Cihan Demirci

The Pathogenesis of Acute Kidney Injury and the Toxic Triangle of Oxygen, Reactive Oxygen Species and Nitric Oxide

Despite the identification of several of the cellular mechanisms thought to underlie the development of acute kidney injury (AKI), the pathophysiology of AKI is still poorly understood. It is clear, however, that instead of a single mechanism being responsible for its etiology, AKI is associated with an entire orchestra of failing cellular mechanisms. Renal microcirculation is the physiological compartment where these mechanisms come together and exert their integrated deleterious action. Therefore, the study of renal microcirculation and the identification of the determinants of its function in models of AKI can be expected to provide insight into the pathogenesis and resolution of AKI. A major determinant of adequate organ function is the adequate oxygen (O(2)) supply at the microcirculatory level and utilization at mitochondrial levels for ATP production needed for performing organ function. The highly complex architecture of the renal microvasculature, the need to meet a high energy demand and the borderline hypoxemic nature of the kidney makes it an organ that is highly vulnerable to injury. Under normal, steady-state conditions, the oxygen supply to the renal tissues is well regulated and utilized not only for mitochondrial production of ATP (mainly for Na reabsorption), but also for the production of nitric oxide and the reactive oxygen species needed for physiological control of renal function. Under pathological conditions, such as inflammation, shock or sepsis, however, the renal microcirculation becomes compromised, which results in a disruption of the homeostasis of nitric oxide, reactive oxygen species, and oxygen supply and utilization. This imbalance results in these compounds exerting pathogenic effects, such as hypoxemia and oxidative stress, resulting in further deterioration of renal microcirculatory function. Our hypothesis is that this sequence of events underlies the development of AKI and that integrated therapeutic modalities targeting these pathogenic mechanisms will be effective therapeutic strategies in the clinical environment.

Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and inflammation.

BACKGROUND The aim of the present study was to test the hypothesis that balanced crystalloid resuscitation would be better for the kidney than unbalanced crystalloid resuscitation in a rat hemorrhagic shock model. METHODS Male Wistar rats were randomly assigned to four groups (n=6/group): (1) time control; (2) hemorrhagic shock control; (3) hemorrhagic shock followed by unbalanced crystalloid resuscitation (0.9% NaCl); and (4) hemorrhagic shock followed by acetate and gluconate-balanced crystalloid resuscitation (Plasma Lyte). We tested the solutions for their effects on renal hemodynamics and microvascular oxygenation, strong-ion difference, systemic and renal markers of inflammation and oxidative stress including glycocalyx degradation as well as their effects on renal function. RESULTS The main findings of our study were that: (1) both the balanced and unbalanced crystalloid solutions successfully restored the blood pressure, but renal blood flow was only recovered by the balanced solution although this did not lead to improved renal microvascular oxygenation; (2) while unbalanced crystalloid resuscitation induced hyperchloremia and worsened metabolic acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevented hyperchloremia, restored the acid-base balance, and preserved the anion gap and strong ion difference in these animals; (3) in addition balanced crystalloid resuscitation significantly improved renal oxygen consumption (increased VO(2), decreased [Formula: see text] ); and (4) however neither balanced nor unbalanced crystalloid resuscitation could normalize systemic inflammation or oxidative stress. Functional immunohistochemistry biomarkers showed improvement in L-FABP in favor of balanced solutions in comparison to the hemorrhagic group although no such benefit was seen for renal tubular injury (measured by NGAL) by giving either unbalanced or balanced solutions. CONCLUSIONS Although balanced crystalloid resuscitation seems superior to balanced crystalloid resuscitation in protecting the kidney after hemorrhagic shock and is certainly better than not applying fluid resuscitation, these solutions were not able to correct systemic inflammation or oxidative stress associated with hemorrhagic shock.

Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion.

The glycolytic enzyme hexokinase (HK) is suggested to play a role in ischemic preconditioning (IPC). In the present study we determined how ischemic preconditioning affects HK activity and HKI and HKII protein content at five different time points and three different subcellular fractions throughout cardiac ischemia-reperfusion. Isolated Langendorff-perfused rat hearts (10 groups of 7 hearts each) were subjected to 35 min ischemia and 30 min reperfusion (control groups); the IPC groups were pretreated with 3 times 5-min ischemia. IPC was without effect on microsomal HK activity, and only decreased cytosolic HK activity at 35 min ischemia, which was mimicked by decreased cytosolic HKII, but not HKI, protein content. In contrast, mitochondrial HK activity at baseline and during reperfusion was elevated by IPC, without changes during ischemia. No effect of IPC on mitochondrial HK I protein content was observed. However, mitochondrial HK II protein content during reperfusion was augmented by IPC, albeit not following the IPC stimulus. It is concluded that IPC results in decreased cytosolic HK activity during ischemia that could be explained by decreased HKII protein content. IPC increased mitochondrial HK activity before ischemia and during reperfusion that was only mimicked by increased HK II protein content during reperfusion. IPC was without effect on the phosphorylation status of HK before ischemia. We conclude that IPC is associated with 1) a biphasic response of increased mitochondrial HK activity before and after ischemia, 2) decreased cytosolic HK activity during ischemia, and 3) cellular redistribution of HKII but not HKI.

