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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Environmental Health, and the Center for Environmental Genetics (CEG), University Cincinnati Medical Center, 2Department of Pathology, Cincinnati Children's Hospital Medical Center, 3Department of Internal Medicine, Division of Nephrology and Hypertension, University Cincinnati Medical Center, and 4Department of Chemistry, University Cincinnati School of Arts and Sciences, Cincinnati Ohio
Submitted 30 July 2006 ; accepted in final form 11 November 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney. kidney; testis; ZIP8; bacterial artificial chromosome
In the "Top 20 Hazardous Substances Priority List" by the Agency for Toxic Substances and Disease Registry and the U.S. Environmental Protection Agency, Cd is ranked seventh (15). Heavy Cd usage began in the 1940s, with large-scale applications in industry (41), mining, and the burning of fossil fuels (4). Principal uses today include nickel-cadmium batteries, pigments, and plastic stabilizers. Major occupational exposures to Cd occur in nonferrous metal smelters, production and processing of Cd alloys and compounds, and, increasingly, in the recycling of electronics (49). Cigarette smoke is by far the greatest source of Cd exposure (51); each cigarette contains 1–2 µg of Cd, and 40–60% of the Cd in inhaled smoke enters the systemic circulation (4, 14, 31). For nonsmokers, the major source (besides second-hand smoke) is ingestion of Cd-contaminated food: shellfish, beef liver and kidney, and grain and cereal products (4, 49).
In humans and other mammals, Cd adversely affects a number of organs and tissues, including lung, pancreas, testis, placenta, and bone, with kidney and liver being the two primary target organs (51). Chronic exposure to low-Cd doses predominantly leads to renal proximal tubular metabolic acidosis and osteomalacia; Cd accumulates primarily in the kidney and is eliminated very slowly with a half-life of 15–20 yr (25). With the rise of world-wide industrialization in combination with a longer life expectancy today, the level of environmental Cd has risen and Cd-induced human disease is of increasing public health concern.
Across all vertebrate taxa, testis is the most sensitive organ to acute Cd-induced damage with irreversible necrosis, occurring at doses below those causing damage to other organs (3). Testicular necrosis occurs after ischemia due to rupture of the microvasculature (9, 10, 17, 18, 28, 39, 40, 48). Certain inbred mouse strains are resistant to Cd-induced testicular damage (33). The Cdm locus was defined as a segment of DNA that confers sensitivity vs. resistance to Cd-induced testicular necrosis (43). Recently, by indirect methods (12), the Cdm locus was correlated with the solute carrier-39a8 (Slc39a8) gene, which encodes the ZIP8 transmembrane transporter protein. Retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures resulted in rvZIP8 cells capable of high rates of Cd influx. ZIP8 in rvZIP8 cells was found to have a Km of 0.62 µM for Cd2+ and 2.2 µM for Mn2+ uptake when tested in HBSS. The ZIP8 endogenous function appears to be an Mn2+-HCO3– symporter, in which Cd is able to highjack and thus gain entrance into cells using a HCO3– gradient (19).
ZIP8 mRNA is expressed at high levels in endothelial cells of the testis vasculature of sensitive strains [DBA/2J (D2) and 129S6/SvEvTac (129)] but at negligible levels in that of resistant strains [C57BL/6J (B6) and A/J] of inbred mice (12). Thus mice having elevated ZIP8 expression in testicular endothelial cells are predisposed to Cd-induced damage to this organ-specific cell type. Endothelial cell injury results in vascular leakage, which includes red cell extravasation and platelet plugging, ultimately causing testicular ischemia, followed by necrosis. Interestingly, although striking differences in ZIP8 expression exist in endothelial cells of the testis vasculature between inbred strains of mice, ZIP8 total mRNA levels are widely distributed in many tissues and do not differ substantially between strains. This observation, in conjunction with data demonstrating no mRNA sequence alterations between sensitive and resistant inbred strains of mice, led to our hypothesis that differential endothelial cell expression of Slc39a8 in blood vessels of the testis is the consequence of a DNA variant site(s) within an intron or in a 5'- or 3'-flanking region cis to this gene.
Cd sensitivity is dominant over the Cd-resistance trait (13, 43). We therefore inserted the Slc39a8 gene, as a bacterial artificial chromosome (BAC) clone that was isolated from the BAC library constructed from the genome of a sensitive strain (e.g., 129), into a resistant (e.g., B6) mouse genome to see whether testicular endothelial cell ZIP8 mRNA and protein would accumulate and whether the phenotype of Cd resistance would revert to Cd sensitivity. This would prove unequivocally that Slc39a8 is Cdm. In addition to the testicular necrosis phenotype in our Cd-treated BAC-transgenic (BTZIP8-3) line, however, we observed acute renal failure that actually preceded damage to the testis by several hours.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Isolation of BAC containing Slc39a8. A BAC carrying the Slc39a8 gene was isolated by PCR screening from the California Institute of Technology BAC 129 mouse genomic library (Invitrogen, Carlsbad, CA). Each membrane from this library carries DNA of 55,296 BAC clones, representing 27,648 unique clones; nine membranes provide eight times coverage of the entire 129 mouse genome. From this library, we isolated several clones containing all, or part, of the Slc39a8 gene; BAC clone 190B6 (Fig. 1A) was used to generate transgenic mice. Determination of the location of this clone in the genome was accomplished by end sequencing, using the universal primer sites in the BAC vector. (It is coincidental that this BAC clone, isolated from a 129 library, has "B6" as part of its name.)
