Search

Long-term treatment with cyclosporine A (CsA) is associated with various types of complications; however, CsA-induced anemia has not been reported. The present study examined the impact of CsA on hematopoietic parameters and intrarenal expression of erythropoietin (EPO) and the EPO receptor (EPOR) in a rat model of chronic CsA nephrotoxicity. Sprague-Dawley rats were fed a low-salt diet (0.05% sodium) and were treated daily for 4 weeks with vehicle (olive oil 1 mL/kg subcutaneously) or CsA (15 mg/kg subcutaneously). The expression of EPO and EPOR was evaluated by immunohistochemistry and immunoblotting, and hematopoietic parameters were assessed by measuring blood hemoglobin and hematocrit levels, and these variables were compared between treatment groups. Renal function, oxidative stress, histopathology (tubulointerstitial fibrosis), apoptotic cell death, and expression of transforming growth factor β-inducible gene-h3 (βig-h3) were also compared between treatment groups. In kidneys from vehicle-treated rats, endogenous EPO and EPOR protein were expressed constitutively in the outer stripe of the outer medulla and the cortex. EPO protein expression decreased significantly in kidneys from CsA-treated rats. By contrast, EPOR expression was higher in kidneys from CsA-treated rats than in vehicle-treated rats. These changes were accompanied by decreases in serum hemoglobin and hematocrit levels and correlated with the number of cells positive for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (r = -0.769, P = .003) and βig-h3 protein expression (r = -0.910, P < .001). Long-term treatment with CsA suppresses renal endogenous EPO expression, resulting in anemia. Increases in apoptotic cell death and βig-h3 expression are closely associated with inhibition of EPO expression in chronic CsA nephrotoxicity., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to verify the estimation and the predictive abilities of serum creatinine (Cr), serum cystatin C (Cys C), and related formulas for acute kidney injury (AKI)., Patients and Methods: Thirty patients who underwent cadaveric donor liver transplantation were enrolled in this prospective study. Glomerular filtration rate (GFR) was assessed by the 99mTc DTPA clearance method and estimated by Cr-predicted clearances (Cockcroft-Gault method [CG] and abbreviated Modification of Diet in Renal Disease equation [MDRD]) as well as by 3 other Cys C-based formulas (Hoek, Filler, and Larsson). AKI was confirmed as GFR<80 mL/min/1.73 m2 in the first posttransplantation week., Results: GFR was significantly correlated with reciprocal Cr, reciprocal Cys C, and the 5 formulas (P<.001 for all). The receiver operating characteristic (ROC) area of Cys C was larger than that of Cr (.937 vs .794, P<.05). ROC area of Hoek, or Filler or Larsson was also larger than that of CG or MDRD (.937, .935, .937 vs .802, .849, P<.05 for all). ROC analysis showed the cutoff values were 1.0 mg/dL for Cr and 1.57 mg/L for Cys C. Hoek, Filler, and Larsson equations all underestimated AKI; their optimal cutoff values should be adjusted to 47, 56, and 44 mL/min/1.73 m2, respectively., Conclusion: Cys C is a better predictor of AKI than Cr. A value of more than 1.57 mg/L might be considered a new definition of AKI.

Objective: To study changes of DNA content in the kidney cellule of rats and relationship with the postmortem interval., Methods: This experiment chose seven parameter of cell nuclear, including the area and integral optical density, determined the changes of DNA content in the kidney cellule of 15 rats at different intervals between 0 and 48 h postmortem with auto-TV-image system., Results: The degradation rate of DNA in nuclear has a certainty relationship to early PMI(in 48 h) of rat, and get binomial regress equation., Conclusion: Determining the quantity of DNA in nuclear should be an objective and exact way to estimate the PMI.

