Molecular dissection of one of the most striking homeostatic responses to hypoxia — the induction of the haematopoietic growth factor erythropoietin — led to the discovery of the transcription factor hypoxia inducible factor (HIF). Unexpectedly, it was found that this system operates in essentially all mammalian cells irrespective of their relevance to erythropoietin production, and that it directs many other responses to hypoxia. The same hypoxic responses can be induced by iron chelators or cobaltous ions, distinctive properties that led to the proposal of a ferroprotein oxygen sensor, which was originally thought to be a haem protein. Work on the HIF signalling pathway has now shown that its transcriptional activity is regulated by the post-translational hydroxylation of specific residues. Hydroxylation is catalysed by a set of oxygen-dependent enzymes that belong to the 2-oxoglutarate-dependent-oxygenase superfamily, which are, in fact, non-haem, Fe2+-dependent enzymes (Schofield CJ and Ratcliffe 2004). Diabetes mellitus is a highly prevalent chronic metabolic disorder that is considered a major health problem in westernized societies. Diabetes, characterized by persistent elevation of blood glucose levels (hyperglycaemia), occurs due to inadequate production of insulin (type 1 diabetes; T1D), or resistance to endogenous insulin usually associated with the metabolic syndrome and obesity (type 2 diabetes; T2D). Despite intensive glycaemic control, individuals with T1D and T2D are predisposed to developing vascular complications, which include cardiomyopathy, atherosclerosis, nephropathy, retinopathy, and neuropathy. Indeed, there is a striking correlation between the incidence of cardiovascular disease and mortality rates in diabetic patients.Although the mechanisms by which diabetes increases cardiovascular complications are incompletely understood, strong supportive evidence from experimental and clinical studies points to the impaired function of the vascular endothelium as a critical inducer of these cardiovascular complications (Cosentino and Luscher 1998, de Haan et al. 2012). Even mild hypoxia can affect HIF signaling, which alters HIF1-α and the HIF target gene and ubiquilates HIF1- α. Even one week of normal oxygen levels cannot remove the ubiquilation of the HIF1- α. These data suggest that HIF signaling may be contributing to the pathogenesis of endothelial dysfunction and that normalization of oxygen levels may not be enough to reduce vascular stress. Chronic hypoxia also generates ubiquilated HIF-1alfa, which through genetic control down regulates glucose transporter (GLUT). Under normal oxygen levels, GLUT 1 is up regulated, returning to its original state. However, we can speculate that these processes of up and down regulation of GLUT 1 exacerbate endothelial dysfunction. In addition to being regulated by hypoxia, the HIF pathway can be modulated by reactive species, nitric oxide, metals, cytokines, growth factor signaling through PI3K and MAPK pathways. In conclusion impairment of HIF-1 dependent responses to hypoxia is a major factor contributing to the impaired vascular responses to ischemia that are associated diabetes (Semenza 2010).
Diabetes leads to chronic renal hypoxia and cellular hypoxia response (mediated by hypoxia-inducible factors) and predisposes to acute kidney injury. Experimental diabetes is associated with a marked decline in renal parenchymal pO2, noted especially in the renal medulla. Palm et al. (Palm et al. 2003), using oxygen microelectrodes, reported reduced renal oxygenation, and their findings were confirmed by blood oxygen level-dependent MRI studies and with the detection of adducts of the hypoxia marker pimonidazole in the renal medulla. Renal hypoxia develops within 1 week after the induction of experimental diabetes with streptozotocin (STZ), leads to the generation of hypoxia-inducible factors (HIF), and is prevented by insulin treatment. Renal hypoxia in the diabetic kidney may represent enhanced oxygen consumption for tubular transport and altered renal microcirculation. Diabetes has been recognized as an independent risk factor for the development of acute kidney injury (AKI) in a variety of clinical settings, such as radiocontrast nephropathy following cardiopulmonary bypass operations or in conditions that lead to renal papillary necrosis. Due to limited morphologic material, the particular structural changes of diabetes-related AKI are still unclear. Conceivably, however, preexisting renal parenchymal hypoxia in the diabetic kidney plays an important role in its predisposition to AKI (Heyman et al. 2008). Chronic renal hypoxia is suspected to play a pathogenic role in the genesis of diabetic nephropathy (DN).
The sensitivity of the kidney to changes in oxygen delivery suggests that hypoxia might be such a determinant. Recent studies have indeed incriminated chronic hypoxia as a possible final common pathway in end-stage kidney injury (Nangaku 2006, Ohtomo et al. 2008). If HIF activation were already maximal under pathological conditions, therapeutic approaches which target HIF might be of little use. In kidney disease, however, this is not the case; HIF activation is suboptimal, and the door to this strategic route is accordingly wide open. Evidence for this comes from a variety of studies. HIF activation is suboptimal in diabetic conditions, and is augmented by treatment with antioxidants (Katavetin et al. 2006, Rosenberger et al. 2008). HIF accumulation in acute ischemia was substantially less than that seen in animals exposed to carbon monoxide, indicating that activation was also submaximal in renal ischemia with complete shutdown of blood flow. In a model of rhabdomyolysis, transcriptional hypoxia adaptation in the most affected tubules was transient and heterogeneous, while proteinuric states may also inhibit optimal HIF activation. Rosenberger et al. (Rosenberger et al. 2006) utilized an ex vivo model of the isolated perfused rat kidney with controlled oxygen consumption and provided evidence for a ‘window of opportunity’ for HIF activation, under moderate sublethal hypoxia, whereas a more severe hypoxia results in suppression/disappearance of HIF and induction of apoptotic cell death.
