Diquat induces renal proximal tubule injury in glutathione reductase-deficient mice

Reactive oxygen species (ROS) have been associated with many human diseases, and glutathione (GSH)-dependent processes are pivotal in limiting tissue damage. To test the hypothesis that Gr1(a1Neu) (Neu) mice, which do not express glutathione reductase (GR), would be more susceptible than are wild-type mice to ROS-mediated injury, we studied the effects of diquat, a redox cycling toxicant. Neu mice exhibited modest, dose- and time-dependent elevations in plasma alanine aminotransferase (ALT) activities, 126+/-36 U/l at 2 h after 5 micromol/kg of diquat, but no ALT elevations were observed in diquat-treated C3H/HeN mice for up to 6 h after 50 micromol/kg of diquat. Histology indicated little or no hepatic necrosis in diquat-treated mice of either strain, but substantial renal injury was observed in diquat-treated Neu mice, characterized by brush border sloughing in the proximal tubules by 1 h and tubular necrosis by 2 h after doses of 7.5 micromol/kg. Decreases in renal GSH levels were observed in the Neu mice by 2 h post dose (3.4+/-0.4 vs 0.2+/-0.0 micromol/g tissue at 0 and 50 micromol/kg, respectively), and increases in renal GSSG levels were observed in the Neu mice as early as 0.5 h after 7.5 micromol/kg (105.5+/-44.1 vs 27.9+/-4.8 nmol/g tissue). Blood urea nitrogen levels were elevated by 2 h in Neu mice after doses of 7.5 micromol/kg (Neu vs C3H, 32.8+/-4.1 vs 17.9+/-0.3 mg/dl). Diquat-induced renal injury in the GR-deficient Neu mice offers a useful model for studies of ROS-induced renal necrosis and of the contributions of GR in defense against oxidant-mediated injuries in vivo.     

Direct effect of cadmium on citrate uptake by isolated rat renal brush border membrane vesicles

High incidence of multiple kidney stone formation has been observed among workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. To study the direct effect of cadmium on citrate uptake by the renal brush border membrane, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cadmium and determined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. Preincubation of BBMV with 2 and 10 mM CdCl₂ for 1 min significantly inhibited citrate uptake compared with that of BBMV without Cd. Analysis of the time course of citrate uptake during 30-min preincubation of BBMV with 0.5 mM Cd also revealed significant reduction of the uptake compared with that of the control BBMV without preincubation. These findings indicate that preincubation of BBMV with cadmium results in time-dependent and concentration-dependent inhibition of citrate uptake.     

Changes in uptake of calcium caused by phytotoxicity of cadmium in Salvinia molesta

The effect of Ca on the uptake of Cd by root and leaves of Salvinia molesta was investigated at different time intervals and under different photoperiods. For detailed study on uptake and interaction, ¹⁵Ca and ¹⁰⁹Cd were used and it was found that there was a higher uptake of ⁴⁵Ca in the root and leaves at 48 h and a concurrent reduction in ¹⁰⁹Cd content at 48 h suggesting alterations in Ca functions due to the phytotoxicity of Cd. Ultrastructural changes due to cadmium toxicity included swirling of thylakoid membranes of the chloroplasts as well as detachment of the tips of trichomes from the leaf.     

镉对去卵巢大鼠骨密度和肾功能的影响

目的：研究镉对去卵巢大鼠骨密度的影响及其与肾脏损伤的关系。方法：将SD大鼠行人工去卵巢术，分别经饮水给不同剂量的镉染毒，24周后处死。用双能量X线骨密度仪（HOLOGIC，QDR－4000）测定股骨颈、股骨中点及腰椎的骨密度，用石墨炉原子吸收法测定血、尿及骨镉，放免法测定血清雌二醇,同时测定镉肾损伤指标尿β2－微球蛋白和尿钙。结果：大鼠去卵巢后，体内雌二醇水平低下，骨密度明显下降。镉染毒可使大鼠股骨颈的骨密度明显降低，且随镉染毒剂量增加呈下降趋势。镉染毒后，大鼠发生肾损伤，尿β2－微球蛋白和尿钙含量升高。同时股骨颈的骨密度与肾损伤指标β2－微球蛋白有显著性相关。结论：镉染毒可降低去卵巢大鼠的骨密度，对股骨颈作用尤为敏感，而且镉引起的骨损伤与镉造成的肾损伤相关。     

