Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

Production of urea from arginine in pars recta and collecting duct of the rat kidney

Urea production from arginine was studied in vitro in the kidney of normal rats in tubule suspensions of the four different renal zones (cortex, outer and inner stripe of outer medulla, and inner medulla), and in individual microdissected nephron segments. Tissue was incubated with L-[guanido-14C]-arginine to measure cellular arginase activity. Addition of urease to the incubate freed 14CO2 from the 14C-urea formed by arginase and released from the cells. CO2 was trapped in KOH and counted. These experiments revealed that significant amounts of urea are produced in the outer stripe and in the inner medulla. This intrarenal urea generation takes place mainly in the proximal straight tubule and in the collecting duct, with increasing activity in these two structures from superficial to deep regions of the kidney. Urea is known to play a critical role in the urinary concentrating process. The fact that some urea can be produced in the mammalian kidney, and that the two structures showing this capacity are straight portions of the renal tubular system descending along the corticopapillary axis suggest that this urea production might play a role in the formation and/or maintenance of the medullary urea concentration gradient.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Location of renal ornithine decarboxylase activity

Renal ornithine decarboxylase (ODC) activity was found to be more prevalent in the medulla of the normal rat kidney than in the cortex. When renal ODC activity was stimulated by ethanol, growth hormone, ACTH, or corticosterone, proportional increases were observed in both medulla and cortex. After hypophysectomy, ODC activities fell equally in both areas of the kidney. The administration of cycloheximide, which is known to cause a rebound increase after six hours in overall renal ODC activity, was followed by an increase of medullary ODC activity while cortical activity remained suppressed.     

Increased H(2)O(2) counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla

A membrane-permeable SOD mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (tempol), has been used as an antioxidant to prevent hypertension. We recently found that this SOD mimetic could not prevent development of hypertension induced by inhibition of renal medullary SOD with diethyldithiocarbamic acid. The present study tested a hypothesis that increased H2O2 counteracts the effects of tempol on renal medullary blood flow (MBF) and Na+ excretion (UNaV), thereby restraining the antihypertensive effect of this SOD mimetic. By in vivo microdialysis and Amplex red H2O2 microassay, it was found that interstitial H2O2 levels in the renal cortex and medulla in anesthetized rats averaged 55.91 +/- 3.66 and 102.18 +/- 5.16 nM, respectively. Renal medullary interstitial infusion of tempol (30 micromol x min-1x kg-1) significantly increased medullary H2O2 levels by 46%, and coinfusion of catalase (10 mg x min-1x kg-1) completely abolished this increase. Functionally, removal of H2O2 by catalase enhanced the tempol-induced increase in MBF, urine flow, and UNaV by 28, 41, and 30%, respectively. Direct delivery of H2O2 by renal medullary interstitial infusion (7.5-30 nmol x min-1x kg-1) significantly decreased renal MBF, urine flow, and UNaV, and catalase reversed the effects of H2O2. We conclude that tempol produces a renal medullary vasodilator effect and results in diuresis and natriuresis. However, this SOD mimetic increases the formation of H2O2, which constricts medullary vessels and, thereby, counteracts its vasodilator actions. This counteracting effect of H2O2 may limit the use of tempol as an antihypertensive agent under exaggerated oxidative stress in the kidney.     

Endogenous Carbamylation of Renal Medullary Proteins

Protein carbamylation is a post-translational modification that can occur in the presence of urea. In solution, urea is in equilibrium with ammonium cyanate, and carbamylation occurs when cyanate ions react with the amino groups of lysines, arginines, protein N-termini, as well as sulfhydryl groups of cysteines. The concentration of urea is elevated in the renal inner medulla compared with other tissues. Due to the high urea concentration, we hypothesized that carbamylation can occur endogenously within the rat inner medulla. Using immunoblotting of rat kidney cortical and medullary homogenates with a carbamyl-lysine specific antibody, we showed that carbamylation is present in a large number of inner medullary proteins. Using protein mass spectrometry (LC-MS/MS) of rat renal inner medulla, we identified 456 unique carbamylated sites in 403 proteins, including many that play important physiological roles in the renal medulla [Data can be accessed at https://helixweb.nih.gov/ESBL/Database/Carbamylation/Carbamylation_peptide_sorted.html]. We conclude that protein carbamylation occurs endogenously in the kidney, modifying many physiologically important proteins.     

