|
Autores:
José Luis CalungaCA *
Zullyt B. Zamora*,
Aluet Borrego*,
Sarahí del Río**
Ernesto Barber3***
Silvia Menéndez*
Frank Hernández*
Teresita Montero**
Dunia Taboada***
*Ozone Research Center, National Center for Scientific Research, Biomedicine Department, P.O.Box 6414, Havana City, Cuba,
**Military Medicine Institute "Luis Díaz Soto" Havana City, Cuba
***Institute of Basic and Preclinical Sciences "Victoria de Girón" Havana City, Cuba
CA Corresponding author@infomed.sld.cu
Chronic renal failure (CRF) represents a world health problem. The aim of this paper is to evaluate the effect of ozone/oxygen gaseous mixture in the renal function, morphology and biochemical parameters, in an experimental model of
CRF.
The effects of ozone/oxygen gaseous mixture were studied in a rat model of subtotal nephrectomy. Plasmatic clearance of p-amino-hippurate and inulin, plasmatic creatinine, urine excretion index (urine volume/water ingested), sodium and potasium excretions, protein excretion in 24 hours and systolic arterial pressure were determinated.
Reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), GSH peroxidase (GSH-Px) and total protein content were analyzed in kidney homogenates. Renal plasmatic flow, glomerular filtration rate, the urine excretion index, the sodium and
potassium excretions and systolic arterial pressure of ozone-treated nephrectomyzed rats were similar to sham rats.
Serum creatinine values and protein excretion in 24 hours of ozone-treated nephrectomyzed rats were decreased comparing with nephrectomyzed rats, still higher than normal values. Histological study demonstrated that animals treated with ozone presented less number of glomerular collapse and tubule degeneration.
Ozone/oxygen mixture induced a significant stimulation in the activity of CAT, SOD and GSH-Px, also an increase in renal GSH concentration was observed. TBARS were significantly increased in nephrectomyzed and ozone-treated nephrectomyzed rats. In this animal model of CRF, ozone rectal administrations produced a delay in the advance of the disease, protecting the kidneys against the deleterious effects of CRF.
Ozone/oxygen mixture induced an increase in the antioxidant system of renal tissue. This behavior suggests ozonetherapy has a protective effect on renal tissue by down regulation of the oxidative stress provoked by ischemia.
Key words: Chronic renal failure, ozone therapy, reactive oxygen species, antioxidant system
CRF represents a world health problem. CRF, once established, goes irreversibly to a final stage, provoking the patient death. In contrast with the capacity of the kidneys to regain function following acute renal injury, renal injury of a more prolonged nature often leads to progressive and irreversible destruction of nephron mass.1
Such reduction of renal mass, in turn, causes structural and functional hypertrophy of surviving nephrons. This compensatory hypertrophy is due to an adaptive hyperfiltration mediated by increase in glomerular capillary pressures and flows. Eventually, these adaptations prove maladaptive, predisposing to sclerosis of the residual glomerular population.1-4 The intrarenal vasculature is the most affected structure, preventing an appropriate blood flow, favoring the glomerular sclerosis.1-5 For that reason, the improvement of the rheological properties of the blood could delay the progression of the CRF.
Glomerulonephritis was the most common initiating cause of CRF in the past. Possibly because of more aggressive treatment of glomerulonephritis, diabetes mellitus and hypertensive renal diseases are now leading causes of CRF. The inexorable course to renal failure often is accompanied by anemia, malnutrition, impaired metabolism of carbohydrates, fats and proteins, impaired platelet function and defective utilization of energy.1
Reactive oxygen species (ROS) play a key intermediary role in the pathophysiologic processes of a wide variety of clinical and experimental renal diseases. It ranges from acute to chronic injuries, making the kidney as the site in which several unrelated diseases involves ROS.6 ROS have been demonstrated to be capable of degrading glomerular basement membrane and inducing glomerular injury, characterized by impaired glomerular filtration and sieving function.7,8
In order to eliminate toxic ROS, cells are equipped with various antioxidant systems. Therefore, the development of tissue injury depends upon the balance between ROS generation and tissue antioxidant defense mechanism.9 Among various antioxidant systems equipped within aerobic cells, three antioxidant enzymes, SOD, GSH-Px and CAT are major mechanisms to reduce local levels of ROS.