The effect of oxygen tension on the cytotoxicity of hydroquinone and selected hydroquinone metabolites to isolated rat renal proximal tubular cells

The cytotoxicity of hydroquinone (HQ) and several of its metabolites was studied using freshly isolated proximal tubular (PT) kidney cells from rats. Incubations were conducted for periods of up to 4 h at 37°C, with cytotoxicity measured either as increased leakage of lactate dehydrogenase or as a decreased energy status, as determined by decreased ratios of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). Incubation atmospheres consisted of either 95% O₂/5% CO₂, to promote cell viability in vitro, or 5% O₂/5% CO₂/90% N₂. Preliminary studies with bovine serum albumin (BSA) added to the incubation media indicated a lack of toxicity for HQ or its metabolites. For the tests discussed in this report, incubations were performed without the addition of BSA. Under 95% O₂ atmospheres, PT cells from male Fischer F344 rats were significantly more sensitive to HQ than those from male Sprague-Dawley (SD) rats, with decreases in ATP to ADP ratios seen as early as 0.5 h at a concentration of 0.5 mM. When incubations were performed under a 5% O₂ atmosphere, 2-(cysteine-S-yl)hydroquinone (Cys-HQ) and HQ toxicities were observed later (3–4 h) in the incubation period, occurred at higher concentrations, were similar in magnitude for the two strains, and were greater for Cys-HQ than for HQ. These results show that variations in oxygen tension can dramatically influence the toxicity of HQ and its metabolites. The specific compounds tested that were cytotoxic at a physiologically relevant oxygen tension (5%) were (in decreasing order of potency): Cys-HQ>2-(glutathion-S-yl)hydroquinone>HQ. These results support an association of toxicity with metabolism through the glutathione pathway, with ultimate toxicity associated with the cysteinyl conjugate. Biochemical characteristics of PT cells from these two strains suggest a significantly greater capacity of cells from the SD rat to respond to oxidative stress.     

Identification of α1-adrenoceptor subtypes in rat renal proximal tubules

Although the existence of α₁-adrenoceptor subtypes has been suggested in the kidney, their characterization in the proximal tubules has not been reported. This study was undertaken to characterize the α₁-adrenoceptor subtypes in the renal proximal tubules. [³H]Prazosin bound to proximal tubules with a K_D of 106 ± 17 pM and a B_max of 152 ± 9 fmol/mg protein. Pretreatment of the tubules with chloroethylclonidine, 100 μM, reduced the B_max of [³H]prazosin binding by about 50%, indicating the presence of the α_1B-adrenoceptor subtype. Competition studies performed with the α-adrenoceptor antagonists, WB 4101, (+)-niguldipine, 5-methylurapidil and phentolamine were shallow and could be distinguished into a high affinity (α_1A) and a low affinity site (α_1B) with an approximately equal distribution of both receptors subtypes. These observations clearly demonstrate the existence of α_1A- and α_1B-adrenoceptor subtypes in the renal proximal tubules.     

Renal handling of cadmium and cadmium-metallothionein: studies on the isolated perfused rat kidney

The isolated kidney perfusion model was used to study the uptake of Cd and metallothionein (MT)-complexed Cd. Cd²⁺ at concentrations above 40 nM strongly depressed the glomerular filtration rate (GFR), whereas MT-complexed Cd (Cd-MT) at concentrations of 0.8–920 nM had no effect on the GFR. In contrast to Cd²⁺, Cd-MT was readily reabsorbed by the kidney and uptake saturation for Cd-MT occured at 240 nM. The maximal transport rate for Cd-MT calculated in this study was 18 pmoles Cd-MT· g^–1·min^–1. The accumulation of Cd in the kidney was more efficient in the experiment using Cd-MT, in which case the Cd kidney contents were about 2–4 times higher than compared to CdCl₂.     