The effect of α-lipoic acid on NOS dispersion in the lung of streptozotocin-induced diabetic rats

Oxidative stress and impaired bioactivity of nitric oxide (NO) play an important role in the organ pathogenesis and angiopathic complications of diabetes mellitus. In this study, we evaluated the effects of alpha-lipoic acid (ALA) on nitric oxide synthase (NOS) in lung tissues. ALA is a strong antioxidant. We wonder how it can affect oxidative stress and NO in the lung cells and vessels of diabetic rats. Wistar rats were divided into four groups; control, diabetic [65 mg/kg streptozotocin (STZ) for 15 days], STZ+ALA-treated (65 mg/kg ALA every 2 days for 15 days), and ALA-only-treated animals. At the end of the experimental period, lipid peroxidation, superoxide dismutase (SOD), and inducible NOS (iNOS) and endothelial NOS (eNOS) distribution were evaluated. Oxidative stress decreased with ALA in diabetic animals, and SOD also increased with ALA. iNOS and eNOS increased in diabetic animals, and ALA prevented iNOS increment in lung tissues. As a result, ALA can prevent some diabetic effects on the lungs and can also protect from vascular damages.

Acute effects of balanced versus unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusion during endotoxemic shock.

ABSTRACT This study was designed to investigate the acute effects of balanced versus unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusions during lipopolysaccharide-induced endotoxemic shock in rats. We tested the hypothesis that balanced colloid resuscitation would be better for the kidney than unbalanced colloid resuscitation. Shock was induced by lipopolysaccharide (10 mg/kg i.v. over 30 min). When mean arterial pressure (MAP) was decreased to 40 mmHg, fluid resuscitation was started with either hydroxyethyl starch (HES130/0.42) dissolved in saline (HES-NaCl) as an unbalanced colloid solution or HES130/0.42 dissolved in Ringer’s acetate (HES-RA) as a balanced colloid solution. Microvascular perfusion in the renal cortex was monitored using laser speckle imaging, and in addition, systemic hemodynamics, renal artery blood flow (RBF), and plasma ion levels were measured. Shock decreased MAP, led to anuria, and worsened all other parameters. Hydroxyethyl starch–NaCl improved MAP (P > 0.05) but did not improve RBF (P > 0.05), metabolic acidosis (P > 0.05), and plasma ion levels (P > 0.05). Hydroxyethyl starch–RA improved MAP (P < 0.05), RBF (P < 0.05), and renal microvascular perfusion (P < 0.05), but did not improve metabolic acidosis (P > 0.05) and plasma ion levels (P > 0.05). Both HES-NaCl and HES-RA treatment could normalize creatinine clearance but not fractional sodium excretion. In endotoxemic rats, balanced colloid (HES) resuscitation was shown to be superior to unbalanced colloid resuscitation in terms of improvement of renal macrovascular and microvascular perfusions. However, whether this results in improved renal function in the long term warrants further study.

Effects of Crataegus microphylla on vascular dysfunction in streptozotocin-induced diabetic rats.

Vascular dysfunction plays a key role in the pathogenesis of diabetic vascular disease. In this study, we aimed to investigate whether chronic in vivo treatment of Crataegus microphylla (CM) extract in diabetic rats induced with streptozotocin (STZ, intraperitoneal, 65 mg/kg) preserves vascular function and to evaluate whether the reduction of inducible nitric oxide synthase (iNOS), proinflammatory cytokines, and lipid peroxidation mediates its mechanisms of action. Starting at 4 weeks of diabetes, CM extract (100 mg/kg) was administrated to diabetic rats for 4 weeks. In aortic rings, relaxation to acetylcholine and vasoreactivity to noradrenaline were impaired, whereas aortic iNOS expression and plasma tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6), total nitrite–nitrate, and malondialdehite levels were increased in diabetic rats compared with controls. Chronic CM treatment significantly corrected all the above abnormalities in diabetic rats. In comparison, pretreatment of the aorta of diabetic rats with N‐[3(aminomethyl) benzyl]‐acetamidine, dihydrochloride (10–5 M), a selective inhibitor of iNOS, produced a similar recovery in vascular reactivity. These results suggest that chronic in vivo treatment of CM preserves endothelium‐dependent relaxation and vascular contraction in STZ‐induced diabetes, possibly by reducing iNOS expression in the aorta and by decreasing plasma levels of TNF‐α and IL‐6 and by preventing lipid peroxidation. Copyright

