Adenosine metabolism in kidney slices under normoxic conditions

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipyridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5'-methylthio-adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.     

Influence of adenine-induced renal failure on tryptophan-niacin metabolism in rats.

To discover the role of the kidney in tryptophan degradation, especially tryptophan to niacin, rat kidneys were injured by feeding a diet containing a large amount of adenine. The kidney contains very high activity of aminocarboxymuconate-semialdehyde decarboxylase (ACMSD), which leads tryptophan into the glutaric acid pathway and then the TCA cycle, but not to the niacin pathway. On the other hand, kidneys contain significant activity of quinolinate phosphoribosyltransferase (QPRT), which leads tryptophan into the niacin pathway. The ACMSD activity in kidneys were significantly lower in the adenine group than in the control group, while the QPRT activity was almost the same, however, the formations of niacin and its compounds such as N1-methylnicotinamide and its pyridones did not increase, and therefore, the conversion ratio of tryptophan to niacin was lower in the adenine group than in the control group. The contents of NAD and NADP in liver, kidney, and blood were also lower in the adenine group. The decreased levels of niacin and the related compounds were consistent with the changes in the enzyme activities involved in the tryptophan-niacin metabolism in liver. It was concluded from these results that the conversion of tryptophan to niacin is due to only the liver enzymes and that the role of the kidney would be extremely low.     

Epsilon-alkyllysinase. New assay method, purification, and biological significance

?-Alkyllysinase (EC 1.5.3.4.) has been solubilized and purified approximately 15-fold from rat kidney. Flavin adenine dinucleotide stimulated the partially purified enzyme preparation. The enzyme produces an equimolar amount of l-lysine and formaldehyde from ?-N-monomethyl-l-lysine while consuming half a mole of oxygen. Based on the determination of radioactive formaldehyde from ?-N-mono[¹⁴C]methyl-lysine a new, highly sensitive assay method has been developed. All available evidence indicates that ?-alkyllysinase is identical to histone demethylase which has been previously reported to be rich in rat kidney mitochondria. The ?-alkyllysinase activity in the liver and kidney of a young rat are relatively low, and reaches the adult level during or before puberty. On the other hand, protein methylase III, which methylates histones using S-adenosyl-l-methionine as methyl donor, is high in the young rat liver and kidney, and decreases to the adult level in a pattern opposing that of ?-alkyllysinase. This opposing pattern of change of protein methylase III and ?-alkyllysinase activity is also found in fast-growing Novikoff hepatoma: While ?-alkyllysinase activity is practically nil in the hepatoma, protein methylase III is very high.     

The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria.

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.     

The comparative content of the mitochondrial phosphoglyceroyl-ATP-containing polymer, purinogen, in rat tissues

Here, we describe an assay for the tissue content of purinogen, a highly phosphorylated labile polymer containing ATP and phosphoglycerate found in the mitochondrial intermembrane space. We report the purinogen content (as adenine nucleotide equivalents) of rat heart and, for the first time, of rat liver, kidney, brain and mixed skeletal muscle. The findings show that purinogen contains very significant proportions of cell adenine nucleotides ranging from 25% of the free pool in brain and skeletal muscle to 135% of it in kidney. The evidence that purinogen may form a controlled intracellular reservoir of inorganic phosphate is briefly discussed.     

Ischemia decreases the content of the adenine nucleotide translocator in mitochondria of rat kidney

The activity of the adenine nucleotide translocator is decreased at ischemia. Studies were undertaken to elucidate changes in the adenine nucleotide translocator by determining its content in mitochondria of ischemic rat kidney. After 60 min of ischemia, the content of the adenine nucleotide translocator amounted only to about 55%, of that measured in control mitochondria. At the same time, the flux control coefficient was increased. These changes paralled the well-known effects of ischemia: the decrease in oxidative phosphorylation and in adenine nucleotides. It is supposed that the decrease in the adenine nucleotide translocatar content accounts, at least partially, for the ischemia-induced impairment of mitochondria.     

Activities of NAD-and NADP-specific isocitrate dehydrogenases in kidney mitochondria of rachitic rat

In the previous research we have demonstrated that rats fed on a rickets-inducing diet show increasing citrate levels in kidney and intestinal mucosa. The study of the enzymes related to citrate metabolism has shown that both NAD+-and NADP+-dependent isocitrate dehydrogenases decrease in kidney mitochondria of rachitic rat. The inhibitory effect of Ca2+ and citrate on the activity of the two dehydrogenases has been also investigated; these metabolites behave as competitive inhibitors against Mg2+ both in normal and in rachitic rats.     