Normal cadmium uptake in microcytic anemia mk/mk mice suggests that DMT1 is not the only cadmium transporter in vivo

Divalent metal transporter 1 (DMT1) is a mammalian iron (Fe) transporter and also transports Cadmium (Cd) in vitro. This study compared Cd absorption in DMT1-dysfunctional MK/Rej-(mk)/(mk) mice (mk/mk mice) and in DMT1-functional, Fe-deficient wild-type (WT) mice, to clarify the role of DMT1 in intestinal Cd absorption in vivo. Mice were given 1 ppm CdCl2 aq in drinking water for 2 weeks, and the concentrations of Cd and Fe in liver, kidney, and intestinal epithelium were subsequently determined. The Fe concentration in intestinal epithelia of WT mice was decreased in proportion to the level of dietary Fe limitation, while Cd accumulation under the same conditions was increased. DMT1 mRNA expression in the small intestine of Fe-deficient WT mice was clearly increased compared to that in Fe-sufficient WT mice. Iron deficiency resulted in up-regulation of Cd uptake in the intestine of Fe-deficient WT mice. The mk/mk mice have a mutation in DMT1 and loss of its function led to decreased intestinal Fe concentration. However, intestinal Cd accumulation was the same as in WT mice and it was also increased in Fe-deficient situation. There is the possibility that an unknown Cd pathway has taken a role on Cd intestinal absorption in vivo and that this pathway is regulated by food Fe concentrations. Therefore, DMT1 is not the sole transporter of intestinal cadmium absorption in vivo.     

Effects of the calcium channel blocker AE0047 on renal function in conscious spontaneously hypertensive rats: Focus on renal proximal tubules

The hypotension induced by peripheral vasodilators is occasionally accompanied by diminished urine formation induced via various anti-diuretic mechanisms. The question of whether an antihypertensive agent has diuretic action is therefore relevant to its usefulness. In the present study, conducted in conscious, spontaneously hypertensive rats (SHR), the lithium clearance technique was used to investigate the effect of intravenously administered AE0047 on renal function; the effect of orally administered AE0047 on uric acid excretion was also investigated. Intravenous injection of AE0047 (10 and 30 μg/kg) produced potent hypotension, accompanied by diuresis and natriuresis. The diuretic action was accompanied by an elevation of FE_Na and FE_Li levels, with minimal change in GFR. Oral administration of AE0047 (1 and 3 mg/kg) led to a dose-related increase in urine volume and in urinary excretion of sodium and uric acid (U_UAV). Simultaneous administration of pyrazinamide, an inhibitor of uric acid secretion in proximal tubules, mitigated the increase in U_UAV. These findings indicate that systemically administered AE0047 produces diuresis and natriuresis, in part via an inhibition of sodium and water reabsorption in the proximal tubules. The action of AE0047 at renal proximal tubular sites thus offers benefit in hypertension therapy. Drug Dev. Res. 41:91–98, 1997. © 1997 Wiley-Liss, Inc.     

Cadmium uptake and induction of metallothionein synthesis in a renal epithelial cell line (LLC-PK1)

LLC-PK₁ cells, an established cell line from pig kidney with proximal tubule properties, were cultivated in vitro at confluence on plastic dishes. They were then exposed (apical side) to inorganic cadmium (CdCl₂, 5 M) for periods ranging between 1 to 24 h. Analysis of the cell supernatant after homogenisation and ultracentrifugation indicated that Cd taken up in the first 3 h was bound to cytosolic high molecular weight proteins, but was redistributed to low molecular weight proteins at later stages. Induction of Cd-metallothionein (Cd-Mt) synthesis, as judged from Cd-Mt binding to a specific anti-Cd-Mt antibody and from the rate of³⁵S-cys incorporation into a specific protein fraction, was apparent 3–6 h after the addition of Cd to the incubation medium.     

