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The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed. [ABSTRACT FROM AUTHOR]

Carcinogenesis is a multistep process that is frequently associated with p513 inactivation. The class 1 carcinogen cadmium (Cd2+) causes renal cancer and is known to inactivate p53. G2/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd2+-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd2+ (50-100 μM) induced DNA damage within 1-6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1-3 h, measured with dihydrorhodamine 123, and G2/M cell cycle arrest at 6 h, which were abolished by the antioxidant a-tocopherol (100 μM). Cd2+-induced G2/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 μM), which relieves G2/M transition block, abolished Cd2+-induced G2 arrest and increased apoptosis. This was accompanied by prevention of Cd2+-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells Cd2+-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G2/M arrest. This may promote survival of premalignant PT cells and Cd2+ carcinogenesis. [ABSTRACT FROM AUTHOR]

The positively charged fluorescent dyes ethidium (Et+) and propidium (Pr2+) are widely used as DNA and necrosis markers. Et+ is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22AI), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (⩽500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCTI/2 are involved. In mouse kidney, perfused Et+ accumulated predominantly in the S2/S3 segments of the PT, but not Pr2+. In cells stably overexpressing human OCTs (hOCTs), Et+ uptake was observed with Km values of 0.8 ± 0.2 μM (hOCT1), 1.7 ± 0.5 μM (hOCT2), and 2.0 ± 0.5 μM (hOCT3), whereas Pr2+ was not transported. Accumulation of Et+ was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP+), cimetidine, and tetraethylammonium (TEA+). For hOCT1 and hOCT2, the IC50 values for MPP+, TEA+, and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et+ uptake by MPP+ and cimetidine was shown to be competitive. Et also inhibited transport of 0.1 μM [3H]MPP+ by all hOCT isoforms with IC50 values between 0.4 and 1.3 μM, and the inhibition of hOCT1-mediated uptake of MPP+ by Et+ was competitive. In Oct1/2-/- mice, Et+ uptake in the PT was almost abolished. The data demonstrate that Et+ is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et and their strong expression in various organs, strict safety guidelines for Et+ handling should be reinforced. [ABSTRACT FROM AUTHOR]

Chronic exposure to Cd2+ causes renal proximal tubular (PT) damage. Cd2+ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd2+ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd2+ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to down-regulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 ± 9.6 and 36.9 ± 14.4% (n = 5-6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 µM CdMT-1 by 35.6 ± 4.2% (n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd2+ toxicity was altered by the effective DMTI shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd2+-induced cell death signaling are not affected by DMTI knock-down. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity. [ABSTRACT FROM AUTHOR]

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMTI) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMTI in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 C1.2 cell line. RT-PCR revealed that mRNAs encoding all four DMTI splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 C1.2 cells. Immunostaining of rat kidney cortex or WKFW-0293 C1.2 cells showed that DMTI protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMTI partially colocalized with the late endosomal/lysosomal proteins LAMPI and cathepsin-L. Using immunogold labeling, DMTI was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 C1.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMTI. In conclusion, renal proximal tubular cells express DMTI in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+. [ABSTRACT FROM AUTHOR]

Cd2+ induces apoptosis of kidney proximal tubule (PT) cells. Mitochondria play a pivotal role in toxic compound induced apoptosis by releasing cytochrome c. Our objective was to investigate the mechanisms underlying Cd2+-induced cytochrome c release from mitochondria in rat PT cells. Using Hoechst 33342 or MTT assay, 10 µM Cd2+ induced ∼5-10% apoptosis in PT cells at 6 and 24 h, which was associated with cytochrome c and apoptosisinducing factor release at 24 h only. This correlated with previously described maximal intracellular Cd2+ concentrations at 24 h, suggesting that elevated Cd 2+ may directly induce mitochondrial liberation of proapoptotic factors. Indeed, Cd2+ caused swelling of energized isolated kidney cortex mitochondria (EC50 ∼9 µM) and cytochrome c release, which were independent: of permeability transition pore (PTP) opening since PTP inhibitors cyclosporin A or bongkrekic acid had no effect. On the contrary, Cd2+ inhibited swelling and cytochrome c release induced by PTP openers (PO43- or H2O2 + Ca2+). The mitochondrial Ca2+ uniporter (MCU) played a key role in mitochondrial damage: 1) MCU inhibitors (La3+, ruthenium red, Ru360) prevented swelling and cytochrome c release; and 2) ruthenium red attenuated Cd2+ inhibition of PO43- -induced swelling. Using the Cd2+-sensitive fluorescent indicator FluoZin-1, Cd2+ was also taken up by mitoplasts. The aquaporin inhibitor AgNO3 abolished Cd2+-induced swelling of mitoplasts. This could be partially mediated by activation of the mitoplast-enriched water channel aquaporin-8. Thus cytosolic Cd2+ concentrations exceeding a certain threshold may directly cause mitochondrial damage and apoptotic development by interacting with MCU and water channels in the inner mitochondrial membrane. [ABSTRACT FROM AUTHOR]