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The size of this BAC clone, as defined by the two BLAST hits on the latest Ensembl database, is therefore 168,722 bp; this is also consistent with pulsed-gel electrophoresis data (not shown).
Generation of BTZIP8 mice. Transgenic mice were created by zygote pronuclear microinjection, performed by the University of Cincinnati Transgenic Mouse Core Facility. Zygotes were derived from (B6D2)F1 heterozygotes crossed with B6 mice. Heterozygous zygotes are larger in size than homozygous zygotes and, hence, have a higher success rate for microinjection. The resistant B6 and the sensitive D2 mice were chosen because marker loci around the Slc39a8 gene had previously been characterized in these inbred strains (12, 13). The BAC DNA was prepared for microinjection, following linearization with PmeI (Fig. 1A). The DNA fragment for microinjection was separated from other restriction fragments by pulsed-field gel electrophoresis, purified, and prepared for microinjection as previously described (27).
Tail-snip DNA from microinjected mice was used to screen for the presence of the transgene, using a primer set at the junction of the BAC vector backbone and the 3'-end of Slc39a8 flanking sequence: the (mouse genomic) BW-1 forward was 5'-AGGCATGATCCCTTCTGTGT-3'; the (vector) Sp6-reverse was 5'-CTATTTAGGTGACACTATG-3'.
This analysis confirmed the presence of the 3'-end of the BAC. Inclusion of the 5'-end of the BAC in transgenic mice was confirmed by sequencing PCR products carrying each of three single nucleotide polymorphisms (SNPs; described below) that differ in 129 mice vs. those in B6 and D2 mice. Celera database SNP names included mCV22353108 (T/C, 129/B6, D2) 2.1 kb upstream from the first exon (BW-17s: 5'-TCCTCACTTGGCTCGTTACC-3'; BW-18r: 5'-TCAAACTGTGGCATTTCATCA-3'); mCV24279118 (C/A, 129/B6, D2) 33 kb upstream from the first exon (BW-15s: 5'-GCTGAACCTCAGCTATTTTCG-3'; BW-16r: 5'-CAGATGGGTAGATGGATGGA-3'); and mCV24279486 (T/C, 129/B6, D2) 65 kb upstream from the first exon (BW-22s: 5'-TGTTTAGCTCTGTGCCACATTT-3'; BW-23r: 5'-GAGCTCTGAGAACCCCACAG-3').
Determination of BAC copy number. The BAC copy number in each BAC-transgenic line was estimated by two convergent approaches. This determination was conducted on mice bred to homozygosity for the B6 endogenous Slc39a8 alleles and containing a single transgene locus. First, from the Celera mouse SNP database plus our sequencing of introns, we identified a restriction fragment-length polymorphism (RFLP; underlined) between B6 and 129 within intron 6 of the Slc39a8 gene: 5'-CCCATGAC(C/T,129/B6)TGCATTCC-3'. The following PCR primers are centered on this restriction site: P115 (5'-AGAAAGCATGAAACCAGAAGC-3') and P116 (5'-ATGCTGGCCAGTTTGCTTAC-3').
AatII digestion was used to cut this PCR fragment. Staining with ethidium bromide allowed UV detection of two fragments, which we then quantified using a Typhoon Phosphorimager (Amersham Biosciences, Piscataway, NJ). Second, we used fluorescent sequencing and the SNaPshot technique to quantify the SNP involved in this RFLP, as previously described (35).
Northern hybridization analysis. Tissues as indicated were harvested and frozen in liquid N2 until use. Total RNA was extracted by using TriReagent and the manufacturer's suggested protocol (Molecular Research Center, Cincinnati, OH). RNA was size-separated on agarose gels and transferred to nylon membranes; the membranes were blocked and hybridized, as previously described (11). For the ZIP8 cRNA probe, it was transcribed from a template of linearized plasmid of the entire ZIP8 open-reading frame in pBlueScript. This plasmid was linearized with BamHI, and T3 RNA polymerase was used to synthesize a cRNA probe of the full-length ZIP8 open-reading frame of 1,386 bp.
Quantitative PCR. Total RNA (2.5 µg) from various tissues as indicated was used as a template for reverse transcription and primed with oligo(dT) using the SuperScript III first-strand kit, according to the manufacturer's recommendations (Invitrogen). PCR primers for the ZIP8 mRNA were 5'-CTCGCCTTCAGTGAGGATGT-3' (forward; BW-24) and 5'-GCTTTGCGTTGTGCTTTCTT-3'(reverse; BW-25). PCR primers for the OAT1 (SLC22A6) mRNA were 5'-GACCAGCCTGCAGAAGGAAC-3' (forward) and 5'-CAGGGATGTGCGAATGATTG-3' (reverse). PCR primers for the DMT1 (SLC11A2) mRNA were 5'-TCAGAGCTCCACCATGACTG-3' (forward) and 5'-TGTGAACGTGAGGATGGGTA-3' (reverse).
Tissues for histology. At the indicated times after treatment, tissues were harvested, cut into small pieces with a razor blade, and fixed in phosphate-buffered 4% paraformaldehyde. For immunofluorescence, mice were anesthetized with avertin and perfused via the heart with 20 ml of PBS and then 20 ml of 4% paraformaldehyde. After dehydration, the tissues were embedded in paraffin. Hematoxylin-stained sections were photographed at several magnifications, using standard procedures.
In situ hybridization. The template for cRNA probes included a portion of the ZIP domain. This sequence is unique to ZIP8 and was generated by PCR using the primers 5'-AATTAACCCTCACTAAAGGGGGATCCGCTATGCCAACCCCGCTG-3' (P081) and 5'-GTAATACGACTCACTATAGGGCATCGATGCAAGATCACAAAGTCCCCT-3' (P082). PCR products contained a 5' T3 polymerase promoter and 3' T7 polymerase promoter for generation of sense and antisense probes, respectively, as described previously (12).