Thus, these enzymes distributed in cytosol and/or mitochondria can abase primary ROS, such as superoxide anion (by SOD) and hydrogen peroxide (by GSH-Px and CAT) before they can interact to form more reactive cytotoxic metabolites (hydroxyl radical or hypochlorous acid, among others). Studies in the past demonstrated that glomerular antioxidant enzymes levels are modulated. Thus, the glomerular antioxidant enzymes are suggested to play an important role in the functional derangement induced by the ROS.10
CRF is associated with depressed SOD and elevated NAD(P)H oxidase expression, which can contribute to oxidative stress by increasing superoxide.11 Another metabolic disturbance associated with CRF is hyperlipidemia, closely related with decreased removal and increase of triacylglycerol production. Up regulation of fatty acid synthase gene expression reveals another factor involved in disturbed lipid metabolism in CRF. It seems that elevated plasma insulin and cytokine concentration could play an important role in the mechanism responsible for the increased FAS gene expression in CRF.12
Taking into account some of the ozone therapeutic properties, such as, antiplatelet activity,13 enhancement of cell energy14 and the increase of the antioxidant defense system,15-22 the aim of this paper is to evaluate the effect of ozone therapy in the renal function, morphology and biochemical parameters that measure oxidative stress in an experimental model of CRF.
Chemicals
All reagents used in determinations of GSH, SOD, CAT, GSH-Px and TBARS were purchased from Sigma Chemicals (St Louis, MO). Other reagents of analytical grade were obtained from normal commercial sources.
Animals
Thirty young female Wistar rats (180-200 g) were maintained in an air filtered and temperature conditioned room (20-22 oC) with a relative humidity of 50-52 %. Rats were fed with standard laboratory chow and water ad libitum and were kept under an artificial light/dark cycle of 12 h. The studies were performed in concordance with the European Union regulations for animal experiments.
Treatment Schedule and Surgical Procedure
Ozone (O3) was generated by an OZOMED equipment (Ozone Research Center, Cuba), from medical grade oxygen by means of a silent electric discharge, representing about 3 % of the gas mixture (O3+O2). The ozone concentration was measured by using an UV spectrophotometer at 254 nm. The ozone dose is the product of the ozone concentration, expressed as mg/L, by the gas (O2 + O3) volume (L).
By knowing the body weight of the rat, the ozone dose is calculated as 0.5 mg/kg.
Surgery was performed as previously described.23 Ventral laparatomy was performed under aseptic conditions after anesthesia (sodium pentobarbital, 30 mg/kg intraperitoneal route). The right kidney was then removed, while two-thirds of the left kidney underwent acute infarction by ligation of two first-order branches of the main renal artery. Recovery from anesthesia and from the surgical procedure was complete within 24 h.
Animals were allocated randomly to 3 experimental groups of 10 animals each:1-Sham group (negative control group), rats underwent a ventral laparatomy under anesthesia as described above. However, only handling of the renal pedicle without the removal of renal mass was performed. 2-NX (positive control group), rats were subjected to 5/6 renal ablation as described above. 3-NX+O3 (ozone group), rats were handled as in group NX but also received, after the damage, 15 sessions of the gas composed of O2 + O3 (2.5-2.6 ml with O3 concentration of 50 mg/ml, representing a dose of 0.5 mg/kg weight), by rectal insufflation once per day.
Sample Preparation
A day before the subtotal nephrectomy, all animals were housed in metabolic cages during 24 h, without food and water ad libitum. In all animals, the weight and the systolic arterial pressure (SAP) in the tail of the animals and protein excretion were measured. All this procedure and measurements were repeated, after the partial nephrectomy, once a week, during 10 weeks, time during which, the CRF continued its evolution.
