Peter Hansell

Reactive oxygen species cause diabetes-induced decrease in renal oxygen tension

Aims/hypothesisAugmented formation of reactive oxygen species (ROS) induced by hyperglycaemia has been suggested to contribute to the development of diabetic nephropathy. This study was designed to evaluate the influence of streptozotocin (STZ)-induced diabetes mellitus, as well as the effects of preventing excessive ROS formation by α-tocopherol treatment, on regional renal blood flow, oxygen tension and oxygen consumption in anaesthetized Wistar Furth rats.MethodsNon-diabetic and STZ-diabetic rats were investigated after 4 weeks with or without dietary treatment with the radical scavenger DL-α-tocopherol (vitamin E, 5%). A laser-Doppler technique was used to measure regional renal blood flow, whilst oxygen tension and consumption were measured using Clark-type microelectrodes.ResultsRenal oxygen tension, but not renal blood flow, was lower throughout the renal parenchyma of diabetic rats when compared to non-diabetic control rats. The decrease in oxygen tension was most pronounced in the renal medulla. Renal cellular oxygen consumption was markedly increased in diabetic rats, predominantly in the medullary region. Diabetes increased lipid peroxidation and protein carbonylation in the renal medulla. Treatment with α-tocopherol throughout the course of diabetes prevented diabetes-induced disturbances in oxidative stress, oxygen tension and consumption. The diabetic animals had a renal hypertrophy and a glomerular hyperfiltration, which were unaffected by α-tocopherol treatment.Conclusions/interpretationWe conclude that oxidative stress occurs in kidneys of diabetic rats predominantly in the medullary region and relates to augmented oxygen consumption and impaired oxygen tension in the tissue.

Diabetes, Oxidative Stress, Nitric Oxide and Mitochondria Function

The role of altered mitochondria function has recently emerged as an important mechanism for the development of diabetic complications. Altered mitochondria function has also been implicated in the ageing process, defective insulin secretion, hypertension, arteriosclerosis, ischemia-reperfusion injury and apoptosis. Normally, the mitochondria are associated with ATP production using primarily pyruvate as the substrate, but recent reports indicate that tissue specific preferences exist. Also, the mitochondria are a substantial source of superoxide production, preferentially during states of elevated intracellular glucose concentrations. The mitochondria function is regulated by several factors including nitric oxide, oxidative stress, mammalian target of rapamycin, ADP and P(i) availability, which result in a complex regulation of ATP production and oxygen consumption, but also superoxide generation. These factors seem to be tissue specific, which warrants a more diverse mechanistic model applying to that specific tissue or cell type. This review presents the basic functions of the mitochondria and focuses on the complex interplay between oxidative stress, nitric oxide and uncoupling proteins in regulating mitochondria function with special focus on diabetes-induced alterations occurring on the mitochondria level.

Determinants of kidney oxygen consumption and their relationship to tissue oxygen tension in diabetes and hypertension

The high renal oxygen (O2) demand is associated primarily with tubular O2 consumption (Qo2) necessary for solute reabsorption. Increasing O2 delivery relative to demand via increased blood flow results in augmented tubular electrolyte load following elevated glomerular filtration, which, in turn, increases metabolic demand. Consequently, elevated kidney metabolism results in decreased tissue oxygen tension. The metabolic efficiency for solute transport (Qo2/TNa) varies not only between different nephron sites, but also under different conditions of fluid homeostasis and disease. Contributing mechanisms include the presence of different Na+ transporters, different levels of oxidative stress and segmental tubular dysfunction. Sustained hyperglycaemia results in increased kidney Qo2, partly due to mitochondrial dysfunction and reduced electrolyte transport efficiency. This results in intrarenal tissue hypoxia because the increased Qo2 is not matched by a similar increase in O2 delivery. Hypertension leads to renal hypoxia, mediated by increased angiotensin receptor tonus and oxidative stress. Reduced uptake in the proximal tubule increases load to the thick ascending limb. There, the increased load is reabsorbed, but at greater O2 cost. The combination of hypertension, angiotensin II and oxidative stress initiates events leading to renal damage and reduced function. Tissue hypoxia is now recognized as a unifying pathway to chronic kidney disease. We have gained good knowledge about major changes in O2 metabolism occurring in diabetic and hypertensive kidneys. However, further efforts are needed to elucidate how these alterations can be prevented or reversed before translation into clinical practice.

Effect of intravenous contrast media on proximal and distal tubular hydrostatic pressure in the rat kidney.