Immunofluorescence and immunohistochemistry.
An antibody to ZIP8, which recognizes a peptide in a large predicted cytoplasmic loop, was prepared and affinity-purified by Bethyl Laboratories (Montgomery, TX). The 16-mer immunogen is contained within a 50-mer covering positions 251–300 of ZIP8: GVTCYANPAVTEPNGHIHFDTVSVVSLQDGKTEPSSCTCLKGPKLSEIGT. For immunofluorescence, tissue sections were cleansed of paraffin, hydrated, and blocked with PBS-Tween 20 + 10% goat serum and 1% BSA overnight at 4°C. Sections were reacted with 
-ZIP8 (1/200) for 4 h at room temperature. After sections were washed and blocked again, sections were incubated with a secondary Alexa Fluor 594-conjugated goat anti-rabbit antibody (Invitrogen) at a dilution of 1:100 for 2 h. To label specifically endothelial cells of the testis, slides were washed and then incubated with a mouse monoclonal anti-CD31 antibody (1/20 dilution; Abcam, Cambridge, MA), followed by detection by a goat anti-mouse Alexa Fluor 488-conjugated secondary antibody (Invitrogen) at a dilution of 1:100 for 2 h. To label specifically epithelial cells of the proximal convoluted tubules (PCT), the slides were washed and then incubated with fluorescein-conjugated Lotus tetragonolobus lectin (Sigma, St. Louis, MO) at a dilution of 1:200 for 30 min at room temperature. Slides were washed and coverslipped with Vectashield 4',6-diamidino-2-phenylindole mounting medium (Vector Laboratories, Burlingame, CA).
For immunohistochemistry, sections were prepared and incubated with primary antibody as above. The primary antibody was detected with the TSA BiotinSystem (catalog no. NEL700A; Perkin-Elmer, Boston, MA), according to the manufacturer's recommendations. In some experiments, peptide preadsorption was used to assess antibody specificity. In these experiments, primary antibody was incubated overnight with or without a 200-fold molar excess of the peptide immunogen, and immunohistochemistry was conducted as above.
Enzyme and small-molecule measurements to assess kidney function.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, following the manufacturer's suggested protocols and using the Hitachi AST (SGOT) and ALT (SGPT) reagent kits, respectively, from Pointe Scientific (Canton, MI). Plasma blood urea nitrogen and serum creatinine phosphokinase were determined with the use of Stanbio enzymatic procedures 2050 and 0910, respectively (Stanbio, Boerne, TX). Before urine was collected, mice were hydrated with 1.0 ml H2O by gavage; urine was collected for the next 2 h from metabolic cages. Urinary and serum creatinine were measured by the Stanbio direct creatinine Liquicolor procedure (no. 0420). Urinary N-acetyl-
-D-glucosaminidase (NAG) was measured by first diluting 10:1 to minimize any effects of enzyme inhibitors; then, NAG was measured via liberation of p-nitrophenylate (23). Urinary 
-glutamyltranspeptidase (GGT) was measured by the use of glutamyl derivatives of aromatic amines as substrate (45); L--glutamyl-p-nitroanilide was added as a substrate (21), with glycylglycine to increase the speed of the reaction (30). The increase in absorbance of p-nitroaniline at 405 nm is proportional to GGT activity (42). Urinary glucose, ketones, protein, blood, and pH were measured with Diascreen-5 reagent strips for urinalysis (Hypoguard, Minneapolis, MN). Blood pressure and heart rate measurements were taken by tail-cuff measurements (BP analysis system model MC 4000; Hatteras Instruments; Cary, NC).
Sample preparation for determining tissue Cd concentrations.
At the indicated times after Cd treatment, tissues (100 mg of kidney, liver, lung and brain) were placed in septum-sealed glass tubes and treated with 0.5 ml of 50% (vol/vol) HNO3 and subjected to microwave digestion (150 W; 1.00-min ramp time, 2.00-min hold time; 250 psi; 170°C), using the CEM Explorer system equipped with the Discover auto-sampler (Matthews, NC) (50). Digested samples were then diluted to 10.0 ml with double-distilled water containing an yttrium internal standard (100 µg/l).
Inductively coupled plasma mass spectrometer.
An Agilent 7500ce (Agilent Technologies, Tokyo, Japan) inductively coupled plasma mass spectrometer equipped with shielded-torch and collision/reaction-cell technology was used for the element-specific detection of Cd. The collision/reaction cell consisted of an octopole-ion guide, operated in "rf-only" mode, which also served for the removal of polyatomic interferences. Forward power was 1,500 W (with shielded torch); plasma gas-flow rate was 15.6 l/min; auxiliary gas-flow rate was 1.0 l/min; carrier gas-flow rate was 1.20 l/min; the nebulizer was glass-expansion microcentric; spray chamber was 2°C (Scott double-channel); sampling depth was 6 mm; sampling and skimmer cones were nickel; dwell time was 0.1 s; isotopes monitored included 55Mn, 66,68Zn, 111,112,113,114Cd, and 89Y (internal standard); and the octopole reaction system used helium (flow optimized before each experiment).
Statistical analysis. Statistical significance between groups was determined by ANOVA among groups and Student's t-test between groups. All assays were performed in duplicate or triplicate and repeated at least twice. Statistical analyses were also performed with the use of SAS statistical software (SAS Institute, Cary, NC) and Sigma Plot (Systat Software, Point Richmond, CA).
