G. Gronow

Hypoxic Renal Tissue Damage by Endothelin-Mediated Arterial Vasoconstriction during Radioangiography in Man

In vivo, a high rate of renal blood flow provides a more than sufficient rate of oxygen supply to the kidney. Limitations in tissue oxygenation may occur under systemic circulatory alterations (i.e. cardiovascular failure, arterioscelorosis), respiratory insuffiency (i.e. lung diseases, hypoxia, extreme anemia, acclimation to high altitude), or during perfusion of isolated organs with blood substitutes (Klause, N., and Gronow, G., 1990; Klause, N., et al., 1990). Hypoxic limitation of renal function at a maintained systemic respiration and circulation is less well understood. One example is tissue hypoxia in the kidney due to a vasoconstrictory side effect of radiocontrast media (RCM). Recent animal experiments support evidence that in the pathophysiology of RCM-induced renal vasoconstriction the balance between production of endothelium derived nitric oxide (NO) and endothelin (ET) is disturbed, whereby NO does not seem to play an important role (Bagnis et al., 1997, Morcos et al., 1997).

Hemoglobin induces cytotoxic damage of glycine‐preserved renal tubules

In isolated tubular segments (ITS) of rat kidney cortex, we studied the effect of hemoglobin (Hb) on reoxygenation damage. All tubules were suspended in Ringer’s solution containing 5‐mm glycine and oxygenated for 30 min with 95% O2:5% CO2, followed by a 30‐min period with 95% N2:5% CO2, and final reoxygenation for 60 min. Untreated tubules served as controls. Different concentrations of free Hb and equivalent amounts of intact erythrocytes were added to the incubation medium. Secondly, we added deferoxamine (DFO) to Hb and erythrocytes. Membrane leakage and lipid peroxidation were measured by lactate dehydrogenase and glutamate dehydrogenase and the development of thiobarbituric acid reactive substances. Cell function was quantified by gluconeogenesis and intracellular potassium accumulation. Hb exerted concentration‐dependent cytotoxic effects indicated by significantly increased enzyme leakage rates, lipid peroxidation and a significantly decreased cell function (P < 0.05), in ITS during hypoxia, and subsequent reoxygenation. Moreover, we found that toxicity of both Fe2+ and Fe3+ ions increased with rising concentration. However, Fe2+ showed a higher tissue toxicity than Fe3+. DFO reduced significantly the reoxygenation damage of free Hb and iron ions. Our data clearly demonstrate a pronounced cytotoxic effect of free Hb in ITS, which critically depended on the reduction state of the iron ions.

Amiodarone pretreatment of organ donors exerts anti‐oxidative protection but induces excretory dysfunction in liver preservation and reperfusion

The continuous shortage of organs necessitates the use of marginal organs from donors with various diseases, including arrhythmia‐associated cardiac failure. One of the most frequently used anti‐arrhythmic drugs is amiodarone (AM), which is given in particular in emergency situations. Apart from its anti‐arrhythmic actions, AM provides anti‐oxidative properties in cardiomyocytes. Thus, we were interested in whether AM donor pretreatment affects the organ quality and function of livers procured for preservation and transplantation. Donor rats were pretreated with AM (5 mg/kg of body weight) 10 minutes before flush‐out of the liver with a cold (4°C) histidine‐tryptophan‐ketoglutarate solution (n = 8). Livers were then stored for 24 hours at 4°C before ex situ reperfusion with a 37°C Krebs‐Henseleit solution for 60 minutes in a nonrecirculating system. At the end of reperfusion, tissue samples were taken for histology and Western blot analysis. Animals with vehicle only (0.9% NaCl) served as ischemia/reperfusion controls (n = 8). Additionally, livers of untreated animals (n = 8) not subjected to 24 hours of cold ischemia served as sham controls. AM pretreatment effectively attenuated lipid peroxidation, stress protein expression, and apoptotic cell death. This was indicated by an AM‐mediated reduction of malondialdehyde, heme oxygenase‐1, and caspase‐3 activation. However, AM treatment also induced mitochondrial damage and hepatocellular excretory dysfunction, as indicated by a significantly increased glutamate dehydrogenase concentration in the effluate and decreased bile production. In conclusion, AM donor pretreatment exerts anti‐oxidative actions in liver preservation and reperfusion. However, these protective AM actions are counteracted by an induction of mitochondrial damage and hepatocellular dysfunction. Accordingly, AM pretreatment of donors for anti‐arrhythmic therapy should be performed with caution. Liver Transpl 15:763–775, 2009.

Hippurate metabolism as a hydroxyl radical trapping mechanism in the rat kidney.