Together, these reports suggest the potential of pharmacologic enhancement of HIF to improve outcomes. Due to the powerful and coordinated response it produces against hypoxia, modulation of HIF activity should be effective in a variety of hypoxic states (Nangaku et al. 2008). A recent study evaluated the impact of acute hypoxic stress in diabetic kidneys as compared to controls and an unexpected finding of tubular cell resistance to AKI in the diabetic kidney, despite preexisting hypoxia, underscores an important role for a protective hypoxia-adaptive response (Heyman et al. 2008). Expectations for HIF activation in not only kidney disease but also conditions involving other organs such as the heart and brain have attracted much recent effort in the development of specific and nontoxic HIF activators (Nangaku et al. 2008). Endothelial dysfunction is a generalized syndrome characterized by attenuated vasodilation, due at least in part to decreased available nitric oxide (NO), a concomitant buildup of reactive species and stimulation of vascular stress pathways (Soriano et al. 2001). Another characteristic of endothelial dysfunction is an altered angiogenic response, which can affect tissue vascularity, immune cell infiltration, blood flow patterns, and wound healing.
If the blood vessels cannot supply the tissues with blood, a state of relative decreased oxygen ensues further contributing to endothelial dysfunction. Hypoxia induces expression of HIF, a transcription factor complex shown to be the master regulator of the cellular response to decreased oxygen levels (Semenza 2003). In addition to being regulated by hypoxia, the HIF pathway can be modulated by reactive species (Haddad et al. 2000, Chandel et al. 2000) and growth factor signaling through phosphatidylinositol 3-kinase and mitogen activated protein (MAP) kinase pathways (Kamat et al. 2007). The pathogenesis of endothelial dysfunction in type 2 diabetes is complex and involves many mechanisms. Visceral obesity, Insulin resistance, hypertension, post prandial hyperlipidemia particularly post prandial hypertriglyceridaemia, fasting and post prandial hyperglycemia result in an increased oxidative stress. Vascular endothelium is very susceptible to oxidative stress damage and this enhanced oxidative stress seen in diabetic individuals in turn causes endothelial dysfunction.
In early stages, insulin resistance and free fatty acids act directly on e-NOS activity and mitochondrial function. This leads to oxidative stress and increase generation of superoxide radicals. Thus, endothelial dysfunction is an early abnormality in diabetes and may play a key role in the micro and macrovascular disease associated with diabetes. Whether endothelial dysfunction causes some of the abnormalities of the metabolic syndrome and whether it can increase the risk of diabetes is unclear and merits further investigation. In a long term study on subjects with endothelial dysfunction it was shown that the risk of developing diabetes increased 6 fold (Hsueh et al. 2004).
Several interventions have been shown to ameliorate endothelial dysfunction in diabetic patients. These include exercise and weight loss, lipid lowering, ACE-inhibition, antioxidant strategies, Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, reducing homocysteine levels, aspirin, reducing hyperglycaemia and more recently nitric oxide co-factors such as tetrahydrobiopterin and modulation of insulin resistance (Madhu 2010). Angiotensin II induces oxidative stress via the activation of NADPH oxidase which damages endothelial cells directly, and results in relative hypoxia due to inefficient cellular respiration. A study showed that prolonged angiotensin II administration reduced renal cortical oxygen tension in association with a reduction of oxygen consumption efficiency and concurrent infusions of tempol, a permeant superoxide dismutase mimetic, blunted these changes (Welch et. al 2005). Thus, angiotensin II induces renal hypoxia through both hemodynamic as well as nonhemodynamic mechanisms (Nangaku and Fujita 2008, Nangaku et al. 2008). The study was designed to explore the role of cobalt chloride induced augmentation on HIF activation in the amelioration of diabetic nephropathy and the associated vascular dysfunction.
There are compelling evidence which show that chronic hypoxia final pathway to ESRD, therapeutic approaches which target the chronic hypoxia should prove effective against a broad range of renal diseases and associated vascular dysfunction. As PHD require iron as a cofactor to hydroxylate the critical prolines on HIF-α, the finding that some of the best-established activators of HIF-1 are chelators of iron is reasonable. Cobalt chloride is among the most well-established iron chelators in the activation of HIF. For exploring the mechanism we had employed CoCl2 in our study as a tool for elucidating the involvement (Nangaku et al. 2008, Norman et. al 2003). While our ultimate goal was to treat chronic hypoxia in diabetic nephropathy induced endothelial dysfunction, we employed a model induced by unilateral nephrectomy followed by STZ treatment in this study to investigate effects of our approach in ameliorating endothelial dysfunction. We tested a hypothesis that administration of cobalt chloride retards the progression of renal failure and associated endothelial dysfunction by improving the levels of eNOS and thus NOx, along with mitigation of oxidative stress with a significant decrement in the plasma glucose levels to which we obtained consistent results.