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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For screening founder transgenic mice by PCR, we used a primer set that amplified the boundary between the vector's SP6 promoter and the 3'-most flanking sequence of clone 190B6. This PCR reaction, which confirmed the presence of the intact 3'-end of clone 190B6, detected three female founder mice, which were designated BTZIP8-5, -6, and -3 (Fig. 1B). Although microsatellite differences within the Slc39a8 gene between B6 and 129 mice exist, none is close to the 5'-end of the BAC used for transgenesis. Sequence analysis revealed a SNP located 19 kb downstream of the PmeI site as the 5'-most sequence difference that exists between 129 and B6 mice in clone 190B6; this PmeI site was used to linearize the BAC. Sequence analysis of this SNP confirmed the presence of transgene sequences very near to the 5'-end of the BAC in all transgenic founder mice; thus we believe that all three founder mice likely have integrated the complete transgene.
Each founder female was crossed with B6 male mice, and the offspring were screened for the presence of the transgene. BTZIP8-5 and BTZIP8-6 mice each delivered three litters, and no transgenic offspring were detected. On the other hand, founder BTZIP8-3 and her offspring have continued to deliver transgenic offspring that do not vary from a 1:1 Mendelian ratio. The official nomenclature for the BAC-transgenic BTZIP8-3 mouse line is TgB6.129/SvJ(Slc39a8)3Neb.
Estimation of BAC copy number. Large DNA fragments carried in BACs often contain sufficient flanking sequences to allow for the formation of independent gene loci (20, 37). This, in turn, leads to transgenes that are expressed similarly to the endogenous gene, in an organ-specific, cell type-specific, and temporal-specific manner; thus expression is usually copy- number-dependent and integration-site-independent. By centering an intronic RFLP within a PCR product, we were able to determine the ratio of B6 (endogenous) vs. 129 (transgene) copies of the Slc39a8 gene in each of the three founder mice (Fig. 1B). The quantitative sequencing SNaPshot technique involves primer extension (of a single allele-specific fluorescent dideoxynucleotide, represented by the diagnostic SNP) followed by fluorescence quantification. The BTZIP8-5 and BTZIP8-6 founders contained 5 ± 1 and 6 ± 1 copies of the transgene Slc39a8, respectively. The BTZIP8-3 founder and offspring have an integrated transgene locus containing 3 ± 1 copies of the transgene Slc39a8, which has continued to follow Mendelian transmission.
Accumulation of ZIP8 mRNA in BTZIP8-3 transgenic mice.
The BTZIP8-3 line was maintained as heterozygotes at the transgene locus, such that (including the two alleles of the endogenous Slc39a8 gene) these BAC-transgenic mice contain a total of 5 ± 1 Slc39a8 gene copies. ZIP8 mRNA levels varied considerably among tissues of the transgenic mouse line (Fig. 2A). Tissue variation was very similar between transgenic mice and nontransgenic littermate controls. Compared with the loading control of total RNA (Fig. 2A), ZIP8 mRNA levels were highest in lung > kidney = testis >> liver > brain = small intestine. When normalized to GAPDH (Fig. 2B), ZIP8 mRNA levels within a given tissue were consistently 2.5 times higher in transgenic than in nontransgenic mice, and these 2.5-fold-enhanced mRNA levels in BTZIP8-3 mice are quite close to levels expected for a gene copy-dependent increase: five copies in transgenic mice vs. two copies in nontransgenic mice. These analyses strongly suggest that the transgene Slc39a8 behaves similarly to the endogenous gene, with regard to tissue-specific expression, and is expressed in a copy-number-dependent manner.
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In situ hybridization of testis from BTZIP8-3 mice (Fig. 3) showed a robust signal in endothelial cells of the testis vasculature. In contrast, nontransgenic littermate control mice showed little expression in these cells, similar to what had been found in the A/J and B6 resistant strains (12). As reported previously, considerable ZIP8 mRNA signal is present throughout the seminiferous tubules in equal amounts in sensitive and resistant strains of mice (12). This finding indicates that, whatever DNA variant site is responsible for expression in the testis vasculature, this mutation does not impact the level of expression in other cell types in the testis that showed no difference between Cd-sensitive and Cd-resistant mouse strains. The same is true for all cell types in other tissues and organs that we have examined.
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Using an anti-ZIP8 antibody, we detected a strong signal in testicular vascular endothelial cells (identified as cells staining with the anti-CD31 endothelial cell-specific antibody) in BTZIP8-3 mice but not in cells of nontransgenic littermate controls (Fig. 4). Peptide preadsorption, using the immunizing peptide and nonimmune IgG used at the same concentration as ZIP8, both resulted in elimination of the ZIP8 immunofluorescent signal (not shown). ZIP8-specific staining was also present in seminiferous tubules of mice of both mouse genotypes, although this is not seen in the high-magnification images shown to highlight blood vessel staining in Fig. 4. Homogeneous seminiferous tubule staining is consistent with the ZIP8 in situ hybridization data (Fig. 3). In BTZIP8-3 mice, the ZIP8 protein was detected in the endothelial cells of capillaries and in areas where larger vessels bifurcate to become capillaries. Endothelial cells clearly express ZIP8 protein on the apical membranes of polarized cells in culture (19), a finding that is consistent with the movement of Cd from plasma into endothelial cells. In summary, sequences contained within the 168.7 kb of BAC clone 190B6 must include the nucleotide(s) needed to specify endothelial cell expression of ZIP8 in the testis vasculature. By extrapolation, this same 168.7 kb on mouse chromosome 3 must contain the mutation that silences this gene in the testis vasculature endothelium of inbred strains that are resistant to Cd-induced testicular damage.