There is strong evidence that estrogens are involved in the etiology, promotion and progression of a variety of cancers, including the cancers of the breast and endometrium. The Syrian hamster estrogen-induced, estrogen-dependent renal neoplasm is a well-established animal model used to elucidate the cellular and molecular mechanisms involved in solely estrogen-induced carcinogenic processes. G(1) cell cycle progression was studied in estrogen-induced early renal tumor foci and in large kidney tumors of castrated male hamsters. Levels of cyclin D1, cyclin E and retinoblastoma (pRb) proteins were higher in these renal neoplasias than in adjacent uninvolved renal tissue and kidneys from untreated, age-matched animals. Of particular interest is the presence of a predominant 35 kDa cyclin E protein variant form in primary renal tumors. In addition, amounts of the phosphorylated forms of cyclin-dependent kinases (cdk) 2 and 4 were decreased, and both RNA and protein levels of p27(kip1) (p27), a cyclin-dependent kinase inhibitor, were markedly higher in early and frank renal tumors than in adjacent uninvolved renal tissue and kidneys of untreated, age-matched animals. These changes in cell cycle components coincided with a rise in renal tumor cell proliferation. Binding of the elevated p27 protein to cyclin E, cdk2 and cdk4, however, was not impaired, suggesting that this cell cycle suppressor protein is functional. In addition, cyclin D1-, cdk2-, cdk4- and cyclin E-associated kinase activities were also lower in these estrogen-induced renal neoplasms than in untreated, age-matched kidneys. Interestingly, when compared with untreated kidney tissue, early and frank renal neoplasms had less of the 62 kDa native form of E2F1 and contained a 57 kDa variant form. Thus we have characterized an unusual deregulation of the cell cycle during estrogen-induced renal tumorigenesis in Syrian hamsters which still allows for estrogen-driven kidney tumor cell proliferation and may contribute to the early genomic instability found.

An estrogen receptor-driven, multistep process for estrogen carcinogenesis in the Syrian hamster kidney is proposed. Because in this species the reproductive and urogenital tracts arise from the same embryonic germinal ridge, it is evident that the kidney has carried over genes that are responsive to estrogens. Using in situ hybridization, overexpression of early estrogen-response genes, i.e., c-myc and c-fos, has been shown to be localized preferentially in early renal tumor foci after 3.5-4.0 months of estrogen treatment. This event coincides with an increased number of S-phase proliferating cell nuclear antigen-labeled cells in these tumor foci, along with a rapid rise in aneuploid frequency in the kidney. Western blot analyses of c-MYC and c-FOS protein products support the overexpression of these genes. Amplification of c-myc, 2.4-3.6-fold, but not of c-fos, was detected in 67% of the primary renal tumors examined, by Southern blot analyses. Consistent chromosomal gains, common to both diethylstilbestrol- and estradiol-induced renal neoplasms, were observed in chromosomes 1, 2, 3, (6), 11, (13), 16, 20, and 21 (chromosome number alterations are indicated in parentheses). Using fluorescence in situ hybridization, the c-myc gene was localized to hamster chromosome 6qb. Chromosome 6 exhibited a high frequency of trisomies and tetrasomies in the kidney after 5.0 months of estrogen treatment and in primary renal tumors. The data presented indicate that estrogen-induced genomic instability may be a key element in carcinogenic processes induced by estrogens.

A cDNA encoding alpha-class glutathione S-transferase Yc (GSTYc) has been isolated from a Syrian hamster kidney library, and its nucleotide sequence (968 bp) has been determined. Analysis of the deduced amino acid sequence revealed a high level of identity between Syrian hamster GSTYc, rat GST Yc1 and Yc2 and mouse GSTYc. Northern-blot experiments demonstrated that Syrian hamster GSTYc expression is tissue-specific. A GSTYc mRNA of approx. 1 kb is expressed in liver, kidney, vas deferens and epididymis. Expression of the GSTYc transcript was not detected in testis or uterus.

Both natural and synthetic estrogens are capable of inducing renal neoplasms in Syrian hamsters with an incidence approaching 100%. Neither the sequence of events nor the mechanisms involved in estrogen carcinogenesis in this model have been established. Results presented here indicate that estrogen induces renal tubular damage in the hamster kidney that is progressive and cumulative. Tubular injury was evident both as abnormal or lost microvilli, accumulation of cytoplasmic lipid droplets, vacuolization, and increases in secondary and tertiary lysosomes after 1.5 months of diethylstilbestrol (DES) treatment. Increasing tubular damage was evidence by the detachment of tubular cells, cell debris, and occluded renal tubular lumens. In an effort to repair proximal tubular damage in the hamster kidney elicited by estrogens, a 4.0-fold increase in proximal tubule BrdU labeling was evident at 4 months of DES or 17 beta-estradiol (E2) treatment and in earlier estrogen treatment periods (1-3 months). During this period, there was a significant increase in aneuploid cells in the hamster kidney, the near diploid frequency increased more than 6.0-fold, and the near tetraploid frequency increased at least 3.0-fold between 1.5 and 3.5 months of estrogen treatment. Based on these data, the early sequence of events leading to estrogen-induced renal neoplastic transformation in the hamster is presented.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.

