Original Contribution
Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

https://doi.org/10.1016/j.freeradbiomed.2012.06.015Get rights and content

Abstract

The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease.

Highlights

► Renal proximal tubule cell (PTC) lysosomes of Hp 2-2 DM mice have iron deposits. ► Renal PTC lysosomal membranes of Hp 2-2 DM mice are oxidized. ► PTC lysosomal membrane integrity of Hp 2-2 DM mice is compromised. ► Vitamin E stabilizes PTC lysosomal membrane integrity in Hp 2-2 DM mice. ► Iron-induced injury may explain increased nephropathy in Hp 2-2 DM humans.

Section snippets

Animal studies

All procedures were approved by the Animal Care Committee of the Technion (protocol number IL-112-11-11). All mice were of a C57B1/6 genetic background. The Hp 2 allele is present only in humans. All other species have only an Hp 1 allele, which is highly homologous with the human Hp 1 allele. Thus, wild-type mice carry the Hp 1 allele (referred herein as Hp 1-1 mice). The construction of the murine Hp 2 allele and the targeting of its insertion by homologous recombination to the murine Hp

Increased iron-rich deposits in lysosomes of proximal tubule cells in Hp 2-2 DM mice

We sought to assess the intracellular localization of iron within the proximal tubules using TEM. TEM demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubule cells (Fig. 1A). EDX and EELS were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich (Fig. 1B). Lysosomes containing these iron deposits were more frequent in the proximal tubule of Hp 2-2 (65±4% of all lysosomes) compared with Hp 1-1 DM mice

Discussion

In this study we have demonstrated that increased lysosomal redox-active iron results in lysosomal membrane injury in renal cells of Hp 2-2 DM mice. These data therefore provide a novel pathophysiological mechanism explaining why the progression to end-stage renal disease is increased in DM individuals with the Hp 2-2 DM genotype. Moreover, the interaction between the vitamin E and the Hp genotype on lysosomal injury suggests that a pharmacogenomic paradigm of selective administration of

Acknowledgments

This work was supported by a grant from the Israel Society Foundation (ISF) to F.M. Nakhoul and in part from Aboutboul Family in memory of Daniel Aboutboul and by grants from NIH (R01DK085226) and the ISF to A.P.L. This work was also generously supported by a grant from the Slava Smolakovski Fund to the Rambam-Atidim Academic Excellence Program. The electron-microscopic analyses were supported by the Dan David Foundation to T.C.I. and I.M. M.L. thanks Gerd Fulda for operating the microscope.

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    Both authors contributed equally to this manuscript.

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