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When we initially had tried to evaluate Cd-induced testicular damage in BTZIP8-3 mice, we treated three BTZIP8-3 mice and three nontransgenic littermate controls and planned to evaluate testicular damage at 24 h. Before the 24-h time point, however, all BTZIP8-3 mice became moribund and had to be euthanized. As observed for Cd-sensitive strains of mice, this was not the case for the nontransgenic littermate controls. We wondered whether the Cd-treated BTZIP8-3 mice were dying from severe damage to the liver, which is a major target organ of acute poisoning by high Cd doses. Cd-treated BTZIP8-3 mice showed modest rises in ALT and AST levels, however, compared with nontransgenic littermate controls (Table 1). Also, no striking histological differences in the liver between Cd-treated BTZIP8-3 and Cd-treated nontransgenic littermate controls were found (data not illustrated). These data indicate that this dose of Cd was not acutely hepatotoxic, and liver damage was certainly not the reason for BTZIP8-3 mice dying and littermates not dying after Cd treatment.
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Histological examination of kidney from BTZIP8-3 mice, 12 h after the subcutaneous injection of Cd, showed injury localized predominantly to the cortical region (Fig. 7), characterized by mild tubular dilatation and moderately extensive vacuolization of cytoplasm in the epithelial cells, mainly from the proximal tubules. In some areas, the vacuoles were more confluent and displayed subnuclear localization. In addition, there was substantial loss of the brush borders. Kidneys from the nontransgenic littermate controls showed minimal injury, localized also to the cortical region and proximal tubules, with significantly less epithelial vacuolization or loss of brush borders. The interstitium and glomeruli, on the other hand, revealed no histological changes in either the BTZIP8-3 mice or nontransgenic littermates.
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Acute kidney failure in response to Cd could be caused by rhabdomyolysis, endotoxic shock, or vascular endothelial damage. Because serum creatinine phosphokinase was not highly elevated (Table 1), rhabdomyolysis is quite unlikely. Although we found a decrease in blood pressure (Table 1), it was not statistically significantly different between Cd-treated BTZIP8-3 mice and littermate controls; this rules out vascular endothelial damage or endotoxic shock and suggests that death, which usually occurred in Cd-treated BTZIP8-3 mice between 15 and 30 h after treatment with the 30-µmol/kg dose, is most likely the result of acute renal failure.
Localization of ZIP8 in kidney. In BTZIP8-3 and littermate control mice not previously treated with Cd, the in situ hybridization signal in kidney (Fig. 8) was high in the tubular cortex and virtually absent in the medulla. Grain density in the tubular cortex of kidneys from BTZIP8-3 mice was higher than that of nontransgenic littermate controls; this is consistent with increased ZIP8 expression in all tissues examined in the transgenic mice (Fig. 2). In general, in situ hybridization signals were highest in cells having morphology consistent with proximal tubular epithelium, although a gradient in expression between the different segments (i.e., S1, S2, S3) was not obvious.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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There is no in vitro or cell culture assay that can prove that Slc39a8 is Cdm; the blood-barrier characteristics of the endothelium of the testis vasculature are unique to any other cell type in the body (6, 22), including the blood-brain barrier. We therefore reasoned that a Slc39a8 allele from a sensitive strain of mice should confer sensitivity to Cd-induced testicular necrosis in a resistant inbred strain. The BT-ZIP8–3 line was generated (Fig. 1) and characterized: ZIP8 mRNA levels in various tissues and quantification of ZIP8 mRNA (Fig. 3) provided strong evidence that the BTZIP8-3 line expresses its Slc39a8 transgenes in the endothelial cells of the testis vasculature. As endpoints for assessing phenotypic reversion and proof the Slc39a8 gene is Cdm, the transgenic mice showed expression of ZIP8 mRNA (Fig. 3) and protein (Fig. 4) in vascular endothelial cells of the testis and grossly displayed exquisite sensitivity to Cd-induced testicular damage (Fig. 5). Histological analysis also confirmed Cd-induced damage in the testis (Fig. 6) but not in the liver (data not shown).
Somewhere within this BAC (Fig. 1) is located the DNA variant site responsible for the phenotype of resistance to Cd-induced testicular necrosis. This Slc39a8 mutation does not appear to be associated with any other strain-specific difference in sensitivity to Cd-induced damage involving organ systems other than the testis (43). Hence, this conclusion is consistent with the ZIP8 transporter possibly being able to mediate Cd injury to organ systems aside from testis. Indeed, the transporter properties of ZIP8 make it a likely candidate for uptake of Cd into cells (12, 19), which is a requirement for Cd damage. Inbred mouse strain-dependent differences in ZIP8 expression are apparently limited, however, to endothelial cells of the testicular vasculature. Indeed, even within the testis, the majority of ZIP8 mRNA resides in cells of the seminiferous tubules, and this localization is strain-independent (12).
It is not clear why the BTZIP8-5 and BTZIP8-6 founders did not produce transgenic offspring, although, compared with the BTZIP8-3 line, these founders contained several more transgene copies. It is thus very likely that "having more than five copies of the mouse Slc39a8 gene" might always lead to embryonic lethality.