Syrian hamsters were treated with diethylstilbestrol (DES), a potent estrogen and kidney carcinogen, or ethinyl estradiol (EE), a strong estrogen but weak carcinogen, for 1-9 months. At monthly intervals their kidneys were studied using light, immunoperoxidase, and electron microscopic techniques. At 5 months, DES-treated animals exhibited interstitial lesions composed of small round cells with a high nuclear:cytoplasmic ratio. Immunoperoxidase and ultrastructural studies showed these cells to be similar to cells in fully formed tumors at 9 months. Early lesions in EE-treated animals (seen as early as 1 month) were dissimilar, these lesions appeared in the deep cortex adjacent to the renal pelvis, where proximal tubules underwent hyperplastic changes, showing columnar cells with large nuclei, occasional mitoses, and sloughing of apical cytoplasm. Cells in early lesions of EE-treated animals did not resemble the fully developed tumor in either immunoperoxidase or ultrastructural features; although with longer treatment these tubular lesions progressed to dysplasia (3-5 months) and severe dysplasia/carcinoma in situ (7 months), they did not form grossly visible tumors during the 9-month study. Both early lesions identified were specific, inasmuch as they were not observed in control animals and animals treated with beta-dienestrol and 17 alpha-estradiol (noncarcinogenic weak estrogens). Animals given a combination of DES and EE showed tubular hyperplasia but not interstitial lesions; this finding was of particular interest because hamsters given this combination of estrogens do not develop gross renal tumors. These results strongly implicate the primitive interstitial cell in the hamster kidney as the cell of origin of the DES-induced neoplasm.

Aryl hydrocarbon hydroxylase (AHH) activity was determined in castrate and intact male Syrian hamster kidney and liver microsomes following in vivo treatment with either diethylstilbestrol (DES) or 17 beta-estradiol as well as other steroid hormones. After 1 month of estrogen treatment, there was a 5-fold decline in AHH activity in castrated hamster kidneys compared with untreated castrate levels. The amount of AHH activity in the kidney was depressed more than 75% of untreated castrate levels even after the estrogen had been withdrawn for 6 days. Consistent with a nearly 2.5-fold higher renal AHH activity observed in intact male hamsters compared to castrates was the finding of a 1.7-fold elevation in the activity of this enzyme after treatment of castrated animals with androgen[5 alpha-dihydrotestosterone (5 alpha-DHT)] for 1 month. Moreover, following withdrawal of estrogen from intact hamsters, the increase in AHH activity in the kidney essentially paralleled the rise in serum testosterone levels. In castrated animals, the depression of AHH activity by estrogen was partially reversed by concomitant 5 alpha-DHT treatment. However, no appreciable changes were seen in liver AHH activity with androgen treatment in the presence or absence of estrogen. Similarly, the level of AHH activity, which was nearly 7- and 14-fold higher than intact and castrate kidney levels, respectively, was not altered by estrogen treatment. Neither progesterone nor cortisone had any effect on the levels of AHH activity in either the kidney or liver. Therefore, AHH activity in the male hamster kidney, but not the liver, is responsive to both estrogens and androgenic hormone.

Only very low levels of peroxidase activity were detected in castrated male hamster kidneys [1.0 +/- 0.8(S.E.) units/g protein], and chronic estrogen administration, either diethylstilbestrol (DES) or 17 beta-estradiol, for 1-5 months did not result in any appreciable increase in this activity. In contrast, hamster liver peroxidase activity was initially 10- to 20-fold higher than kidney levels, and chronic estrogen treatment for similar periods resulted in up to a 9-fold elevation in the activity of this enzyme. Moreover, the level of liver peroxidase activity in both intact and in 5 alpha-dihydrotestosterone-treated castrate hamsters was 2-fold higher than castrate-untreated values. Pure renal carcinoma induced after 9 months of estrogen treatment exhibited peroxidase values similar to those found in hamster livers [124 +/- 27 (S.E.) units/g protein] following estrogen treatment. When administered concomitantly with DES, tamoxifen significantly reduced the elevated levels of liver peroxidase activity observed after 2 months of DES treatment alone. A high affinity (KA = 0.10 X 10(9)M-1) estrogen receptor was found in liver cytosols of DES-treated hamsters which had increased slightly from untreated castrate levels.