The effect of big endothelin-1 in the proximal tubule of the rat kidney

1. An obligatory step in the biosynthesis of endothelin-1 (ET-1) is the conversion of its inactive precursor, big ET-1, into the mature form by the action of specific, phosphoramidon-sensitive, endothelin converting enzyme(s) (ECE). Disparate effects of big ET-1 and ET-1 on renal tubule function suggest that big ET-1 might directly influence renal tubule function. Therefore, the role of the enzymatic conversion of big ET-1 into ET-1 in eliciting the functional response (generation of 1,2-diacylglycerol) to big ET-1 was studied in the rat proximal tubules.
2. In renal cortical slices incubated with big ET-1, pretreatment with phosphoramidon (an ECE inhibitor) reduced tissue immunoreactive ET-1 to a level similar to that of cortical tissue not exposed to big ET-1. This confirms the presence and effectiveness of ECE inhibition by phosphoramidon.
3. In freshly isolated proximal tubule cells, big ET-1 stimulated the generation of 1,2-diacylglycerol (DAG) in a time- and dose-dependent manner. Neither phosphoramidon nor chymostatin, a chymase inhibitor, influenced the generation of DAG evoked by big ET-1.
4. Big ET-1-dependent synthesis of DAG was found in the brush-border membrane. It was unaffected by BQ123, an ET_A receptor antagonist, but was blocked by bosentan, an ET_A,B-nonselective endothelin receptor antagonist.
5. These results suggest that the proximal tubule is a site for the direct effect of big ET-1 in the rat kidney. The effect of big ET-1 is confined to the brush-border membrane of the proximal tubule, which may be the site of big ET-1-sensitive receptors.

     

Co-exposure to lead increases the renal response to low levels of cadmium in metallurgy workers

Purpose

Research on the effect of co-exposure to Cd and Pb on the kidney is scarce. The objective of the present study was to assess the effect of co-exposure to these metals on biomarkers of early renal effect.

Methods

Cd in blood (Cd-B), Cd in urine (Cd-U), Pb in blood (Pb-B) and urinary renal biomarkers, i.e., microalbumin (μ-Alb), beta-2-microglobulin (β₂-MG), retinol binding protein (RBP), N-acetyl-β-d-glucosaminidase (NAG), intestinal alkaline phosphatase (IAP) were measured in 122 metallurgic refinery workers examined in a cross-sectional survey.

Results and conclusions

The median Cd-B, Cd-U, Pb-B were: 0.8 μg/l (IQR = 0.5, 1.2), 0.5 μg/g creatinine (IQR = 0.3, 0.8) and 158.5 μg/l (IQR = 111.0, 219.3), respectively. The impact of Cd-B on the urinary excretion of NAG and IAP was only evident among workers with Pb-B concentrations ≥75th percentile. The association between Cd-U and the renal markers NAG and RBP was also evidenced when Pb-B ≥75th percentile. No statistically significant interaction terms were observed for the associations between Cd-B or Cd-U and the other renal markers under study (i.e., μ-Alb and β2-MG). Our findings indicate that Pb increases the impact of Cd exposure on early renal biomarkers.     

Occupational exposure to cadmium: effect on metallothionein and other biological indices of exposure and renal function

The effect of duration of employment at a North American cadmium smelter on urinary metallothionein (MT), total protein, ₂-microglobulin (2-MG), glucose, cadmium, copper and zinc of 53 men was studied. The levels of all urinary parameters increased with the duration of employment. Smoking history did not affect any of the above parameters studied. Although age was responsible for some of the changes noted in protein, glucose and ₂-MG levels, its effect on MT and cadmium was insignificant. All urinary parameters were significantly related with each other. The relationship of elevated urinary MT levels with respect to renal dysfunction was also examined. Subjects with abnormal renal function excreted significantly higher amounts of MT than did those with normal renal function. The results confirm not only that occupational exposure to cadmium over long periods results in renal dysfunction but also that urinary MT could be used to monitor exposure and ultimately the appearance of the renal dysfunction.     

Proliferative responses observed following vancomycin treatment in renal proximal tubule epithelial cells

Vancomycin (VAN) is a glycopeptide antibiotic used to treat gram-positive infections. Nephrotoxicity is a common side effect observed with vancomycin therapy. However, the mechanism of vancomycin-induced nephrotoxicity has not been fully characterized. In this study we examined the effect of vancomycin on cellular proliferation in renal proximal tubule cells. A dose- and time-dependent increase in cell number and total cellular protein was observed following vancomycin exposure. Vancomycin exposure also caused an increase in BrdU incorporation followed by the accumulation of renal proximal tubule cells in G₂/M phase of the cell cycle. These effects were inhibited by pretreatment with the mitogen-activated protein kinase inhibitor, PD098059, suggesting an association between the cell proliferative effect of VAN and the induction of the mitogen-activated protein kinase signaling pathway. Mitochondrial function in renal proximal tubule cells was assessed using oxygen consumption and ATP concentrations. We observed an increase in oxygen consumption and ATP concentrations following short-term exposure to vancomycin. Together, our data suggest that vancomycin treatment produces alterations in mitochondrial function that coincide with a cell proliferative response in renal proximal tubule epithelial cells.     