Northern hybridization analysis indicated that ZIP8 is expressed at high levels in kidney (Fig. 2). This is of great interest with regard to Cd-induced disease in humans because the kidney proximal tubule has been known for decades to be the most critical cell target for chronic Cd exposure. We demonstrated that the highest amounts of ZIP8 mRNA (Fig. 8) and protein (Fig. 9) are expressed in the renal proximal tubules and that expression is greater in the BTZIP8-3 line than in the nontransgenic littermate. Moreover, Cd levels are twofold greater in the BTZIP8-3 mouse than in the nontransgenic littermate. Thus a gene-dose increase (from two copies of the Slc39a8 gene to five copies) heightens Cd uptake in the renal proximal tubules, following acute administration of this metal. The outcome is acute renal failure, and the various signs indicating renal proximal tubular damage actually supersede testicular necrosis by several hours.
DMT1 (NRAMP2; official name SLC11A2) is a proton-coupled symporter of several divalent metal ions, including Fe2+, Zn2+, Mn2+, Co2+, Cd2+ and Ni2+, and plays a critical role in the pathogenesis of iron deficiency and overload disorders (38). One group (16) showed membrane-bound SLC11A2 was localized in the collecting ducts more than in the PCT or DCT, and a second group (8) showed it was localized at the brush border and apical pole of epithelial cells of the PCT. Calcium channels have also been postulated to transport Cd in kidney, but there are no definitive data to date to support that this transport occurs in the PCT (7). There is evidence that Cd is transported by SLC11A2 in the DCT (16), which is not the primary target for Cd in the kidney. The Km of ZIP8 for Cd is 0.62 µM in mouse cell cultures using HBSS (19), which is half as much as the Km of SLC11A2 for Cd when the transporter mRNA was expressed in Xenopus laevis oocytes (36). Thus SLC11A2 also transports Cd with high affinity, a transport quality likely essential for the uptake of low levels of environmental Cd. We believe, therefore, that the most relevant transporter for Cd-induced pathophysiological changes will be the high-affinity transporter with the cell-type-specific localization that fits the target cell population. In this regard, ZIP8 seems a better candidate for mediating kidney toxicity because it is expressed in proximal tubules, whereas SLC11A2 is reported to be expressed in DCT. Herein, we show that gene-dose increases in Slc39a8 can indeed negatively impact proximal tubular damage, further suggesting an important role for ZIP8 in this cell type.
If basolateral entry of Cd into the PCT cell is important, one potential transporter would be OAT1 (SLC22A6), which has been shown in Madin-Darby canine kidney cells stably transfected with human SLC22A6 to mediate the inward transport of Hg2+ in the form of Cys-S-Hg-S-Cys and other similar complexes (52). Chronic Cd treatment of rats leads to a decreased Vmax of the Na+/H+ exchange antiporter-3 in the PCT (2), but we believe this inhibitory effect is a consequence of Cd poisoning of proteins in the cell (most likely due to excess Cd, taken up by the cell via ZIP8). In the present study, we have found no compensatory upregulation of either SLC11A2 or SLC22A6 mRNA, when comparing untreated BTZIP8-3 mice with nontransgenic littermates.
In humans, Cd-induced renal disease occurs, following Cd accumulation in the cells comprising the S1 and S2 segments of the PCT over time, until Cd concentration in these cells reaches a threshold for Cd damage (44). No system that measures acute Cd toxicity (including this report) accurately duplicates this process. It has previously been reported that Cd-metallothionein (Cd-MT) complexes are extremely nephrotoxic, relative to CdCl2, and Cd-MT has been hypothesized to be important in Cd-induced nephrotoxicity (46). Studies with Mt1/2(–/–) knockout mice have shown that Cd-MT is not necessary for PCT to be the target cell population for chronic Cd-induced toxicity; in fact, Mt1/2(–/–) mice are more sensitive to chronic Cd-induced nephrotoxicity than wild-type mice (32). These data do not mean that Cd-MT is not an important source of Cd in the kidney, in organisms that express MT, but the data show that Cd-MT complexes are not necessary. Furthermore, as noted above, data suggest that Cd-induced nephrotoxicity requires the accumulation of Cd in the PCT rather than the acute inhibition of cellular transport (46). Thus any model for Cd-induced nephrotoxicity should include progressive Cd uptake that is not acutely toxic but rather, with time, leads to the chronic slow accumulation Cd in the PCT. Any transporter involved in this process must possess cell-type-specific expression and membrane localization, consistent with the transporter being interposed between the milieu that contains Cd and the target cell type. ZIP8 is an apical transporter with high expression in lung, kidney, and testis; thus ZIP8 is perhaps a strong candidate for uptake of Cd from the airways in the lungs and, as we show in the present report, for uptake of Cd by testis vasculature endothelial cells and reuptake of Cd from the glomerular filtrate into the PCT epithelial cells.
The molecular form of Cd carried in the blood might be an important determinant in the mechanism of Cd toxicity. No one knows the mechanisms by which Cd-MT complexes lead to Cd accumulation in the PCT. Transporters such as ZIP8 may be important for this uptake, as well as the free Cd, which itself is likely associated with thiol groups within the plasma (1). In the present report, we have demonstrated that ZIP8 is present in the PCT and that a modest gene-dose overexpression of ZIP8 in the PCT renders these cells sensitive to acute Cd-induced nephrotoxicity. Together with data that demonstrate that ZIP8 is a high-affinity rogue Cd transporter (19), ZIP8 is an excellent candidate for the transporter that allows Cd to accumulate in the PCT during chronic Cd intoxication. Whether ZIP8 is important for chronic Cd intoxication, leading to such clinical disorders as Fanconi syndrome, will require further experimentation.