Hippurate (Hip) is considered to be the end product of benzoate (BA) metabolism. However, the kidney is able to metabolize Hip. Although only Hip but no BA is present in the blood, rat urine contains under normal conditions less Hip (about 0.4 mM) than BA (about 4.5 mM) and of hydroxylated derivatives of BA (hydroxy-BAs = HB and dihydroxy-BAs = DHB). Generation of HBs and DHBs is the result of radical substitution by free OH radicals (·OH). Thus, rate of synthesis of HBs and DHBs may reflect the production rate of ·OH in the kidney. ·OH generation is elevated following ischemic stress. Therefore, production of HBs and DHBs can be expected to be elevated in postischemic injury. The validity of this assumption was tested in vitro on isolated tubular segments and in vivo in the rat. Metabolism of Hip at 0.1 mmol l–1 (0.1 mM) as well as of BA resulted in enlarged production of both HBs (especially 3-HB and 4-HB) and of DHBs (especially 2,6-DHB). Production of 2,3- and especially of 2,5-DHB was elevated in the presence of high concentration (1.0 mM) of salicylate (2-HB) only. In vivo both in acute (120 min) and in chronic (5 days) experiments ligation of one renal artery for 30 respectively 60 min resulted in enlarged excretion of HBs and DHBs, especially of 2,6- and 3,5-DHB. This finding is noteworthy since (a) formation of 2,6-DHB necessitates as precursor salicylate which could not be detected in our experiments and (b) the spontaneous attack of ·OH upon the benzol ring would prefer the positions 2,3- 2,5- and 3,4-. Therefore, the existence of regulating factor(s) guiding OH groups to definite positions is a distinct possibility. These results indicate that metabolism of Hip leading to hydroxylated BAs may be a renoprotective mechanism against attack of ·OH in reoxygenated renal tissue.

Effect of hydroxyl radical scavengers in renal cortical cells.

In renal cortical cells the reversibility of posthypoxic cellular function may be limited by their high demand for metabolic energy. Irreversible structural and functional damage may be detected after 15–20 min of normothermic oxygen deprivation (Gronow et al., 1984, 1990). Furthermore, cellular defense mechanisms may fail in the reoxygenation period due to the exhaustion of radical scavengers and antioxidants. Adenosine, the hypoxic break-down product of ATP will provide xanthine/hypoxanthine for an increased accumulation of superoxide radicals via xanthinoxidase (Palier and Neumann, 1991; Zager et al., 1995; Sogabe et al., 1996). Finally, posthypoxic cellular recovery may be limited in the kidney by the formation of reactive oxygen species (ROS). Among these the univalent reduction of hydrogen peroxide leads to the formation of the hydoxyl radical (OH), a highly reactive biological oxidant.

Cytoprotective Effect of Isotonic Mannitol at Low Oxygen Tension

The beneficial effect of mannitol infusion in postischemic kidneys remains unresolved. Contradictory reports may have originated from at least 3 different mechanisms: a) arterial vasodilation, b) osmotic support of hypoxic cellular volume regulation, and c) scavenging of hydroxyl radicals in the reoxygenation period. To exclude vascular effects we tested at 37 degrees C in hypoxic (PO2 less than 1 mmHg) and reoxygenated isolated tubular cells of rat kidney cortex cellular function, i.e. intracellular K+ accumulation (K+), posthypoxic lactate gluconeogenesis (GNG), loss of membrane-bound tau-Glutamyltransferase (tau GT), and formation of a lipid peroxidation product, malondialdehyde (MDA). Mannitol (M) was added to a Ringer incubation medium in variable concentration either without (hypertonic M) or with omission of equiosmolar amounts of NaCl (isotonic M). K+ and GNG were significantly supported, and tau GT-loss markedly suppressed in a range of 10-50 mmol/l hypertonic and isotonic M. At higher concentrations no improvement (isotonic) or even deleterious effects (hypertonic) of M occurred. Beneficial effects of lower concentrations of M (10 mmol/l) were not correlated to anaerobic glycolysis, and 1 as well as 10 mmol/l M induced a comparable and significant reduction in posthypoxic MDA-formation. This effect was most pronounced when M was only added in hypoxia, indicating leakiness of cellular membranes in hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen Transport to Renal Tissue: Effect of Oxygen Carriers

