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 vitamin E to Hp 2-2 DM individuals may offer considerable renal protection similar to that recently demonstrated for cardiovascular disease [22
There exists prior evidence that proximal tubule cell lysosomal iron overload is a common pathogenic feature of early diabetic nephropathy [23
]. Nankivell and colleagues demonstrated a dramatic increase in lysosomal iron deposition in biopsies from individuals with DN as compared to normal controls [23
]. Moreover, the amount of proximal tubule cell iron deposition was highly correlated with the degree of loss of renal function in these individuals. While the Hp genotype of these individuals in this prior study was not reported this prior study demonstrates the relevance of the findings of the current study done in mice to DN in man.
Proximal tubule cell lysosomal iron accumulation has also been demonstrated in human biopsy specimens from individuals with early diabetic nephropathy, although the Hp-type relationship in these specimens was not investigated. As lysosomes were derived from whole kidneys, theoretically the compromise in lysosome membrane integrity demonstrated in this study may have occurred in any renal cell type. However, we assert that the changes described here in lysosomal structure and function are specific for proximal tubule cells. We have shown that the lysosomal injury is due to iron accumulating in the lysosome. The focus on proximal tubule cells is based on prior data [11
] showing that Perl’s iron stain is present only in proximal tubule cells and not in the glomerulus and its constituent cells nor in other tubular or interstitial cells. It is axiomatic that in the absence of a Perl’s stain there is no possibility that cells other than the proximal tubular cells are iron overloaded in the Hp 2-2 DM mice. Furthermore, these other renal cell types do not have iron deposits by EM as we have shown here in proximal tubule cells.
The source of excess proximal tubule iron in Hp 2-2 DM is Hb [20
]. In Hp 2-2 DM the normal clearance mechanism via the CD163 receptor [24
] for the Hp–Hb complex is severely impaired with an approximate 5-fold increase in the half -life of the complex [22
]. Consequently the steady-state levels of the complex are higher in Hp 2-2 DM [22
]. Moreover, in DM the glomerular filtration barrier is perturbed, thereby allowing complexes such as Hp–Hb to cross the barrier. Proximal tubule cubulin and megalin have both been demonstrated to bind to Hb in the glomerular filtrate, mediating Hb uptake into proximal tubule cells, and may serve as an entry point into these cells for the Hp–Hb complex [25
]; however other siderophores such as neutrophil gelatinase-associated lipocalin may also mediate this process [26
]. The endocytosed Hp–Hb complex is then targeted to the lysosome where it is degraded [25
]. As the half-life of Hp 2-2-Hb and Hp 1-1-Hb in the lysosome does not appear to be different[27
], the differences observed in the steady-state amount of proximal tubule iron in mice with the Hp 1-1 or Hp 2-2 genotypes results from the greater flux of iron through the renal proximal tubule cell in Hp 2-2.
The form in which excess iron is found in lysosomes of Hp 2-2 DM mice appears to be as cross-linked iron-rich protein complexes such as ferritin, hemosiderin, and lipofuscin based on the characteristic features of these complexes on TEM [15
]. This excess iron in Hp 2-2 DM renal proximal tubule lysosomes is redox active and may therefore result in cellular toxicity. The lysosome membrane appears to be better geared for oxidative insults than other cellular membranes, as it is enriched with antioxidants (vitamin E); the vitamin E content of the lysosome membrane is 40 times greater than that of the cytoplasmic membrane [28
]. While the lysosome may be geared to deal with oxidants such as iron, the unique properties of the Hp 2-2–Hb complex in the setting of DM may overwhelm its antioxidative defenses [29
]. The Hp 2-2–Hb complex may act as a Fenton reagent, and in the setting of DM, peroxide production may be sufficiently high to promote a large increase in hydroxyl radical formation via Fenton chemistry [31
Lysosomal injury has recently been suggested as a new pathophysiological paradigm playing a major role in a wide range of chronic diseases ranging from atherosclerosis to neurodegenerative disease [32
]. A loss of lysosomal membrane integrity would be expected to lead to the ability of degradative enzymes present in the lysosome to enter the cytosol and wreak havoc, leading to cell injury and death. We propose that any cell that endocytoses the Hp–Hb complex may develop lysosomal injury but with the exception of macrophages, no other cell type other than the proximal tubule cell has a mechanism to endocytose this complex. Consistent with this hypothesis, we have recently demonstrated that macrophages release cathepsin D (an intralysosomal enzyme) into the cytoplasm after taking up Hp 2-2–Hb complexes (unpublished observations) and this may explain the association of the Hp type with atherosclerotic plaque rupture in DM [33
]. The loss of lysosomal integrity would also be expected to interfere with the maintenance of the acidic pH gradient present in the lysosome and therefore interfere with lysosome degradative functions conceivably leading to a defect in autophagy [32
In conclusion, we have provided evidence for a novel mechanism whereby the Hp genotype may predispose to renal injury in the setting of DM. Elimination of excess proximal tubule iron, or its dangerous oxidative potential, possibly by a combination of iron chelating agents [34
] and/or antioxidants [35
] may thus prove helpful in the prevention of DN in DM individuals with the Hp 2-2 genotype.