It is difficult to dissociate impaired energy production or other biochemical defects from a defective transporter system. For example, just as chronic Cd causes a decreased Vmax of the rat Na+/H+ exchange antiporter-3 (2), we postulate that ZIP8-mediated reabsorption of Cd might also poison the NBC1 transporter, causing decreased HCO3– reuptake, which would lead to metabolic acidosis. Whether functional mutations in the (mouse or human) SLC39A8 gene might exist and cause disease and how pivotal the ZIP8 transporter might be (to the various inherited and acquired forms of renal Fanconi syndrome) are long-range goals in our laboratories.
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Portions of these data were presented at the 45th Annual Meeting of the Society of Toxicology, San Diego, CA (March, 2006).
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2. Ahn DW, Chung JM, Kim JY, Kim KR, Park YS. Inhibition of renal Na+/H+ exchange in cadmium-intoxicated rats. Toxicol Appl Pharmacol 204: 91–98, 2005.[CrossRef][Web of Science][Medline]
3. Alsberg CL, Schwartze EW. Pharmacological action of cadmium. J Pharmacol Exp 13: 504–505, 1919.
4. ATSDR. Toxicological Profile for Cadmium. Atlanta, GA: Agency for Toxic Substances and Disease Registry, 1999.
5. Barbier O, Jacquillet G, Tauc M, Cougnon M, Poujeol P. Effect of heavy metals on, and handling by, the kidney. Nephron Physiol 99: 105–110, 2005.[CrossRef]
6. Bart J, Groen HJ, van der Graaf WT, Hollema H, Hendrikse NH, Vaalburg W, Sleijfer DT, de Vries EG. An oncological view on the blood-testis barrier. Lancet 3: 357–363, 2002.[CrossRef][Web of Science]
7. Bridges CC, Zalups RK. Molecular and ionic mimicry and the transport of toxic metals. Toxicol Appl Pharmacol 204: 274–308, 2005.[CrossRef][Web of Science][Medline]
8. Canonne-Hergaux F, Gros P. Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice. Kidney Int 62: 147–156, 2002.[CrossRef][Web of Science][Medline]
9. Chiquoine AD. Observations on the early events of cadmium necrosis of the testis. Anat Rec 149: 23–35, 1964.[CrossRef][Medline]
10. Clegg EJ, Carr I. Changes in the blood vessels of the rat testis and epididymis produced by cadmium chloride. J Pathol Bacteriol 94: 317–322, 1967.[CrossRef][Web of Science][Medline]
11. Dalton TP, Dieter MZ, Matlib RS, Childs NL, Shertzer HG, Genter MB, Nebert DW. Targeted knockout of Cyp1a1 gene does not alter hepatic constitutive expression of other genes in the mouse [Ah] battery. Biochem Biophys Res Commun 267: 184–189, 2000.[CrossRef][Web of Science][Medline]
12. Dalton TP, He L, Wang B, Miller ML, Jin L, Stringer KF, Chang X, Baxter CS, Nebert DW. Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis. Proc Natl Acad Sci USA 102: 3401–3406, 2005.
13. Dalton TP, Miller ML, Wu X, Menon A, Cianciolo E, McKinnon RA, Smith PW, Robinson LJ, Nebert DW. Refining the mouse chromosomal location of Cdm, the major gene associated with susceptibility to cadmium-induced testicular necrosis. Pharmacogenetics 10: 141–151, 2000.[CrossRef][Web of Science][Medline]
14. Elinder CG, Kjellstrom T, Lind B, Linnman L, Piscator M, Sundstedt K. Cadmium exposure from smoking cigarettes: variations with time and country where purchased. Environ Res 32: 220–227, 1983.[Medline]
15. Fay RM, Mumtaz MM. Development of a priority list of chemical mixtures occurring at 1,188 hazardous waste sites, using the HazDat database. Food Chem Toxicol 34: 1163–1165, 1996.[CrossRef][Web of Science][Medline]
16. Ferguson CJ, Wareing M, Ward DT, Green R, Smith CP, Riccardi D. Cellular localization of divalent metal transporter DMT1 in rat kidney. Am J Physiol Renal Physiol 280: F803–F814, 2001.
17. Gunn SA, Gould TC, Anderson WA. The selective injurious response of testicular and epididymal blood vessels to cadmium and its prevention by zinc. Am J Pathol 42: 685–702, 1963.[Web of Science][Medline]
18. Gupta RK, Barnes GW, Skelton FR. Light-microscopic and immunopathologic observations on cadmium chloride-induced injury in mature rat testis. Am J Pathol 51: 191–205, 1967.[Web of Science][Medline]
19. He L, Girijashanker K, Dalton TP, Reed J, Li H, Soleimani M, Nebert DW. ZIP8, member of the solute-carrier-39 (SLC39) metal-transporter family: characterization of transporter properties. Mol Pharmacol 70: 171–180, 2006.
20. Heintz N. BAC to the future: the use of BAC-transgenic mice for neuroscience research. Nat Rev Neurosci 2: 861–870, 2001.[CrossRef][Web of Science][Medline]
21. Henry RJ, Chiamori N, Golub OJ, Berkman S. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am J Clin Pathol 34: 381–398, 1960.[Web of Science][Medline]
22. Holash JA, Harik SI, Perry G, Stewart PA. Barrier properties of testis microvessels. Proc Natl Acad Sci USA 90: 11069–11073, 1993.
23. Horak E, Hopfer SM, Sunderman FW Jr. Spectrophotometric assay for urinary N-acetyl--D-glucosaminidase activity. Clin Chem 27: 1180–1185, 1981.
