect of BPCBG on the5-CF uptake was higher than that of DTPA-CaNa3with the chelator concentration (For100μM, P<0.01), further suggesting that the hOAT3/hOAT4-mediated uptake of BPCBG was stronger than that of DTPA-CaNa3. Western Blot results showed that hOAT4protein expressing was much more reduced in hOAT4knockdown HEK293cells than in vector HEK293cells and HEK293cells. However, there was no marked difference of the hOAT3/hOAT4-mediated uptake of5-CF between hOAT4knockdown cells and vector cells, indicating that5-CF intake may be transported through hOAT3compensatory function. In contrast, after cells were pre-treated with BPCBG for24h, the uptake of5-CF was significantly lower in hOAT4knockdown cells than in vector cells (P<0.001).Furthermore, pre-treatment of vector cells with BPCBG obviously increased more5-CF intake than without BPCBG pre-treatment (P<0.001), suggesting that BPCBG can enter into the cells and the intracellular BPCBG and extracellular5-CF could exchanged through hOAT4resulting to an increasing uptake of5-CF, and then BPCBG could be secreted by hOAT4. In the hOAT4knockdown cells and vector cells, DTPA-CaNa3had no obvious effect on the uptake of5-CF, suggesting that DTPA-CaNa3couldn’t be secreted by hOAT4. After HEK293cells were exposed to1.4mM uranyl acetate dihydrate for48h, intake of5-CF was not markedly reduced perhaps because uranium didn’t injure the function of transport protein in cell membrane. Therefore, BPCBG transported into HEK293cells could significantly reduce uranium-induced micronuclei formation and apoptosis (P<0.05and P<0.05), while co-administration of chelating agents and probenecid could not reduce uranium-induced cell injury because probenecid inhibited the uptake of BPCBG into HEK293cells. As chelating agent control, DTPA-CaNa3could not decrease cell damage compared with uranium exposure alone control, no matter whether with or without probenecid treatment.Conclusion BPCBG can be transported by hOATl, hOAT3or hOAT3/hOAT4into renal cells and secreted by hOAT4from the renal cells and therefore protect cells against uranium-induced cell injury; while DTPA-CaNa3can be transported by hOAT3or hOAT3/hOAT4not by hOATl into renal cells and its affinity for hOAT3or hOAT3/hOAT4was much lower than that of BPCBG, hence the uptake of DTPA-CaNa3into renal cells was much lower than that of BPCBG and it didn’t secret by hOAT4, which may be one of reasons for DTPA-CaNa3having no protective effect on uranium-induced cell damage. Our data provide the evidence that transport efficacy of aminocarboxylic type chelating agents mediated by hOAT1, hOAT3or hOAT3/hOAT4reduce with the number of aminocarboxylic acid groups.