The time of the study was not prolonged for more than 10 weeks, avoiding the unpredictable death due to the final stage of the CRF. In the last day of evolution, plasmatic clearance of p-amino-hippurate (PAH) and inulin, in order to know the renal plasma flow (RPF) and the glomerular filtration rate (GFR), respectively, were determined using the method of unique injection (no urine) and the multicompartimental analysis of the plasmatic concentration curves in 9 blood samples.24 Also, the urine excretion index (urine volume/water ingested), sodium and potasium excretions were determined in order to know tubular work. Creatinine in serum was determined in the final blood sample obtained by intracardiac puncture (2 ml of blood were extracted).
Thereafter the animals were euthanized by ether anesthesia. The kidneys were dissected and immediately frozen at -20°C until analysis could be completed.
Biochemical determinations
PAH and inulin were determined in deproteinated plasma samples by cadmium sulfate, using for PAH a photocolorimetric technique, modified by Smith and Tinkelstein.25 Inulin was measured by the direct method of resorcinol without alkaline treatment,26 corresponding calibration curves were used.27 Proteins were calculated by the Biuret photocolorimetric technique using a Spectrophotometer (Shimadzu).28 Potassium and sodium urine concentrations were measured for the calculation of the excretions of both substances in a Carning flame photometer (model 400), using the method described by Oser.29
Creatinine in plasma was measured in deproteinated filtrates by the method of sodium tungstate, using for its valoration the method of picric acid modified by Brot.30
Kidney homogenates were obtained using a tissue homogenator Ultraturrax T-25 Polytron at 4°C. The homogenates were prepared by using a 100 mM KCl buffer (pH 7) containing EDTA 0.3 mM (1:10 w/v) for GSH, TBARS, GSH-Px and SOD determinations (Buffer 1). The homogenates were spun down with a centrifuge at 600g for 60 min at 4°C. The supernatants were taken for biochemical determinations.
Kidney homogenates for CAT enzymatic assay were carried out using 50 mM phosphate buffer (pH 7) containing 1% Triton X-100 (1:9 w/v) (Buffer 2). The homogenates were centrifuged at 600g for 60 min at 4 °C and the supernatants were used for the CAT assay.
GSH was determined by a slightly modified version of the method of Beutler,31 using an spectrophotometer. One mL of the kidney homogenate, as described before, was mixed with 1.5 mL of 5 % metaphosphoric acid and centrifuged at 3000g for 10 min at room temperature. Fifty hundred µL of this acidic supernatant was mixed with 2 mL of 0.2 M phosphate buffer and 0.25 mL of 0.04 % 5,5'-Dithio-bis(-2-nitrobenzoic acid).
Absorbance of the yellow solution was measured at 412 nm within 10 min. A molar extinction coefficient of 13.6 M cm-1 that describes the formation of the thiolate anion by the reaction of sulfhydril groups with5, 5'-Dithio-bis-2-nitrobenzoic acid (DTNB) at 412 nm was used to quantify GSH.
Enzymatic activity of SOD was determined by a modified version of the method of Minami and Yoshikawa.29 One unit of SOD enzymatic activity is equal to the amount of enzyme that diminishes the initial absorbance of nitro blue tetrazolium by 50 %.
CAT was determined according to the method of Evans and Diplock.32 The enzyme activity is expressed as the first order constant that describes the decomposition of H2O2 at room temperature.
Enzymatic activity of GSH-Px was measured using a modified version of the method of Thonson et al.33. The enzyme activity is expressed as international units of enzymatic activity/mg of protein. International units are expressed as µmoles of hydroperoxides transformed/min/mL of enzyme.
This assay was used to estimate TBARS levels as described by Ohkawa et al.34 The absorbance of 3 mL of the colored layer was measured spectrophotometrically at 532 nm, using 1,1,3,3-tetraethoxypropane as standard.
Protein concentrations were determined by the method of Lowry,35 using bovine serum albumin as standard.
Histological study
Samples of rat kidneys were taken and fixed in 10 % neutral buffered formalin, processed and embedded in paraffin. A pathologist unaware of the treatment schedule examined the histological sections, stained with hematoxylin and eosin.
