The role of oxygen free radicals in the development of chronic renal failure.

This study examined whether there is increased production of oxygen free radicals during chronic renal failure. Rats subjected to 3/4 nephrectomy and sham operated controls were killed after 3 weeks. Lipid extracts of plasma and renal tissue were examined by HPLC and kidney specimens were also analyzed by EPR spectroscopy. The redox capacity of blood was assessed using nitroblue tetrazolium and plasma ascorbate levels were measured with HPLC. There was no detectable renal production of oxygen free radicals in rats with chronic renal failure. Kidney parenchymal content of other oxidants and the oxidant:reductant ratio were similar in control and uremic animals. The plasma redox capacity and ascorbate levels were elevated in uremic rats. We conclude that early in the course of chronic renal failure, there is not excessive production of oxygen free radicals. There is accumulation of reductants, primarily ascorbate, in the plasma of uremic animals.     

Effect of propranolol on glycerol induced acute renal failure in rats

The effect produced by propranolol, administered for a prolonged period of time and in large doses, on renal function in rats has been studied, as well as the modifications induced by this treatment in an experimental model of acute renal failure, and the effects of a single dose of propranolol given 1 hour before provoking failure. Propranolol, administered chronically, causes sodium and water retention and increases creatinine clearance. Acute renal failure induced by glycerol in rats treated for 7 days with propranolol is less severe than the one produced in untreated animals. In this ARF model, a single dose of propranolol does not seem to have a protective effect.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

Calcium and phosphate metabolism in uranyl nitrate-induced acute renal failure

Plasma calcium regulation in uranyl nitrate-treated dogs was studied. A discrete hypercalcemia was observed without significant changes in the level of plasma ionized calcium. Serum phosphate increased markedly following uranyl nitrate treatment. A sharp rise of iPTH was demonstrated. Uranyl nitrate-induced acute renal failure in dogs was found to be a useful model for studying the mechanisms regulating calcium and phosphate metabolism.     

Effect of prostaglandin inhibition on glomerular filtration rate in normal and uremic rabbits

Studies were performed to assess the effect of alterations in prostaglandin biosynthesis on glomerular filtration rate in rabbits with normal renal function and after surgical reduction of renal mass. In normal animals, the administration of either of two cyclo-oxygenase inhibitors resulted in a 53% reduction in urine prostaglandin E excretion, but no change in creatinine clearance. Creatinine clearance rates were almost 71% lower in the uremic animals when compared to the animals with normal renal function. Despite the reduction in renal mass, urine prostaglandin E excretion rates in the uremic animals were over twice that seen in normal rabbits. When factored by either glomerular filtration rate or remaining renal mass, urine prostaglandin E excretion rates in uremic rabbits when compared to normal animals were increased more than 9-times and 4-times respectively. Administration of cyclo-oxygenase inhibitors in the uremic animals resulted in a 71% decrease in urine prostaglandin E excretion and, unlike the non-uremic animals, a 53% fall in creatinine clearance. These findings suggest that intact renal prostaglandin biosynthesis is a necessary factor in the homeostatic adaptive mechanisms which maintain the glomerular filtration rate in animals with decreased renal mass.     

Molsidomine, a nitric oxide donor and L-arginine protects against rhabdomyolysis-induced myoglobinuric acute renal failure

Rhabdomyolysis-induced myoglobinuric acute renal failure accounts for about 10-40% of all cases of acute renal failure (ARF). Nitric oxide and reactive oxygen intermediates play a crucial role in the pathogenesis of myoglobinuric acute renal failure (ARF). This study was designed to investigate the effect of molsidomine and L-arginine in glycerol induced ARF in rats. Six groups of rats were employed in this study, group I served as control, group II was given 50% glycerol (8 ml/kg, intramuscularly), groups III and IV were given glycerol plus molsidomine (5 mg/kg, and 10 mg/kg p.o. route respectively) 60 min prior to the glycerol injection, group V animals were given glycerol plus L-arginine (125 mg/kg, p.o.) 60 min prior to the glycerol injection, and group VI received L-NAME (10 mg/kg, i.p.) along with glycerol 30 min prior to glycerol administration. Renal injury was assessed by measuring plasma creatinine, blood urea nitrogen, creatinine and urea clearance. The oxidative stress was measured by renal malondialdehyde levels, reduced glutathione levels and by enzymatic activity of catalase, reduced glutathione and superoxide dismutase. Tissue and urine nitrite levels were measured as an index of total nitric oxide levels. Glycerol treatment resulted in a marked decrease in tissue and urine nitric oxide levels, renal oxidative stress and significantly deranged the renal functions along with deterioration of renal morphology. Pre-treatment of animals with molsidomine (10 mg/kg) and L-arginine 60 min prior to glycerol injection markedly attenuated fall in nitric oxide levels, renal dysfunction, morphological alterations, reduced elevated TBARS and restored the depleted renal antioxidant enzymes. The animals treated with L-NAME along with glycerol further worsened the renal damage observed with glycerol. As a result, our results indicate that molsidomine and L-arginine may have beneficial effects in myoglobinuric ARF.     