Role of Arf1 in endosomal trafficking of protein-metal complexes and cadmium-metallothionein-1 toxicity in kidney proximal tubule cells

Cadmium (Cd) is nephrotoxic. Circulating Cd-metallothionein complexes (CdMT) are filtered by the kidney, reabsorbed by proximal tubule cells (PTC) via receptor-mediated endocytosis, and trafficked to lysosomes which results in apoptosis. ADP-ribosylation factors (Arfs) regulate vesicular trafficking. Arf1 is traditionally associated with the secretory pathway, but has been recently found involved in endocytotic trafficking in PTC. Hence, the role of Arf1 was investigated in MT-1 and transferrin (Tf) endocytosis, and in CdMT-1-induced cell death in a PTC line by overexpressing Arf1-wildtype (WT) or dominant-negative mutant Arf1-T31N. Endogenous Arf1 distribution in PTC was punctate throughout the cytosol, but was not detected in the plasma membrane. Arf1 colocalized with markers for sorting to late endosomes (Rab7, CLC6). Arf1 weakly overlapped with the late endosomal/lysosomal marker CLC7, but not with markers for early (Rab5, CLC5) and recycling endosomes (Rab11). Arf1-T31N significantly attenuated CdMT-1 toxicity by ∼60% when compared to Arf1-WT. However, overexpression of Arf1-T31N did not prevent internalization of Alexa Fluor 546-coupled Tf or MT-1 which accumulated in an EEA1-positive early endocytotic compartment, but not in Arf1-WT overexpressing cells. We conclude that Arf1 is involved in trafficking of protein-metal complexes, including CdMT, to late endosomes/lysosomes in renal PTC.     

The receptor subtype mediating the action of angiotensin II on intracellular sodium in rat proximal tubules

1. An investigation was undertaken to explore the subtype of receptor involved in mediating the actions of angiotensin II on intracellular sodium content in suspensions of isolated proximal tubules of the rat.
2. Intracellular sodium content of the proximal tubules was measured with ²³Na n.m.r. spectroscopy and under these conditions basal sodium content of the tubular cells was 69.04±1.73 nmol mg⁻¹ dry weight and the ATP levels, at 8.3±0.9 nmol ATP mg⁻¹ protein, were consistent with active respiration by the tissue.
3. In the presence of 10⁻⁴ M PD123319, a selective non-peptide AT₂ receptor antagonist, intracellular sodium levels rose from steady state by 30% (P<0.01; n=7) within 10 min of exposure to angiotensin II 10⁻¹¹ M. Over the subsequent 30 min steady state levels were re-established. Administration of angiotensin II 10⁻¹¹ M, in the presence of the selective AT₁ receptor antagonist, losartan at either 10⁻⁶ M (n=5) or 10⁻⁴ M (n=6), was without effect on intracellular sodium levels, which were significantly different (P<0.001) from those observed when PD 123319 was present.
4. Angiotensin II 10⁻⁵ M, administered to the tubular suspension in the presence of 10⁻⁴ M PD123319, decreased (P<0.01, n=6) intracellular sodium content by 16% in the first 5 min, but in the following 25 min returned to steady state levels. However, in the presence of losartan 10⁻⁴ M, angiotensin II 10⁻⁵ M had no effect on intracellular sodium content which was markedly different (P<0.001) from that obtained in the presence of PD123319.
5. These findings show that at both the high and low concentrations of angiotensin II, its modulation of intracellular sodium levels within the proximal tubule cells is mediated via the activation of AT₁ receptors. The intracellular mechanism underlying this effect remain to be investigated.

