Toxicity of the kidney proximal tubule by cadmium-metallothionein: Role of lipocalin-2 receptor-mediated endocytosis, vesicle trafficking and lysosomal DMT1
Final Report Abstract
The transition metal ion cadmium (Cd2+) is a significant environmental contaminant due to its presence in phosphate fertilizers used in agriculture. Cd2+ is taken up by the body via crops and through the food chain. The kidneys and liver together contain ~85% of the Cd body burden. More than 60% is found in the kidneys where Cd has a biological half-life of 20-30 years. The differential distribution of Cd in various organs and the long biological half-life in the kidney may explain the increased sensitivity of the kidney to Cd. This may also account for an increased incidence of chronic kidney disease in populations with chronic low Cd exposure (e.g. due to smoking) and even of end-stage renal failure. As a result of Cd’s tendency to accumulate in the proximal tubule (PT), the kidney is usually the primary critical target organ of chronic Cd toxicity in the body. But Cd is not only toxic to PT cells, but also to glomeruli and distal tubules. The major form of Cd found in the circulation is a complex with apo-thionein (CdMT), which is taken up and stored by tubule cells to cause kidney injury. We therefore aimed 1) to characterize mechanisms of receptor-mediated endocytosis of CdMT in kidney tubules other than via megalin/cubilin (the primary uptake mechanism for CdMT in PT), in particular to determine the contribution of the lipocalin-2 receptor (Lip2-R), a protein of unclear function that is expressed in the kidney, in CdMT nephrotoxicity; 2) to investigate the role of small GTP-binding protein ARF1 in trafficking of CdMT to lysosomes; and 3) to extend our understanding of the role of the divalent metal transporter 1 (DMT1) in CdMT nephrotoxicity, especially to identify interacting partners of DMT1, which may participate in transport and delivery of Cd2+ to cellular targets, such as mitochondria. We discovered that Lip2-R is mainly expressed in the distal nephron (distal convoluted tubules and inner medullary collecting ducts) in rodent kidney. Additional studies have also demonstrated Lip2-R in distal intestinal segments, in particular the colon, of rodents and humans. Using cultured cells, Lip2-R turned out to be a high-affinity (EC50 10-100 nM) multiligand receptor for proteins/protein-metal complexes (e.g. lipocalin-2/24p3/NGAL, transferrin, albumin, metallothioneins, phytochelatins) that is implicated in endocytosis/transcytosis of its cargo. Evidence was also provided that ARF1 is mainly located in sorting to late endosomes (using the markers Rab7 and CLC6) and weakly overlaps with the late endosomal/lysosomal marker CLC7, but not with markers for early (Rab5, CLC5) and recycling endosomes (Rab11). Functional studies using ARF1-wildtype and a non-functional mutant ARF1 showed that ARF1 is involved in trafficking of protein-metal complexes (CdMT, transferrin) to late endosomes/lysosomes in renal PT cells. We further discovered that DMT1 is expressed in the outer mitochondrial membrane where it interacts with the protein Tom6 and with an inner mitochondrial membrane protein (COXII). DMT1 may be important for the mitochondrial import of Fe2+, Mn2+, Cu2+ (and possibly toxic Cd2+), thus mediating key mitochondrial functions, e.g. the synthesis of heme, iron-sulfur clusters for electron transport or mitochondrial redox enzymes, such as superoxide dismutases. Moreover, our studies on rats with a mutation of DMT1 suggest that dysfunction of mitochondrial DMT1 may accelerate the deterioration of renal tubular function in ageing animals. The localization of DMT1 in mitochondria is not limited to the kidney and therefore may be relevant in other tissues as well. This discovery may affect current models how potentially damaging, redox-active Fe2+ is handled by cells and discussions how Fe2+ is targeted to mitochondria. Studies are underway to clarify the impact of Lip2-R on the function of cells, tissues and organisms in health and disease, in particular to characterize physiological functions of the Lip2-R, e.g. in adaptation to osmotic stress, and its role in kidney diseases, such as acute kidney injury associated with hemoglobinuria. Furthermore, mitochondrial DMT1 could have relevance for a variety of diseases, including Parkinson’s disease and need to be investigated in future studies.
Publications
- (2010) Catch me if you can! Novel aspects of cadmium transport in mammalian cells. Biometals 23:857-875
Thévenod, F.
- (2011) Role of Arf1 in endosomal trafficking of protein-metal complexes and cadmium-metallothionein-1 toxicity in kidney proximal tubule cells. Toxicology Letters 203:210-218
Wolff, N.A., Lee, W.-K., Thévenod, F.
- (2012). The lipocalin-2 (24p3/NGAL) receptor is expressed in the distal nephron and mediates protein endocytosis. Journal of Biological Chemistry 287:159-169
Langelueddecke C., Roussa, E., Fenton, R.A., Wolff, N.A., Lee, W.-K., Thévenod, F.
- (2013) Expression and function of the lipocalin-2 (24p3/NGAL) receptor in rodent and human intestinal epithelia PLoS ONE 8:e71586
Langelueddecke C., Roussa, E., Fenton, R.A., Thévenod, F.
(See online at https://doi.org/10.1371/journal.pone.0071586) - (2014). Differential transcytosis and toxicity of the hNGAL receptor ligands cadmium-metallothionein and cadmiumphytochelatin in colon-like Caco-2 cells: Implications for in vivo cadmium toxicity Toxicology Letters 226:228-235
Langelueddecke, C., Lee, W.-K., Thévenod, F.
(See online at https://doi.org/10.1016/j.toxlet.2014.01.049) - (2014). Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1). FASEB Journal, 2014 28:5, 2134-2145
Wolff, N.A., Ghio, A.J., Garrick, L.M., Garrick, M.D., Zhao, L., Fenton, R.A., Thévenod, F.
(See online at https://doi.org/10.1096/fj.13-240564)