Effects of parathyroidectomy on tissue calcium,phosphorus, magnesium,and copper concentrations in aluminum-loaded uremic rats

Rats were subjected to a two-stage 5/6 nephrectomy and treated with Al for 2 and 4 wk with a cumulative dose of 4.2 and 8.4 mg of Al, respectively. Other animals were parathyrectomized (PTx) and loaded with 8.4 mg of Al for 4 wk. Total Al, Ca, P, Mg, and Cu contents were analyzed in the liver, kidney, and bone by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that Al given to growing uremic rats significantly increased the content of Al in the liver, kidney, and bone. Moreover, Al treatment increased the liver and kidney Ca levels and decreased the Ca and P values in bone. Previous parathyroidectomy significantly reduced Al accumulation within organs and changes in the Ca and P levels in the bone, liver, and kidney. The result was not influenced by different degrees of renal failure.     

Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure.

We investigated L-kynurenine distribution and metabolism in rats with experimental chronic renal failure of various severity, induced by unilateral nephrectomy and partial removal of contralateral kidney cortex. In animals with renal insufficiency the plasma concentration and the content of L-tryptophan in homogenates of kidney, liver, lung, intestine and spleen were significantly decreased. These changes were accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate-limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3-dioxygenase activity was unchanged. Conversely, the plasma concentration and tissue content of L-kynurenine, 3-hydroxykynurenine, and anthranilic, kynurenic, xanthurenic and quinolinic acids in the kidney, liver, lung, intestine, spleen and muscles were increased. The accumulation of L-kynurenine and the products of its degradation was proportional to the severity of renal failure and correlated with the concentration of renal insufficiency marker, creatinine. Kynurenine aminotransferase, kynureninase and 3-hydroxyanthranilate-3,4-dioxygenase activity was diminished or unchanged, while the activity of kynurenine 3-hydroxylase was significantly increased. We conclude that chronic renal failure is associated with the accumulation of L-kynurenine metabolites, which may be involved in the pathogenesis of certain uremic syndromes.     

Dopamine and TSH secretion in uremic male rats

The role of dopamine in the dysregulation of TSH secretion in uremic male rats was investigated using the dopamine antagonist, pimozide. In order to obviate the effect of weight loss due to uremia-induced anorexia as a cause of altered TSH secretion in uremia, we also studied a group of normal animals whose food intake was restricted and who demonstrated weight loss comparable to that of the uremic animals. Baseline TSH concentrations were not significantly different in the normal, uremic or starved animals. Pimozide administration produced no change in the baseline TSH concentrations in any of the groups of rats. The peak TSH response to TRH (5 micrograms IV) was significantly blunted in the uremic animals compared to the normal controls and the starved animals. Pimozide administration did not alter the peak TRH-stimulated TSH response in either the normal animals or the starved animals. However, the peak TRH-stimulated TSH response was significantly increased in the uremic animals and was comparable to the peak TSH response seen in the pimozide-untreated control animals. The data suggest that experimental renal failure in rats is associated with diminished sensitivity of the thyrotroph to TRH stimulation, and that this blunted sensitivity may be dopamine-dependent since it can be abolished by pharmacologic dopamine blockade.     

Impaired binding of low density lipoprotein to hepatic membranes from uremic guinea pigs.

Chronic renal failure is associated with abnormalities in lipoprotein metabolism that may contribute to premature atherosclerosis and early mortality in patients on dialysis. In previous studies, we found that plasma clearance of radiolabelled low density lipoprotein (LDL) was retarded in nephrectomized guinea pigs left with one-sixth of normal functioning renal mass. To elucidate potential mechanisms of delayed LDL clearance, we compared binding of LDL to hepatic membranes from both normal and uremic guinea pigs. One hundred micrograms of the 8000-100,000 X g hepatic microsomal protein was incubated with 125I-labelled normal guinea pig LDL (10-150 micrograms/mL) for 1 h at 37 degrees C, and the membrane washed and pelleted by centrifugation in a Beckman Ti 42.2 rotor. Parallel incubations with excess unlabelled LDL were done to determine specific binding. LDL specific binding to uremic hepatic membranes was significantly impaired compared with normal ones. The major abnormality, as determined by Scatchard transformation of the binding data, was a reduction of the apparent maximal binding of LDL to uremic membranes, with an average Bmax of 4.1 micrograms/mg protein compared with 6.6 micrograms/mg protein for normal hepatic microsomes. The affinity of LDL for uremic liver membranes was only slightly diminished with a mean apparent Kd of 35.2 micrograms/mL in comparison with 21.8 micrograms/mL for normal liver membranes. These results provide a biochemical explanation for the diminished LDL clearance in uremia and may account for the dyslipidemia of renal failure.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