The isolated perfused rat kidney introduced by Weiss and colleagues in 1959 has become a commonly used tool in the field of renal physiology and pharmacology (Weiss, Passow and Rothstein, 1959; Little and Cohen, 1974; Ross, 1978; Maack, 1986). In view of technical complications such as blood clotting and the release of vasoactive factors most authors preferred a hyperoxygenated (PO2~660 mmHg) balanced salt solution instead of blood as a perfusate. However, recent experiments with the isolated Ringer-perfused rat kidney indicate that oxygen transport to renal tissue has become a central question: due to a steep gradient of oxygen partial pressure in the outer medullary region of mammalian kidneys (Leichtweiss et al., 1969; Baumgartl et al., 1972) the poor oxygen binding capacity of hyperoxygenated salt solutions induced functional and morphological lesions in distinct renal tissue zones (Alcorn et al., 1981; Brezis et al., 1984; Schurek and Kriz, 1985). The aim of the present experiments was to compare the effects of three different oxygen carriers on function and tissue integrity of the isolated perfused rat kidney. The following served as oxygen carriers: a) coupled haemoglobin, b) washed erythrocytes, and c) perfluorocarbons. Perfusions performed with hyperoxygenated Ringer solutions (PO2~660 mmHg) served as a control. Our data indicate that renal function (perfusion flow rate, glomerular filtration rate, absolute and fractional Na+ reabsorption) as well as parameters of tissue integrity (i.e. the loss of enzymes, and tissue water content) are maintained best with an erythrocyte suspension and, for a limited time period, with coupled haemoglobin in the perfusate. They are reasonably maintained during Ringer perfusion, but are severely impaired in the presence of a perfluorocarbon emulsion.

Restriction of Hypoxic Membrane Defect by Glycine Improves Mitochondrial and Cellular Function in Reoxygenated Renal Tubules

The hypoxic tolerance of renal tubular cells may be improved by specific amino acids. In studies on the protective role of glutathione Weinberg and co-workers (1987) observed that not the tripeptide GSH, but one of its components, the amino acid glycine, suppressed hypoxic alterations in isolated tubular segments of rabbit renal cortex. Later experiments confirmed the cytoprotective role of short-chained, non-essential, neutral amino acids such as glycine or alanine (Heyman et al., 1992). The exact mechanism of glycine protection, however, remains unclear. Some authors postulated an unspecific (Baines et al., 1990) or ligand-bound (Weinberg et al., 1990) membrane-stabilizing effect of glycine and related compounds. Recently we reported that glycine supports cellular volume regulation in renal cortical cells at low extracellular oxygen tension (PO 2 < 1 mm Hg) as well as in a subsequent reoxygenation period (Gronow et al., 1990).

Benzoate hydroxylation: a measure of oxidative stress in divers.

Hyperoxia may facilitate the formation of reactive oxygen species. Recent experiments indicated signs of oxidative stress after 3.5 h hyperoxic diving. We analyzed in the urine of healthy, 100% O2-breathing male volunteers before and after 45 min seawater diving (170 kPa) or 30 min resting at 280 kPa in a pressure chamber (HBO) for sub-fractions of hydroxybenzoate (HB), monohydroxybenzoate (MHB), and of dihydroxybenzoate (DHB). Measurements were performed by HPLC and electrochemical or UV-detection. Additionally, urinary concentrations of thiobarbituric acid-reactive substances (TBARS) and of creatinine (CREA) were analyzed by standard colorimetric assays. During HBO treatment, TBARS, DHB, 2,4-DHB, and 3,4-DHB increased significantly. MHB and CREA did not change. 2,4- and 3,4-DHB-alterations correlated with changes in TBARS. Diving induced urine dilution and stimulated oxygen consumption. Urinary TBARS and HB rose significantly higher during diving at 170 kPa than during HBO at 280 kPa. A different pattern in urinary sub-fractions of DHB could be observed in divers: 2,6 > 2,3 > 2,5 > 3,4. Changes in 2,6- and 2,5-DHB correlated significantly with alterations in TBARS. 2,6-DHB probably indicated renal oxidant stress similar to previously described animal experiments. It is concluded that analyzing urinary HB may provide a sensitive measure to quantify and qualify oxidant stress in divers.

Diminution of Histidine-Induced Reoxygenation Damage By Glycine in Posthypdxic Renal Cells

Free oxygen radicals are generated in cells at inner membranes of mitochondria, in the endoplasmatic reticulum, and in the cytosol. Under physiological conditions and at normal oxygen tension, these oxygen species are detoxified by protective enzymes (e.g. superoxide dismutase, SOD, and catalase) as well as by electron accepting scavengers like α-tocopherol or polyalcohols (Chance et al., 1979). In hyperoxia or during reperfusion, however, tissue activities of protective enzymes and/or the capacity of oxygen radical scavanging cell constituents becomes insufficient, and free oxygen radicals oxidize cellular macromolecules and membranes (Baker et al., 1985; Gronow et al., 1989).