Statistical analysis
Using the OUTLIERS preliminary tests for detection of error values started the statistical analysis. Afterward the Anova method (one way analysis of variance) was used followed by homogeneity variance test (Bartlett-Box). In addition, a multiple comparison test was used (Duncan test) and for the comparison of two groups, the Student's t test was done. Results are presented as mean ± standard deviation (SD). Different letters indicate a statistical significance of at least p<0.05.
At the end of the study (10 weeks after the partial nephrectomy), animals treated with ozone presented: SAP figures lower than the positive control group, but higher values with respect to negative control group. Urine excretion index, in the ozone group, was similar to the negative control group, however, positive control group presented lower values respect to the others groups (Table 1).
At the end of the study, protein excretion figures, in the ozone group, were higher than the negative control group, but lower than the positive control group. Potasium and sodium excretion values, in negative control and ozone groups were similar, but lower in the positive group with respect to negative and ozone groups (Table 2).
At the end of the study, RPF and GFR presented higher figures in the ozone group respect to the others. The lowest values of RPF and GFR were achieved in positive control group (Table
3)
Histological injuries were 13 and 100 % in the ozone and positive control groups, respectively (Table 4).
The histological findings were glomerular collapse, tubule degeneration, cortical-medullar hemorrhages, dilatation of convoluted tubules, glomerular capsule dilatation and renal injury, among others. Positive control group presented those manifestations in 100 % of animals, however in the ozone groups were presented in 10-20 %.
Subtotal nephrectomy induced a significant increase in TBARS (p=0.0253) of 144 %, whereas applications of ozone/oxygen gaseous mixture after subtotal nephrectomy increase TBARS in 90 % (p=0.0253) over the levels of NX group.
SOD activity was significantly decreased in 55 % (p=0.0143) in NX group, but in NX+O3 group, 39 % of the enzymatic activity was recovered (p=0.0253). Ozone therapy after subtotal nephrectomy induced a total increase in SOD activity of 94 % (p=0.0281). A similar behavior was observed for CAT enzymatic activity. In NX group there was a significant decrease of 50 %, whereas in NX+O3 group was observed a recovery of 218 % (p=0.0143) in the enzymatic activity of CAT, indicating a total increase in CAT activity of 268 % (p=0.0034).
GSH concentration and GSH-Px enzymatic activity were not significantly affected by subtotal nephectomy in this animal model. But when fifteen intrarectal applications of ozone/oxygen gaseous mixture were applied we observed a significant stimulation of GSH-Px and an increase in GSH concentration. Renal GSH concentration was significantly increased in 87 % (p=0.0253) whereas GSH-Px activity was increased in 39 % (p=0.0253) ( Table 5).
Animals submitted to the subtraction of 5/6 of the total renal mass moved forward the installation of the CRF, demonstrated by the increase of SAP, plasma creatinine, protein excretion, decrease of potasium and sodium excretions, as well as the presence of renal damage in the histological study.
This behavior is still more pronounced in the positive control group, where the renal damage achieved 100 %.
The results have shown, at the end of the study, that the animals treated with ozone had the highest figures of RPF and GFR, as well as lower figures of proteinuria, plasma creatinine concentration, higher urine excretion and lower SAP in comparison with the positive control group. These results can be linked to the ozone antiplatelet activity,10 diminishing blood viscocity, that could produced a decrease in the friction between the blood and the glomerular vascular walls, decreasing the flow resistance, increasing the RPF and GFR
The flow rise contributes to diminish the endothelial injuries and the glomerular collapse, avoiding the tubular hypoxia, the hemorrhages and the release of several proinflammatory cytokines.36-40
A depression in SOD expression was reported previously11 for the same CRF model that we used, that could be correlated with the observed diminution in the activity of SOD and CAT, which can contribute to oxidative stress by increasing superoxide and hydrogen peroxide generation.
As it was reported before,14-22 ozone therapy is an oxidative approach that provoking brief moments of oxidative stress may stimulate antioxidant system to fight against the phenomenon per se.
Thus, the significant increase in lipid peroxidation, measured in the form of TBARS, was expected after the surgical procedure, followed by fifteen ozone applications. In spite of the significant increase in TBARS, an increase in antioxidant status was seen in NX+O3 group.