24. IARC. Meeting of the IARC working group on beryllium, cadmium, mercury and exposures in the glass manufacturing industry. Scand J Work Environ Health 19: 360–363, 1993.[Web of Science][Medline]
25. Jarup L. Cadmium overload and toxicity. Nephrol Dial Transplant 17, Suppl 2: 35–39, 2002.[Abstract]
27. Jiang Z, Dalton TP, Jin L, Wang B, Tsuneoka Y, Shertzer HG, Deka R, Nebert DW. Toward the evaluation of function in genetic variability: characterizing human SNP frequencies and establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus. Hum Mutat 25: 196–206, 2005.[CrossRef][Web of Science][Medline]
28. Johnson MH. The effect of cadmium chloride on the blood-testis barrier of the guinea pig. J Reprod Fertil 19: 551–553, 1969.
29. Joly V, Bergeron Y, Bergeron MG, Carbon C. Endotoxin-tobramycin additive toxicity on renal proximal tubular cells in culture. Antimicrob Agents Chemother 35: 351–357, 1991.
30. Kulhanek V, Dimov DM. A new useful modification for the determination of -glutamyltranspeptidase activity. Clin Chim Acta 14: 619–623, 1966.[Medline]
31. Lewis GP, Coughlin LL, Jusko WJ, Hartz S. Contribution of cigarette smoking to cadmium accumulation in man. Lancet 1: 291–292, 1972.[Web of Science][Medline]
32. Liu J, Liu Y, Habeebu SS, Klaassen CD. Susceptibility of MT-null mice to chronic CdCl2-induced nephrotoxicity indicates that renal injury is not mediated by the CdMT complex. Toxicol Sci 46: 197–203, 1998.
33. Lucis OJ, Lucis R. Distribution of cadmium-109 and zinc-65 in mice of inbred strains. Arch Environ Health 19: 334–336, 1969.[Web of Science][Medline]
34. National Toxicology Program. Ninth Report on Carcinogens. National Toxicology Program. Research Triangle Park, NC: National Institute of Environmental Health Sciences, 2000, p. 1–85.
35. Norton N, Williams NM, Williams HJ, Spurlock G, Kirov G, Morris DW, Hoogendoorn B, Owen MJ, O'Donovan MC. Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools. Hum Genet 110: 471–478, 2002.[CrossRef][Web of Science][Medline]
36. Okubo M, Yamada K, Hosoyamada M, Shibasaki T, Endou H. Cadmium transport by human NRAMP2 expressed in Xenopus laevis oocytes. Toxicol Appl Pharmacol 187: 162–167, 2003.[CrossRef][Web of Science][Medline]
37. Ristevski S. Making better transgenic models: conditional, temporal, and spatial approaches. Mol Biotechnol 29: 153–163, 2005.[CrossRef][Web of Science][Medline]
38. Rolfs A, Bonkovsky HL, Kohlroser JG, McNeal K, Sharma A, Berger UV, Hediger MA. Intestinal expression of genes involved in iron absorption in humans. Am J Physiol Gastrointest Liver Physiol 282: G598–G607, 2002.
39. Schlaepfer WW. Sequential study of endothelial changes in acute cadmium intoxication. Lab Invest 25: 556–564, 1971.[Web of Science][Medline]
40. Setchell BP, Waites GM. Changes in the permeability of the testicular capillaries and of the "blood-testis barrier" after injection of cadmium chloride in the rat. J Endocrinol 47: 81–86, 1970.
41. Stoeppler M. Metals and their compounds in the environment. In: Cadmium, edited by Weinheim ME. New York: VCH, 1991, p. 803–851.
42. Szasz G. A kinetic photometric method for serum -glutamyltranspeptidase. Clin Chem 15: 124–136, 1969.[Abstract]
43. Taylor BA, Heiniger HJ, Meier H. Genetic analysis of resistance to cadmium-induced testicular damage in mice. Proc Soc Exp Biol Med 143: 629–633, 1973.[CrossRef][Medline]
44. Thevenod F. Nephrotoxicity and the proximal tubule. Insights from cadmium. Nephron Physiol 93: 87–93, 2003.
45. Tiez NW. Clinical Guide to Laboratory Tests. Philadelphia, PA: Saunders, 1990.
46. Tsuruoka S, Sugimoto K, Muto S, Nomiyama K, Fujimura A, Imai M. Acute effect of cadmium-metallothionein on glucose and amino acid transport across the apical membrane of the rabbit proximal tubule perfused in vitro. J Pharmacol Exp Ther 292: 769–777, 2000.
47. Waisberg M, Joseph P, Hale B, Beyersmann D. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192: 95–117, 2003.[CrossRef][Web of Science][Medline]
48. Waites GM, Setchell BP. Changes in blood flow and vascular permeability of the testis, epididymis and accessory reproductive organs of the rat after the administration of cadmium chloride. J Endocrinol 34: 329–342, 1966.
49. World Health Organization. Cadmium: environmental health criteria. WHO Report 134: 117–120, 1992.
50. Yathavakilla SV, Shah M, Mounicou S, Caruso JA. Speciation of cationic selenium compounds in Brassica juncea leaves by strong cation-exchange chromatography with inductively-coupled-plasma mass spectrometry. J Chromatogr A 1100: 153–159, 2005.[CrossRef][Web of Science][Medline]
51. Zalups RK, Ahmad S. Molecular handling of cadmium in transporting epithelia. Toxicol Appl Pharmacol 186: 163–188, 2003.[CrossRef][Web of Science][Medline]
52. Zalups RK, Ahmad S. Transport of N-acetylcysteine S-conjugates of methylmercury in Madin-Darby canine kidney cells stably transfected with human isoform of organic anion transporter-1. J Pharmacol Exp Ther 314: 1158–1168, 2005.
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