Sequential inactivation of reactive oxygen species by combined overexpression of SOD isoforms and catalase in insulin-producing cells

Insulin-producing cells show very low activity levels of the cytoprotective enzymes catalase, glutathione peroxidase, and superoxide dismutase. This weak antioxidative defense status has been considered a major feature of the poor resistance against oxidative stress. Therefore, we analyzed the protective effect of a combined overexpression of Cu,ZnSOD or MnSOD together with different levels of catalase. Catalase alone was able to increase the resistance of transfected RINm5F insulin-producing tissue culture cells against H(2)O(2) and HX/XO, but no protection was seen in the case of menadione. In combination with an increase of the MnSOD or Cu,ZnSOD expression, the protective action of catalase overexpression could be further increased and extended to the toxicity of menadione. Thus, optimal protection of insulin-producing cells against oxidative stress-mediated toxicity requires a combined overexpression of both superoxide- and hydrogen peroxide-inactivating enzymes. This treatment can compensate for the constitutively low level of antioxidant enzyme expression in insulin-producing cells and may provide an improved protection in situations of free radical-mediated destruction of pancreatic beta cells in the process of autoimmune diabetes development.     

Sexual dimorphism in oxidant status in spontaneously hypertensive rats

Male spontaneously hypertensive rats (SHR) have a blunted pressure-natriuresis relationship and enhanced oxidative stress compared with female SHR. Furthermore, oxidative stress contributes to abnormal renal Na+ handling and renal damage in hypertension. The aim of this study was to determine whether a sex difference exists in renal inner medullary hydrogen peroxide (H2O2) levels and/or antioxidant systems in SHR and the influence of sex steroids on these systems. Thirteen-week-old intact and gonadectomized male and female SHR were placed in metabolic cages for 24-h urine collection. Renal inner medullas were isolated for antioxidant activity assays and Western blot analysis or for measurements of H2O2 using Amplex Red. Studies verified that male SHR had greater Na+ reabsorption compared with female SHR. Male SHR had enhanced urinary excretion of H2O2 compared with female SHR. Gonadectomy decreased H2O2 excretion in males and increased H2O2 excretion in females, suggesting that testosterone stimulates total body oxidative stress and estrogen suppresses levels of total body oxidative stress. There was not a sex difference in inner medullary H2O2 levels. Male SHR had a testosterone-dependent increase in inner medullary SOD activity, and both intact and gonadectomized males had high levels of inner medullary catalase activity compared with females. The results of this study showed that there was a sexual dimorphism in Na+ handling and oxidant status. We hypothesize that there is a testosterone-sensitive increase in whole body reactive oxygen species production that results in a compensatory increase in the inner medullary antioxidant capability possibly to normalize Na+ handling.     

Putative osmolytes in the kidney of the Australian brush-tailed possum,Trichosurus vulpecula

The Australian brush-tailed possum, Trichosurus vulpecula, is capable of producing a moderately concentrated urine, at least up to 1300 mOsm l(-1). Kidneys of adult animals fed in captivity on a normal diet with ready access to water were analysed. The inner medullary regions were found to have moderately high concentrations of sodium (outer medulla, 367+/-37; inner medulla 975+/-93 mmol kg(-1) dry wt.), chloride (outer medulla 240+/-21; inner medulla 701+/-23 mmol kg(-1) dry wt.) and urea (outer medulla, 252+/-62; inner medulla, 714+/-69 mmol kg(-1) protein). When the animals were fed on a 'wet diet', amounts of these substances in the outer medulla and cortex were reduced, although with the exception of urea these changes were not significant. There were highly significant changes in amounts of Na(+), Cl(-) and urea in the inner medulla (Na(+), 566+/-7; Cl(-), 422+/-9 mmol kg(-1) dry wt.; urea 393+/-84 mmol kg(-1) protein). Likewise, the inner medulla of animals fed a 'dry diet' with limited access to water showed highly significant increases in the same substances (Na(+), 1213+/-167; Cl(-), 974+/-137 mmol kg(-1) dry wt.; urea, 1672+/-98 mmol kg(-1) protein). Inositol was found in the outer medulla (224+/-90 mmol kg(-1) protein) and inner medulla (282 mmol kg(-1) protein) as was sorbitol (outer medulla, 62+/-20; inner medulla, 274+/-72 mmol kg(-1) protein). Both these polyols were reduced in amount in renal tissue from 'wet diet' animals, and increased in 'dry diet' animals, but the changes were not statistically significant. The methylamines, betaine and glycerophosphorylcholine (GPC), showed a similar pattern, but both were significantly elevated in the inner medulla of 'dry diet' animals (betaine 154+/-57 to 315+/-29 mmol kg(-1) protein; GPC 35+/-7 to 47+/-10 mmol kg(-1) protein). It was concluded that in this marsupial the concentrating mechanism probably functions in a similar way to that in higher mammals, and that the mechanism of osmoprotection of the medulla of the kidney involves the same osmolytes. However, the high ratio of betaine to GPC in the inner medulla resembles the situation in the avian kidney.     