The effect of i.v. injection of contrast media (CM, 1 600 mg I/kg b.w.) on proximal and distal tubular hydrostatic pressure (PTHP, DTHP) in the rat was investigated using a micropuncture technique. The PTHP and DTHP after injection of diatrizoate, iohexol, ioxaglate, or mannitol returned to control values within approximately 20 min. However, following iotrolan injection PTHP was still elevated above control levels after 35 min while DTHP remained elevated throughout the experiment (50 min). Iotrolan has a lower osmotic potential than the other CM when given in equivalent iodine doses. The concentration of iotrolan may thus increase more along the tubules than the other CM and consequently lead to a higher viscosity of urine, resulting in increases in PTHP and DTHP. The high intratubular pressure induced by iotrolan may explain our previous findings of reduced single nephron glomerular filtration rate caused by this CM.

Effects of Intravenous Contrast Media on Cortical and Medullary Blood Flow in the Rat Kidney

The effect of slow intravenous infusion of contrast medium (CM) (1600 mg I/kg body weight) on cortical blood flow (BF) and medullary BF in rat kidneys was investigated by laser-Doppler flowmetry on either renal cortex or exposed renal papillas (inner medulla). The effect on cortical BF was evaluated after infusion of either ioxaglate, iohexol, or ioxithalamate. Mannitol and Ringers solution were used as control substances. The effect on medullary BF was examined after infusion of either ioxaglate, iohexol, iopamidol, ioxithalamate, or mannitol. BF was measured continuously during a 30-minute control period and a 60-minute experimental period, starting with the CM infusion. Cortical BF was unchanged in the ioxaglate group and significantly increased in the iohexol, ioxithalamate, and mannitol groups (P less than .05). Medullary BF was moderately increased in the ioxaglate group (P less than .05) but moderately decreased in the groups that received iohexol, iopamidol, ioxithalamate, or mannitol (P less than .05). The reduction in medullary BF following infusion of the ratio 3.0 nonionic CM and of the ratio 1.5 ionic CM might be one contributory mechanism to the pathogenesis of CM nephropathy, especially in the presence of microangiopathy in the kidney.

Polyol-pathway-dependent disturbances in renal medullary metabolism in experimental insulin-deficient diabetes mellitus in rats

Aims/hypothesisThe renal medullary region is particularly vulnerable to reduced oxygen concentration because of its low blood perfusion and high basal oxygen consumption. This study investigated renal metabolic changes in relation to the previously observed decreased oxygen tension in streptozotocin-induced diabetic rats.MethodsBlood perfusion, oxygen tension and consumption, interstitial pH, and glycolytic and purine-based metabolites were determined in the renal cortex and the medulla of non-diabetic and diabetic animals by, respectively, laser Doppler flowmetry, oxygen and pH microelectrodes, and microdialysis. The importance of increased polyol pathway activity for the observed alterations was investigated by daily treatment with the aldose reductase inhibitor AL-1576 throughout the course of diabetes.ResultsThe diabetes-induced decrease in renal oxygen tension, due to augmented oxygen consumption, did not result in manifest hypoxia in either the cortical or the medullary region, as evaluated by microdialysis measurements of purine-based metabolites. The profound alterations in medullary oxygen metabolism were, however, associated with an increased lactate : pyruvate ratio and a concomitantly decreased pH. Notably, the renal medullary changes in oxygen tension, oxygen consumption, lactate : pyruvate ratio and pH were preventable by inhibition of aldose reductase.Conclusions/interpretationSubstantial metabolic changes were observed in the renal medulla in diabetic animals. These disturbances seemed to be mediated by increased polyol pathway activity and could be prevented by inhibition of aldose reductase.

Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells

We have previously reported increased O(2) consumption unrelated to active transport by tubular cells and up-regulated mitochondrial uncoupling protein (UCP)-2 expressions in diabetic kidneys. It is presently unknown if the increased UCP-2 levels in the diabetic kidney results in mitochondrial uncoupling and increased O(2) consumption, which we therefore investigated in this study. The presence of UCP-2 in proximal tubular cells was confirmed by immunohistochemistry and found to be increased (western blot) in homogenized tissue and isolated mitochondria from kidney cortex of diabetic rats. Isolated proximal tubular cells had increased total and ouabain-insensitive O(2) consumption compared to controls. Isolated mitochondria from diabetic animals displayed increased glutamate-stimulated O(2) consumption (in the absence of ADP and during inhibition of the ATP-synthase by oligomycin) compared to controls. Guanosine diphosphate, an UCP inhibitor, and bovine serum albumin which removes fatty acids that are essential for UCP-2 uncoupling activity, independently prevented the increased glutamate-stimulated O(2) consumption in mitochondria from diabetic animals. In conclusion, diabetic rats have increased mitochondrial UCP-2 expression in renal proximal tubular cells, which results in mitochondrial uncoupling and increased O(2) consumption. This mechanism may be protective against diabetes-induced oxidative stress, but will increase O(2) usage. The subsequently reduced O(2) availability may contribute to diabetes-induced progressive kidney damage.

