Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

Abstract

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-β2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.

Smoking-by-genotype interaction in type 2 diabetes risk and fasting glucose

“…These CUBN observations point to plausible mechanisms, namely depressed levels of vitamin B12 and HDL cholesterol, through which smoking might interact with cubilin to cause T2D. Cigarette smoking impairs cubilin-mediated renal protein reabsorption through cadmium and other contaminants, which form complexes with proteins that have high affinity for cubilin and accumulate in the proximal tubule [59]. A mendelian randomization study found an association between a genetic instrument for low vitamin B12 levels (including one CUBN variant) and higher fasting glucose levels and lower pancreatic beta-cell secretory function, as measured by HOMA-β, but not with higher odds of T2D [60].…”

Cadmium and Lead Exposure, Nephrotoxicity, and Mortality

“…Through systemic circulation, CdMT and CdPC of dietary origin and intestinal and hepatic CdMT [ 126 ] all reach the kidneys, where they are filtered by glomeruli by virtue of their small molecular weights [ 8 ]. Once they pass the glomerular membrane into the tubular lumen, they are then subsequently reabsorbed by proximal tubular epithelial cells as these cells are equipped with protein internalization mechanisms [ 131 , 132 , 133 , 134 ]. Due to a lack of biologically active mechanisms to eliminate excess metals, nearly all reabsorbed Cd is retained in the kidneys.…”

“…In summary, ingested Cd is mostly reabsorbed and sequestered in kidneys [ 126 , 131 , 132 , 133 , 134 ], while ingested Pb is taken up and retained in bone [ 135 ]. Only a small fraction of Cd and Pb in the kidneys is excreted in urine ( Section 3.4 , Figure 2 ).…”

In Vitro Evaluation of the Effects of Cadmium on Endocytic Uptakes of Proteins into Cultured Proximal Tubule Epithelial Cells

“…This could not be ascribed simply to the increase in dead cells after Cd exposure, since no effects were observed in the endocytosis efficiencies of albumin or transferrin ( Figure 4) and only the surviving cells that were not detached from the plates during Cd exposure were used for flow cytometry analyses. Although a few studies have investigated the effects of Cd on the interactions of LMW proteins with megalin/cubilin systems in cultured renal cells [18,19], the present study utilized flow cytometry for quantitatively evaluating the endocytosis efficiency of the proteins and showed the effects of Cd on the uptakes of LMW proteins in cultured PTECs.…”

“…The reabsorption of luminal biomolecules including β 2 -MG and MT by PTECs is mediated by megalin-dependent endocytosis at the apical membrane of PTECs [ 11 , 12 , 13 , 14 ]. However, many studies on Cd cytotoxicity have focused on the mechanisms of cell lethality, including apoptosis caused by Cd [ 15 , 16 , 17 ], and only a few studies have examined Cd’s direct effects on the efficiency of protein reabsorption by PTECs [ 18 , 19 ], especially under conditions where PTECs are surviving in the presence of Cd.…”