     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Antimicrobial activity of isolate HL-12 against Clavibacter michiganensis subsp. michiganensis in the presence of cadmium

An actinomycete, strain HL-12, that was isolated from a farmland on the Huajiachi campus of Zhejiang University was capable of inhibiting the growth of Clavibacter michiganensis subsp. michiganensis (Cmm) and was identified as a member of Streptomyces. Its antimicrobial activity against Cmm was measured using the agar plate sensitivity method in pure culture and evaluated by the inhibition ratio of Cmm in soil. The inhibitory activity of strain HL-12 against Cmm following exposure to low concentrations of Cd was greater than the inhibitory activity following exposure to high concentrations of Cd both in liquid culture and in soil. A stronger inhibition was also seen following a 24 h preculture in the presence of Cd in liquid culture. The growth of Cmm in soil was stimulated at low concentrations of Cd (<5.0 mg Cd kg⁻¹ dry soil) but inhibited when cultured in high concentrations of Cd (5.0 and 10.0 mg Cd kg⁻¹ dry soil). A higher inhibition ratio of strain HL-12 against Cmm, which was over 40% after soil incubation for 2 weeks, was observed following exposure to low concentrations of Cd (<5.0 mg Cd kg⁻¹ dry soil).     

Luminal transport of thiol S-conjugates of methylmercury in isolated perfused rabbit renal proximal tubules

Lumen-to-cell transport, cellular accumulation, and toxicity of l-cysteine (Cys), glutathione (GSH) and N-acetylcysteine (NAC) S-conjugates of methylmercury (CH₃Hg⁺) were evaluated in isolated, perfused rabbit proximal tubular segments. When these conjugates were perfused individually through the lumen of S₂ segments of the proximal tubule it was found that Cys-S-CH₃Hg and GSH-S-CH₃Hg were transported avidly, while NAC-S-CH₃Hg was transported minimally. In addition, 95% of the ²⁰³Hg taken up by the tubular cells was associated with precipitable proteins of the tubule, while very little was found in the acid-soluble cytosol. No visual cellular pathological changes were observed during 30 min of study. Luminal uptake of Cys-S-CH₃Hg was temperature-dependent and inhibited significantly by the amino acids l-methionine and l-cystine. Rates of luminal uptake of GSH-S-CH₃Hg were twice as great as that of Cys-S-CH₃Hg and uptake was inhibited significantly (74%) by the presence of acivicin. When 2,3-bis(sulfanyl)propane-1-sulfonate (DMPS) was added to the bathing or luminal fluid, luminal uptake of Cys-S-CH₃Hg was diminished significantly. Overall, our data indicate that Cys-S-CH₃Hg is likely a transportable substrate of one or more amino acid transporters (such as system B^0,+ and system b^0,+) involved in luminal absorption of l-methionine and l-cystine along the renal proximal tubule. In addition, GSH-S-CH₃Hg appears to be degraded enzymatically to Cys-S-CH₃Hg, which can then be taken up at the luminal membrane. By contrast NAC-S-CH₃Hg and Cys-S-CH₃Hg (in the presence of DMPS) are not taken up avidly at the luminal membrane of proximal tubular cells, thus promoting the excretion of CH₃Hg⁺ into the urine.     

Cadmium alters the localization of N-cadherin,E-cadherin,and beta-catenin in the proximal tubule epithelium

Recent studies on proximal tubule-derived cells in culture have shown that Cd has relatively specific damaging effects on the cadherin-dependent junctions between the cells. The objective of the present study was to determine whether Cd can affect cadherin-dependent junctions in the proximal tubule epithelium in vivo. Male Sprague-Dawley rats received subcutaneous injections of Cd (0.6 mg/kg in isotonic saline, 5 days per week for up to 6 weeks). One day each week, 24-h urine samples were collected and analyzed for protein and creatinine. After 5-6 weeks, the Cd-treated animals developed significant proteinuria, with no change in creatinine excretion. Visualization of pan-cadherin immunoreactive materials by immunoperoxidase labeling showed that Cd caused a marked reduction in the intensity of cadherin labeling associated with the apical and the basolateral surfaces of the epithelial cells of the proximal tubule, but no change in the pattern of cadherin labeling in other segments of the nephron. Results of studies utilizing specific antibodies against N-cadherin, E-cadherin, and beta-catenin showed changes in the localization of all three molecules in the proximal tubule. Assessment of cell membrane integrity with trypan blue and ethidium homodimer showed no overt evidence of death in the proximal tubule epithelial cells. Additional results showed that Cd caused only a slight increase in the total levels of glutathione and no significant peroxidation of membrane lipids, indicating only a modest level of oxidative stress. These results indicate that Cd can disrupt cadherin-dependent cell-cell junctions in the proximal tubule, and they raise the possibility that a loss of cadherin-mediated adhesion may contribute to the nephrotoxic effects of Cd.