Kidney Hypoxia, Attributable to Increased Oxygen Consumption, Induces Nephropathy Independently of Hyperglycemia and Oxidative Stress

Diabetic nephropathy is strongly associated with both increased oxidative stress and kidney tissue hypoxia. The increased oxidative stress causes increased kidney oxygen consumption resulting in kidney tissue hypoxia. To date, it has been difficult to determine the role of kidney hypoxia, per se, for the development of nephropathy. We tested the hypothesis that kidney hypoxia, without confounding factors such as hyperglycemia or elevated oxidative stress, results in nephropathy. To induce kidney hypoxia, dinitrophenol (30 mg per day per kg bodyweight by gavage), a mitochondrial uncoupler that increases oxygen consumption and causes kidney hypoxia, was administered for 30 consecutive days to rats. Thereafter, glomerular filtration rate, renal blood flow, kidney oxygen consumption, kidney oxygen tension, kidney concentrations of glucose and glycogen, markers of oxidative stress, urinary protein excretion, and histological findings were determined and compared with vehicle-treated controls. Dinitrophenol did not affect arterial blood pressure, renal blood flow, glomerular filtration rate, blood glucose, or markers of oxidative stress but increased kidney oxygen consumption, and reduced cortical and medullary concentrations of glucose and glycogen, and resulted in intrarenal tissue hypoxia. Furthermore, dinitrophenol treatment increased urinary protein excretion, kidney vimentin expression, and infiltration of inflammatory cells. In conclusion, increased mitochondrial oxygen consumption results in kidney hypoxia and subsequent nephropathy. Importantly, these results demonstrate that kidney tissue hypoxia, per se, without confounding hyperglycemia or oxidative stress, may be sufficient to initiate the development of nephropathy and therefore demonstrate a new interventional target for treating kidney disease.

Activation of Hypoxia-Inducible Factors Prevents Diabetic Nephropathy

Hyperglycemia results in increased oxygen consumption and decreased oxygen tension in the kidney. We tested the hypothesis that activation of hypoxia-inducible factors (HIFs) protects against diabetes-induced alterations in oxygen metabolism and kidney function. Experimental groups consisted of control and streptozotocin-induced diabetic rats treated with or without chronic cobalt chloride to activate HIFs. We elucidated the involvement of oxidative stress by studying the effects of acute administration of the superoxide dismutase mimetic tempol. Compared with controls, diabetic rats displayed tissue hypoxia throughout the kidney, glomerular hyperfiltration, increased oxygen consumption, increased total mitochondrial leak respiration, and decreased tubular sodium transport efficiency. Diabetic kidneys showed proteinuria and tubulointerstitial damage. Cobalt chloride activated HIFs, prevented the diabetes-induced alterations in oxygen metabolism, mitochondrial leak respiration, and kidney function, and reduced proteinuria and tubulointerstitial damage. The beneficial effects of tempol were less pronounced after activation of HIFs, indicating improved oxidative stress status. In conclusion, activation of HIFs prevents diabetes-induced alteration in kidney oxygen metabolism by normalizing glomerular filtration, which reduces tubular electrolyte load, preventing mitochondrial leak respiration and improving tubular transport efficiency. These improvements could be related to reduced oxidative stress and account for the reduced proteinuria and tubulointerstitial damage. Thus, pharmacologic activation of the HIF system may prevent development of diabetic nephropathy.

Differentiating between effects of streptozotocin per se and subsequent hyperglycemia on renal function and metabolism in the streptozotocin-diabetic rat model

The animal model with streptozotocin (STZ)‐induced diabetes mellitus is associated with progressive renal disturbances. The aim of this study was to differentiate between toxic effects of STZ and the effect of hyperglycemia. Previous studies have been limited to investigating the influence of STZ on glomerular filtration rate (GFR), albuminuria and renal morphology. The present study presents a new approach when transplanting β‐cells to cure the STZ‐treated animals and extends the evaluation to include both renal function and oxygen metabolism.