Estrogen 2- and 4-hydroxylase (ESH), a microsomal enzyme which mediates the formation of catechol estrogens, has been studied in the kidneys of castrated male Syrian hamsters, a species uniquely susceptible to induction of renal carcinomas by both steroidal and stilbene estrogens. The apparent Km for estrone was 17.0 microM, and Vmax was 0.5 pmol per mg protein per min for ESH in renal microsomes derived from castrated hamsters. Different steroidal estrogen substrates exhibited decreasing catechol formation with hamster kidney microsomal preparations in the following order: estrone greater than d-equilenin greater than 17 beta-estradiol greater than equilin greater than ethynyl estradiol greater than estriol. Except for beta-dienestrol, the stilbene estrogens revealed levels of catechol formation that were similar to 17 beta-estradiol. These findings provide a rationale for the weak carcinogenic activity of ethynyl estradiol, estriol, and beta-dienestrol, since they were poor substrates for hamster renal ESH and for the relatively potent carcinogenic activity of the distal metabolite of diethylstilbestrol, indenestrol B/A, which exhibited substantial levels of o-hydroxylation when used as a substrate. Interestingly, ESH activity was significantly greater in the hamster kidney compared to corresponding rat tissue, and catechol estrogen formation was found to be 2.5- to 19-fold higher in the hamster kidney compared to the rat, using various steroidal and stilbene estrogen substrates. Moreover, the finding that a 3.5- to nearly 6-fold decrease, compared to untreated levels, in catechol formation in kidneys but not in livers of alpha-naphthoflavone-exposed hamsters, depending on the steroidal or stilbene estrogen substrate used, is consistent with the belief that the catechol estrogen pathway is pertinent to events leading to estrogen-induced renal tumorigenesis in the hamster.

The activity of microsomal estrogen 2-/4-hydroxylase enzyme (ESH), which mediates the formation of catechol estrogens, was determined in the hamster kidney and liver under different endocrine states and after treatment with diethylstilbestrol (DES) and 5 alpha-dihydrotestosterone alone or in combination. Our results indicate that at least 64% of the renal ESH activity is localized in the kidney cortex. Employing either estrone or 17 beta-estradiol as substrate, a significant decline in renal ESH activity was observed after castration, with estrone remaining the more active substrate. In contrast, hepatic ESH activity, which is about 2.0- to 2.5-fold higher than the kidney enzyme, was not altered after gonadectomy using either estrogen substrate. A further reduction in renal ESH activity was found in DES-treated castrated hamsters when estrone was used. Androgen treatment resulted in a nearly 2-fold increase in kidney ESH activity using either estrogen substrate. Animals treated concomitantly with DES and 5 alpha-dihydrotestosterone exhibited catechol estrogen formation similar to untreated castrate hamster kidney microsomes. In contrast, hamster liver ESH activity was unaffected by androgen treatment. HPLC profiles of the catechol estrogen monomethyl ethers confirm these changes. Hamster kidney ESH activity in females was only 5-7% of that in intact males. Ovariectomy resulted in a 3-fold increase in the activity of this microsomal enzyme with either estrogen substrate. ESH activity was substantially increased in uteri of intact animals after androgen treatment. These data clearly demonstrate that ESH activity is under androgen control, particularly in the hamster kidney of both sexes, and may be pertinent in understanding the antagonism of this hormone in estrogen-induced renal tumorigenesis.

Catechol-O-methyltransferase (COMT) [EC 2.1.1.6] is a ubiquitous cytosolic enzyme which has a pertinent role in the inactivation of both natural and synthetic catechol estrogens in mammalian tissues. We have compared the COMT activity in mouse, hamster and rat kidney, liver and red blood cells and examined the kinetic characteristics of this enzyme in the latter two species using various catechol estrogens as substrates. Results presented here indicate that the ratios of COMT activity in the kidney versus the liver of the rat and mouse are nearly identical, 0.48-0.52, whereas there is a 29-fold ratio between the level of COMT activity in these two tissues in the hamster. In red blood cells, the level of COMT activity is 4- and 12-fold lower in the hamster compared to mouse and rat, respectively. When the kinetic characteristics of this enzyme were assessed in the hamster and rat kidney and liver, except for 2-hydroxymoxestrol which had an apparent Km value of 15-48 microM, the other catechol estrogen substrates exhibited Km values ranging from 1-10 microM. Generally, the Vmax values were markedly higher in the rat kidney and liver than those observed in corresponding hamster tissues. The significantly lower COMT activity in the hamster liver and red blood cells suggests that under chronic estrogen treatment at high doses, the concentration of catechol estrogens in these tissues may exceed the capacity of COMT to effectively catalyse their O-methylation into inactive metabolites. The resulting accumulation of catechol estrogens may contribute to the estrogen carcinogenicity observed in the hamster liver and kidney. Additionally, when 2-hydroxyestrone was used as a substrate, the estrogen-induced renal carcinoma exhibited only 8.6% of the COMT activity found in the normal kidney.