Effect of glycerol-induced acute renal failure and di-2-ethylhexyl phthalate on the enzymes involved in biotransformation of xenobiotixs.

The effects of di-(2-ethylhexyl)-phthalate (DEPH) on the levels of cytochrome P-450 and b5 monooxygenases were studied in the rat kidney and liver in acute renal failure induced by glycerol. Intramuscular injection of glycerol (50%,10 ml x kg(-1)) to rats produced proximal tubular damage and acute renal failure. The indicators of renal function, serum urea and creatinine significantly increased (480 and 350 percent, respectively). In control and glycerol-treated animals DEPH had no significant effect on the concentrations of serum urea and creatinine. Twenty-four hours after glycerol injection the total amount of cytochrome P-450 and b5 significantly decreased in renal but increased in liver microsomal fractions. Moreover, 48 and 72 hours after glycerol injection the level of cytochrome P-450 and b5 significantly increased in both organs. A single dose of DEPH (2 ml x kg(-1), i.p.) also elevated the total cytochrome P-450 and b5 in control animals. This enhancing effect of DEPH was additive to that of glycerol in glycerol-induced acute renal failure. These results indicate that DEPH and glycerol evoked pathological changes may affect the metabolism of xenobiotics plus endogenous hormones in the liver and in kidney.     

Elimination of endogenous toxin, creatinine from blood plasma depends on albumin conformation: site specific uremic toxicity & impaired drug binding

Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state.     

Role of glomerular nitric oxide in glycerol-induced acute renal failure

Myoglobinuric acute renal failure remains one of the least understood clinical syndromes and the mediators involved remain obscure. The aim of the present study was to assess the role of nitric oxide in glycerol-induced acute renal failure under normal conditions and after uninephrectomy. Acute renal failure was induced in rats by injection of 50% glycerol (10 mL x kg(-1) body weight). Half of the animals were subjected to uninephrectomy two days before glycerol injection. Two days after the induction of acute renal failure, glomeruli from some animals were isolated and glomerular nitrite production was measured. Another group of animals was used for acute clearance studies. In this case, the effect of infusing either L-NAME or L-arginine was assayed. Glomerular nitrite production was significantly decreased in glycerol-induced acute renal failure. Glomeruli from uninephrectomized animals showed an increase in nitrite production, both in normal conditions and after glycerol injection, as compared with glomeruli from non-nephrectomized animals. L-NAME infusion worsened renal function in all the study groups, but more slowly in animals with glycerol-induced acute renal failure than in control rats. In uninephrectomized animals L-NAME reduced renal function more than in animals with two kidneys. In conclusion, in this model of acute renal failure the decrease in glomerular nitric oxide production plays an important role in the decrease in renal function. After uninephrectomy, an increase in glomerular nitric oxide synthesis plays a protective role against glycerol-induced acute renal failure.     

Changes in plasma glycerol level in rabbits during fatigue-induced stress

The lipolytic response to fatigue-induced stress was studied in fed and fasted rabbits and in fed propranolol-treated rabbits. The initial plasma glycerol level was higher and the increase in glycerol after stress was lower in fasted as compared to fed animals. Propranolol lowered the initial glycerol level and attenuated the fatigue-induced rise in glycerol concentration. The data suggest that in rabbits, as in other species, catecholamines increase lipolysis through stimulation of beta-adrenoceptors. The increment in alpha-adrenergic responsiveness during fasting may contribute to decreased glycerol response to stress observed in fasted rabbits.     