Purine-phosphoribosyltransferase activities in rat and mouse tissues and in Ehrlich ascites-tumour cells

1. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase in extracts of rat and mouse liver, brain, spleen, heart and kidney, of rat bone marrow and of Ehrlich ascites-tumour cells have been measured. The specific activity of adenine phosphoribosyltransferase in tumour cells (3mμ-moles of nucleotide formed/min./mg. of protein) was between 15 and 60 times the activity of any mouse tissue examined. Hypoxanthine-phosphoribosyltransferase activity was greater in extracts of tumour cells than in mouse tissues, but the difference was not great. 2. The specific activities of both adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase in ascites-tumour cells decreased respectively by 57% and 36% 2 days after the cells had been inoculated into mice. After 4 days of tumour growth the specific activities of both enzymes increased, reaching a maximum at 10 days. 3. The activity of adenine phosphoribosyltransferase in rat-liver extracts increased steadily up to 4 days after partial hepatectomy, and was still above the control value after 14 days. Hypoxanthine-phosphoribosyltransferase activity in extracts of regenerating rat liver did not increase until the second day after operation and had begun to decrease by the fourth day. 4. From the results it was concluded that adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase may be physiologically important enzymes in rapidly growing tissues, and that inhibitors of the former enzyme have potential value in chemotherapy.     

The Importance of Iron,Calcium and Free Radicals in Reperfusion Injury: An Overview of Studies in Ischaemic Rabbit Kidneys

An overview of a series of experiments attempting to link iron and calcium redistribution and release of free fatty acids with falls in pH and adenine nucleotide levels during cold storage of rabbit kidneys is presented. The data reviewed strongly suggest that these events are inextricably linked to subsequent reperfusion injury. Circumstantial evidence incriminating iron was provided by experiments showing that iron chelation decreased reperfusion injury after warm (WI) and cold ischaemia (CI) in rat skin flap and rabbit kidney models. Evidence for a role for calcium was provided when it was found that a calcium channel blocking agent added to the saline flush solution before storage inhibited lipid peroxidation, whereas chemicals which caused release or influx of calcium into the cell exacerbated oxidative damage. Additional involvement of breakdown products of adenine nucleotides was suggested by the protection from lipid peroxidation afforded by allopurinol. Involvement of calcium-activated phospholipase A, was strongly suggested by increases In free fatty acids during cold storage and both this increase and lipid peroxidation were inhibited by addition of dibucaine to the storage solution.     

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Differences in the altered energy metabolism of hemorrhagic shock and hypoxemia.

The effect of hemorrhagic shock, hypoxemia, and anoxia on the levels of adenine and pyridine nucleotides of liver and kidney was assessed. ATP levels in liver and kidney of animals in shock or animals subjected to 7 min of anoxia decreased by 85 and 73%, respectively. Under hypoxic conditions (arterial PO2 AT 18 MMHg), the decrease was only 62 and 48% in liver and kidney, respectively. Tissue NAD levels decreased and NADH levels increased during shock but were found to be essentially unaltered during experimental hypoxemia. Thus, shock produced greater alterations in adenine and pyridine nucleotides than did hypoxemia alone, indicating that stagnant hypoxemia due to shock is more deleterious to energy metabolism than is severe hypoxemia with an otherwise normal circulation. The results also suggest that if an anterial PO2 OF 18 MMHg represents the initial stages of tissue hypoxia, then tissue ATP levels are a more sensitive indicator of this than NAD levels.     

Riboflavin metabolism in the hypothyroid human adult

It had been shown that thyroxine regulates the conversion of riboflavin to riboflavin mononucleotide and flavin adenine dinucleotide (FAD) in laboratory animals. In the hypothyroid rat, the flavin adenine dinucleotide level of the liver decreases to levels observed in riboflavin deficiency. We have shown that in six hypothyroid human adults, the activity of erythrocyte glutathione reductase, an accessible FAD-containing enzyme, is decreased to levels observed during riboflavin deficiency. Thyroxine therapy resulted in normal levels of this enzyme while the subjects were on a controlled dietary regimen. This demonstrates that thyroid hormone regulates the enzymatic conversion of riboflavin to its active coenzyme forms in the human adult.     