The increase in renal TBARS might not be related with an increase in renal damage because the remarkable increased in antioxidant system surpass this effect, inducing a general status of antioxidant protection.
Renal damage in this model of CRF might correspond with generation of ROS species, such as superoxide and hydrogen peroxide. Renal GSH concentration and GSH-Px activity might not be directly affected by subtotal nephrectomy in this model. However, ozone therapy induced a significant increase in renal GSH amount and GSH-Px activity (p=0.0253), besides a remarkable stimulation of SOD and CAT activity, that surpass the increase in lipid peroxidation.
This suggests that ozone tharapy has a protective effect on renal tissue, by up regulation of the antioxidant system, protecting against the oxidative stress provoked by ischemia.
In the other hand, it had been demonstrated that ozone is able to regulate the calcium levels, maintaining its homeostasis, avoiding any damage to the cell structure.41.
Also, it is possible that the ozone therapy effect, with the stimulation of the antioxidant defense system,12-16 protected the tissues against the oxidative stress present in CRF,6 being in correspondence with the positive histological results obtained.
In this animal model of CRF, rectal administrations of ozone produced a delay in the advance of the disease, protecting the kidneys against the deleterious effects present in the CRF. Consequently, whenever possible, ozone therapy may become an important therapy to improve the quality of life of patients suffering of
CRF.
1. Fauci, A.S., Braunwald, E., Isselbacher, K.J., Wilson, J.D., Martin, J.B., Kasper, D.L., Hauser, S.L. and Longo, D.L. Chronic Renal Failure. In: McGraw-Hill Companies Inc. Editor. Harrison's Principles of Internal Medicine. New York, USA: McGraw-Hill Companies Inc; 1998.p.1513 1518.
2. Lane, P.H. Long.term furosemide treatment in normal rat:dissociation of glomerular hypertrophy and glomerulosclerosis. Am J. Kid. 1999;33(6):10581065.
3. Fine, L.G. Adaptation of renal tubule in uremia. Kidney Int 1982;22:546552.
4. Slomowitz, L. Tubuloglomerular feedback in chronic renal failure. Nephron 1987;45(4):264269.
5. Sheldin, D.W., Gusbech, G. The kidney physiology and physiopathology. In: Roven Press Editor. The kidney physiology and physiopathology. New York, USA: Roven Press;1985.p.19011905.
6. Ichikawa, I., Kiyama, S., Yoshioka, T. Renal antioxidant enzymes: Their regulation and function. Kidney Int 1994; 45:19.
7. Yoshioka, T., Ichikawa, I. Glomerular dysfunction induced by polymorphonuclear leukocyte-derived reactive oxygen species. Am J Physiol 1989;257(Renal Fluid Electrol. Physiol. 26):F53F59.
8. Yoshioka, T., Moore-Jarret, T., Ichikawa, I., Yared, A. Reactive oxygen species of extra-renal origin can induce massive functional proteinuria, Kidney Int 1990; 37:497502.
9. Kawamura, T., Yoshioka, T., Bills, T., Fogo, A., Ichikawa, I. Glucocorticoid activates glomerular antioxidant enzymes and protects glomeruli from oxidant injuries. Kidney Int 1991;40:291301.
10. Yoshioka T, Bills T, Moore-Jarrett T, Greene HL, Burr IM, Ichikawa I. Role of antioxidant enzymes in renal oxidant injury. Kidney Int 1990;38:282287.
11. Vaziri ND, Dicus M, Ho ND, Boroujerdi-Rad L, Sindhu RK. Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency. Kidney Int 2003;63(1):179184.
12. Szolkiewicz M, Nieweglowski T, Korczynska J, Sucajtys E, Stelmanska E, Goyke E, Swierczynski J, Rutkowski B. Upregulation of fatty acid synthase gene expression in experimental chronic renal failure.Metabolism 2002;51(12):16051610.
13. Matsuno, K., Miura, T., Shinriki, N. The effect of ozone on platelet activation, Proceedings of the 13th Ozone World Congress (Kyoto, Japan) 1997;3:178184.
14. Bocci, V. Ozone as a bioregulator. Pharmacology and toxicology of ozonetherapy today. Journal of Biological Regulators and Homeostatic Agents 1997;10(2/3):153.