Fully Balanced Fluids do not Improve Microvascular Oxygenation, Acidosis and Renal Function in a Rat Model of Endotoxemia

ABSTRACT The expectation of fluid therapy in patients with septic shock is that it corrects hypovolemia, with the aim of restoring tissue perfusion and oxygenation and organ function. This study investigated whether different types of resuscitation fluids were effective in improving renal microcirculatory oxygenation, acidosis, oxidative stress, and renal function in a rat model of endotoxemic shock. Five groups of rats were used: a sham group, a lipopolysaccharide (LPS) group, and three LPS groups that received 30 mL/kg/h of 0.9% sodium chloride (0.9% NaCl), a new bicarbonate buffered crystalloid solution closely resembling the composition of plasma (FB-Cxt) or a hydroxyethyl starch-ringer acetate solution. Systemic hemodynamic variables, renal blood flow, microvascular oxygenation, oxidative/nitrosative stress, and renal function were measured. LPS-induced shock was only partially resolved by fluid administration. Animals became arterially hypotensive despite adequate central venous pressure. Hydroxyethyl starch-ringer acetate was more effective at improving arterial pressures and renal blood flow than 0.9% NaCl or FB-Cxt. Fluids had marginal effects on pH and HCO3− levels irrespective of the buffer, or on renal &mgr;PO2 and dysfunction. Colloids increased the markers of renal oxidative stress (P < 0.001), whereas unbalanced crystalloids increased the markers of nitrosative stress during sepsis (P < 0.01). Endotoxemia-induced acidosis and decreases in renal &mgr;PO2 or renal injury were not corrected solely by fluid resuscitation, irrespective of the buffer of the fluid. Our study supported the idea that fluids must be supplemented by other compounds that specifically correct renal inflammation and oxygenation to be effective in resolving septic shock-induced renal failure.

Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury

Abstract Background and objectives: Acute kidney injury (AKI) is a clinical condition associated with a degree of morbidity and mortality despite supportive care, and ischemia/reperfusion injury (I/R) is one of the main causes of AKI. The pathophysiology of I/R injury is a complex cascade of events including the release of free oxygen radicals followed by damage to proteins, lipids, mitochondria, and deranged tissue oxygenation. In this study, we investigated whether the antioxidant ascorbic acid would be able to largely prevent oxidative stress and consequently, reduce I/R-related injury to the kidneys in terms of oxygenation, inflammation, and renal failure. Materials and methods: Rats were divided into three groups (n = 6/group): (1) a time control group; (2) a group subjected to renal ischemia for 60 min by high aortic occlusion followed by 2 h of reperfusion (I/R); and (3) a group subjected to I/R and treated with an i.v. 100 mg/kg bolus ascorbic acid 15 min before ischemia and continuous infusion of 50 mg/kg/hour for 2 h during reperfusion (I/R + AA). We measured renal tissue oxidative stress, microvascular oxygenation, renal oxygen delivery and consumption, and renal expression of inflammatory and injury markers. Results: We demonstrated that aortic clamping and release resulted in increased oxidative stress and inflammation that was associated with a significant fall in systemic and renal hemodynamics and oxygenation parameters. The treatment of ascorbic acid completely abrogated oxidative stress and inflammatory parameters. However, it only partly improved microcirculatory oxygenation and was without any effect on anuria. Conclusion: The ascorbic acid treatment partly improves microcirculatory oxygenation and prevents oxidative stress without restoring urine output in a severe I/R model of AKI.

Arteriolar changes in nitric oxide activity and sensitivity during the course of streptozotocin-induced diabetes.

Nitric oxide (NO) may play an important role in the pathogenesis of diabetic microangiopathy. However, arteriolar changes in NO activity and sensitivity to NO may be dependent on both the type of arteriole and the duration of diabetes. Therefore, we assessed, in the in situ spinotrapezius muscle preparation of streptozotocin-diabetic rats and of controls, inside diameters of A2-A4 arterioles and the reactivity to topically applied acetylcholine and nitroprusside, before and after N(G)-nitro-L-arginine (L-NNA) at 2, 4, 6 and 12 weeks of diabetes. In A2 arterioles, basal diameters and the contribution of NO to basal diameter were not affected during the course of streptozotocin-induced diabetes. However, the maximal response to acetylcholine in these arterioles was attenuated after 2 until 4 weeks, and from 4 weeks on a sustained decrease in reactivity to sodium nitroprusside was observed. In A3 arterioles, both the basal diameter and the contribution of NO to basal diameter were decreased after 2 weeks and increased after 6 weeks, while the response to sodium nitroprusside was unaffected. In A4 arterioles, a significant increase in basal diameter was observed after 6 weeks only. Thus, this study shows that streptozotocin-induced diabetes causes microvascular changes in NO activity and sensitivity that depend on the type of arteriole. For each order of arteriole, these changes show a specific pattern during the course of diabetes.