Localization of Smad6 and Smad7 in the Rat Kidney and Their Regulated Expression in the Anti-Thy-1 Nephritis

Under normal conditions, kidney expresses Smad6 and Smad7 most abundantly among the organs of the body. To understand the physiological roles of these Smad expressions in the kidney, we first identified the sites of Smad6 and Smad7 expression in the rat kidney by in situ hybridization. The expression of Smad7 in the rat kidney was only observed in the glomeruli, while Smad6 was expressed in both the glomeruli and thick ascending limb of Henle's loop. In order to investigate whether Smad6 and 7 are also involved in the negative feedback loop of TGF-β signaling in vivo, we examined the changes of mRNA levels of these Smads in the glomeruli of rat anti-Thy1 (1–22–3) nephritis, a model where the expression of TGF-β in the glomeruli has been shown to be most up-regulated from day 4 to 14 after the antibody injection. Unexpectedly, 7 days after injection, the levels of Smad6 and Smad7 did not increase but rather decreased to ∼70% of the levels on day 0. During that period, Smad7 immunostaining was observed in the glomerular endothelial cells (GEN) where Smad3 immunostaining was also observed. This suggested that Smad7 expression was not augmented by the TGF-β signal in GEN in vivo in anti-Thy-1 nephritis. The absence of up-regulation of these inhibitory Smads may be involved in the pathogenesis of anti-Thy-1 nephritis.     

The metabolism of organochlorine compound by microsomal enzymes of the shag (Phalacrocorax aristotelis).

1. The activities of microsomal enzymes of adult male shags (Phalacrocorax aristotelis) towards the organochlorine substrates HHDN, HCE and Heom were compred with those of microsomal enzymes of the adult male Wistar rat. 2. Liver homogenates showed similar epoxide hydrase activity to kidney homogenates in the shag, but in the rat liver preparations was much more active than the kidney preparation. 3. Liver microsomes of the shag showed smaller than 8% of the epoxide hydrase activity and smaller than 14% of the hydroxylating capacity of liver microsomes from the rat. 4. The relatively low activity of these enzymes is probably the main reason why the shag has been found to contain relatively high levels of dieldrin in ecological studies.     

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.     

Purification of a mammary-derived growth factor from human milk and human mammary tumors

A growth factor, mammary-derived growth factor 1 (MDGF1), has been purified to apparent homogeneity from human milk. The factor is a pepsin-sensitive, reducing agent-insensitive protein with a molecular mass of 62 kDa and a pI of 4.8. An apparently identical factor has been isolated from human mammary tumors, suggesting that MDGF1 might be made by and act as an autocrine growth factor for mammary cells. High affinity receptors for MDGF1 have been detected on mouse mammary cells, normal rat kidney cells, and A431 epidermoid cells (KD = 2 X 10(-10) M). MDGF1 at picomolar levels stimulates the growth of mammary cells and greatly amplifies their production of collagen, apparently via elevating collagen mRNA levels, an effect that is demonstrated for normal rat kidney cells. The responsiveness of mammary cells to MDGF1 is attenuated when the cells are grown on a basement membrane collagen substratum, a component of the extracellular matrix upon which these cells normally rest in vivo. MDGF1 thus may regulate the production of new basement membrane as mammary epithelium invades the stroma during proliferation.     

Inflammatory response and matrix metalloproteinases in chronic kidney failure: Modulation by adropin and spexin

Chronic kidney disease is a major global public health problem. The peptide hormones adropin and spexin modulate many physiological functions such as energy balance and glucose, lipid and protein metabolism. However, it is unclear whether these peptides may exert effects on renal damage, tissue remodeling, and inflammatory conditions. In view of the limited information, we aimed to investigate the effect of adropin and spexin on matrix metalloproteinase and inflammatory response genes a rat model of adenine-induced chronic kidney failure. Chronic kidney failure was induced in rats by administering adenine hemisulfate. Renal function was determined in an autoanalyzer. Histopathological modifications were assessed by H&E staining. mRNA expression levels of ALOX 15, COX 1, COX 2, IL-1β, IL-10, IL-17A, IL-18 IL-21, IL-33, KIM-1, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, NGAL, TGFβ1, TIMP-1, and TNFα in kidney tissue were measured by qPCR. Our results showed an increase of 24-h urine volume, serum creatinine, BUN, and urine protein levels in group with adenine-induced CKF. Adropin and spexin treatments decreased urine protein and 24-h urine volume. Renal damage, TIMP-1, IL-33, and MMP-2 increased after CKF induction, while COX 1, MMP-9, and MMP-13 levels were significantly reduced. Furthermore, KIM-1, TIMP-1, IL-33, and MMP-2 were downregulated by spexin treatment. Renal damage, NGAL, TIMP-1 IL-17A, IL-33, MMP-2, and MMP-3 decreased after adropin treatment, while MMP-13 levels were upregulated. Treatment with adropin+spexin decreased KIM-1, NGAL, TIMP-1, IL-1β, IL-17A, IL-18, IL-33, ALOX 15, COX 1, COX 2, TGFβ1, TNFα, MMP-2, MMP-3, and MMP-7, but increased MMP-13 levels. Our findings revealed that inflammatory response and MMP genes were modulated by adropin and spexin. These peptides may have protective effects on inflammation and chronic kidney damage progression.