Differential action of methylselenocysteine in control and alloxan-diabetic rabbits

Antidiabetic action of inorganic selenium compounds is commonly accepted. Since in diet selenium mainly exists as selenoamino acids, potential hypoglycemic properties of methylselenocysteine (MSC) were investigated in four groups of rabbits: untreated and MSC-treated control animals as well as alloxan-diabetic and MSC-treated diabetic rabbits. MSC (at a dose of 1 mg/kg body weight) was administered daily for 3 weeks via intraperitoneal injection. The data show, that in MSC-treated control animals plasma glucose concentration was diminished, while plasma urea and creatinine levels as well as urine albumin content were elevated and necrotic changes occurred in kidney-cortex. Decreased GSH/GSSG ratios in blood, liver and kidney-cortex were accompanied by increased glutathione peroxidase and glutathione reductase activities and a diminished renal γ-glutamylcysteine synthetase activity. Death of 50% of control animals was preceded by a dramatic decline in blood glucose concentration. Surprisingly, in MSC-treated diabetic rabbits, plasma glucose levels were either normalized or significantly decreased. Blood and liver GSH/GSSG ratios were increased and renal functions were markedly improved, as indicated by a diminished albuminuria and attenuated histological changes characteristic of diabetes. However, after administration of MSC to diabetic rabbits plasma urea and creatinine levels as well as renal GSH/GSSG ratios were not altered. In view of MSC-induced marked accumulation of selenium in kidneys and liver of control rabbits, accompanied by a decline in blood glucose level, disturbance of glutathione homeostasis and kidney-injury, application of MSC in chemotherapy needs a careful evaluation. On the contrary, MSC supplementation might be beneficial for diabetes therapy due to an improvement of both glycemia and renal function.     

The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast.

The interaction of uranyl ions with inorganic pyrophosphatase from baker's yeast was investigated by measurement of their effect on the protein fluorescence. Fluorescence titrations of the native enzyme with uranyl nitrate show that there is a specific binding of uranyl ions to the enzyme. It was deduced that each subunit of the enzyme binds one uranyl ion. The binding constant was estimated to be in the order of 10(7) M-1. The enzyme which contains a small number of chemically modified carboxyl groups was not able to bind uranyl ions specifically. The modification of carboxyl groups was carried out by use of a water soluble carbodiimide and the nucleophilic reagent N-(2,4-dinitro-phenyl)-hexamethylenediamine. The substrate analogue calcium pyrophosphate displaced the uranyl ions from their binding sites at the enzyme. From the results it is concluded that carboxyl groups of the active site are the ligands for the binding of uranyl ions.     

Intestinal absorption of L-ascorbic acid in rats with renal failure

We studied L-ascorbic acid absorption in rats subjected to subtotal nephrectomy (renal failure (RF) group) and compared the results with those obtained in sham-operated normal animals and those pair-fed with their azotemic counterparts (PF group). In vivo recirculating perfusion and in vitro everted sac techniques were employed. The in vitro experiments were repeated substituting buffer within the serosal compartment with pooled sera from uremic and normal individuals. L-Ascorbic acid absorption in vivo in the RF group was significantly lower than those found in normal control and PF groups. In contrast, the in vitro mucosal to serosal transport was increased in the RF and PF groups when compared with the normal control group, suggesting increased permeability to L-ascorbic acid in the former groups. The disparity between in vivo and in vitro results in the RF animals is indicative of some inhibitory influence present in the intact uremic animals. However, experiments comparing the effect of uremic with normal sera on in vitro transport failed to reveal any suppressive effect of uremic chemical environment. In addition, serum ascorbic acid was reduced in PF and RF groups when compared with the normal control animals, thereby excluding elevated blood level as a cause of impaired absorption in intact animals with RF. In conclusion, in vivo jejunal absorption of L-ascorbic acid is impaired in rats with RF despite evidence of increased in vitro permeability. The latter appears to be mediated by reduced nutrient intake and weight loss. The inhibitory influence present in vivo could not be reproduced by incubation with uremic sera in vitro.     

Polyamines in renal failure

Summary. The levels of polyamines (putrescine, spermidine and spermine) and polyamine oxidase in plasma of patients with chronic renal failure were determined. The level of putrescine was increased but the level of spermine was decreased in the plasma of these patients. The patients also had increased plasma polyamine oxidase activity leading to increased degradation of spermine. As acrolein was a major toxic compound produced from spermine by polyamine oxidase, the levels of free and protein-conjugated acrolein in plasma were also measured. Acrolein levels were enhanced in plasma of patients with chronic renal failure. The accumulated acrolein found as protein conjugates was equivalent to 170 μM, which was about 5-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by spermine oxidase in plasma. An increase in putrescine, spermine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. After patients with chronic renal failure had undergone hemodialysis, their levels of plasma polyamines, spermine oxidase and acrolein returned towards normal. It is likely that acrolein produced from spermine accumulates in the blood due to decreased excretion into urine and may function as a uremic “toxin”.