Developmental immunolocalization of heat shock protein 70 (HSP70) in epithelial cell of rat kidney

During renal development the cells in the medulla are exposed to elevated and variable interstitial osmolality. Heat shock protein 70 (HSP70) is a major molecular chaperone and plays an important role in the protection of cells in the renal medulla from high osmolality. The purpose of this study was to establish the time of immunolocalization and distribution of HSP70 in developing and adult rat kidney. In addition, changes in HSP70 immunolocalization following the infusion of furosemide were investigated. In adult animals, the HSP70 was expressed in the medullary thin ascending limb of Henle's loop (ATL) and inner medullary collecting duct (IMCD). In developing kidney, HSP70 immunoreactivity was first detected in the IMCD of the papillary tip on postnatal day 1. From four to 14 days of age, HSP70 was detected in the ATL after transformation from thick ascending limb, beginning at the papillary tip and ascending to the border between the outer and inner medulla. The immunolocalization of HSP70 in both the ATL and IMCD gradually increased during two weeks. The gradual increase in HSP70 was associated with an increase in its mRNA abundance. However, furosemide infusion resulted in significantly reduced HSP70 immunolocalization in the IMCD and ATL. These data demonstrated that the expression of HSP70 was closely correlated with changes in interstitial osmolality during the development of the kidney. We suggest that HSP70 protects ATL and IMCD cells in the inner medulla from the stress of high osmolality and may be involved in the transformation of the ATL of the long loop of Henle during renal development.     

Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity and reactive oxygen species production in MDCK-I renal cells and intact mice

Cisplatin-induced oxidative stress can cause liver and kidney damage, thus limiting therapeutic efficacy. Thus, in the present study, since Rhus verniciflua Stokes (RVS) containing flavonoids has antioxidant effects, we investigated whether it can protect cisplatin-induced toxicity in vitro and in vivo, The in vitro effects of RVS on the cell viability and reactive oxygen species (ROS) production were investigated using cisplatin-treated Madin–Darby Canine kidney (MDCK)-I renal cells. Its in vivo effects were also studied in BALB/c mice inoculated with CT-26 colon adenocarcinoma cells and treated with cisplatin with or without RVS. Liver and renal functions were assessed together with indices of tissue oxidation. RVS prevented cisplatin-induced cytotoxicity and ROS release against MDCK-I cells. RVS alone exerted modest antitumor activity against CT-26 cells. When used concurrently with cisplatin, RVS prevented the increases in serum blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and NO, while reducing liver and kidney tissue MDA content, and increasing catalase, glutathione (GSH), and superoxide dismutase (SOD) activities. Moreover, the antitumor efficacy of cisplatin was not altered by concurrent administration of RVS. These findings demonstrate that RVS prevents cisplatin-induced toxicity in vitro and in vivo via an antioxidant activity without hurting its antitumor effectiveness, suggesting that RVS can be usefully applied to the neoplastic patients as a combined chemopreventive agent with cisplatin.     

Signaling and gene regulation by urea in cells of the mammalian kidney medulla

Signaling by urea, although incompletely understood, is relevant both to cells of the mammalian kidney inner medulla and to all cells of the organism in the setting of advanced renal failure with its attendant accumulation of urea in the systemic circulation. The molecular events initiated by urea stress are distinct from those occurring in response to hypertonic stress; urea activates a characteristic subset of signaling events, which are in large part specific to cultured renal tubular epithelial cells. Interestingly, urea is protective of hypertonic NaCl-inducible apoptosis in this model. Details of this phenomenon are reviewed. The effect of urea has been likened to that of either hypertonicity or of a peptide mitogen. In preliminary expression array analyses, the profile of genes activated by urea stress in renal medullary cells, however, was found to be unique.     

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.