15. Barber, E., Menéndez, S., León, O.S., Barber, M.O., Merino, N., Calunga, J.L.,Cruz, E., Bocci, V. Prevention of renal injury after induction of ozone tolerance in rats submitted to warm ischemia. Mediators of Inflammation 1999;8:37-41.
16. Hernández, F., Menéndez, S., Wong, R. Decrease of blood cholesterol and stimulation of antioxidative response in cardiopathy patients treated with endovenous ozone therapy. Free Rad Biol Med 1995;19:115-119.
17. León, O.S., Menéndez, S., Merino, N., Castillo, R., Sam, S., Pérez, L., Cruz, E., Bocci, V. Ozone oxidative preconditioning: a protection against cellular damage by free radicals. Mediators of Inflammation 1998;7:289-294.
18. Peralta, C., León, O.S., Xaus, C., Prats, N., Jalil, E.C., Planell, E.S., Puig-Parellada, P., Gelpí, E., Roselló-Catafau, J. Protective effect of ozone treatment on the injury associated with hepatic ischemia-reperfusion: antioxidant-prooxidant balance. Free Rad Res 1999;31:191-196.
19. Candelario-Jalil, E., Mohammed-Al-Dalain, S., León, O.S., Menéndez, S., Pérez-Davidson, G., Merino, N., Sam. S., Ajamieh, H.H. Oxidative preconditioning affords protection against carbon tetrachloride-induced glycogen depletion and oxidative stress in rats. J Appl Toxicol 2001; 21:297-301.
20. Ajamieh H., Merino N., Candelario-Jali E., Menéndez S., Martínez G., Re L., Giutiani A. and León O.S. "Similar protective effect of ischemic and ozone oxidative preconditionings in liver ischaemia/reperfusion injury", Pharmacological Research, 2002. 45(4):333-339.
21. Al-Dalain S.M., Martínez G., Candelario-Jalil E., Menéndez S., Re L., Giuliani A. and León O.S. "Ozone treatment reduces markers of oxidative and endothelial damage in an experimental diabetes model in rats", Pharmaceutical Research, 2001. 44(5):391-396.
22. Ajamieh H. H., Menéndez S., Martínez-S'anchez G., Candelario-Jalil E., Re L., Giuliani A., and León Fernández O.S. Effects of ozone oxidative preconditioning on nitric oxide generation and cellular redox balance in a rat model of hepatic ischaemia-reperfusion. Liver International 2004,24:55-62.
23. Anderson S, Meyer TW, Rennke HG, Brenner BM. Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass. J Clin Invest 1985;76:612617.
24. Folin, O. A system of blood analysis. J Biol Chem 1949;38:81
25. Smith, H.W, Tinkelstein, N. The renal clearance of substituted hipuric acid derivatives and other aromatic acids in dog and man. J Clin Invest 1945;24:388393.
26. Schreiner, G. Determination of inulin by means of resorcinol. Proc Soc Exper Biol and Med 1950;70:726730.
27. Jamison, R.L., Massly, R.H. The uramic concentrating mechanism. N Eng J Med 1976;295:10591067.
28. Oser, B.L. Determination of sodium and potassium. In: MacGraw-Hill, editor. Hawk´s Physiological Chemistry. New York, USA : MacGraw-Hill; 1965.p.11401145.
29. Oser, B.L. Biuret test for protein determination. In: MacGraw-Hill, editor. Hawk´s Physiological Chemistry. New York, USA : MacGraw-Hill; 1965.p.179184.
30. Brot, J. The renal clearance endogenous creatinine in man. J Clin Invest 1948;27:645652.
31. Beutler F, Duron O, Mikus B. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;16:882887.
32. Minami M, Yoshikawa H. A simplified assay method of superoxide dismutase activity for clinical use. Clin Chim Acta 1979;92:337343.
33. Evans C, Diplock AT. Techniques in Free Radical Research. In: Burtin RH and Knippenberg PH, editor. Laboratory Techniques in Biochemistry and Molecular Biology. The Netherlands: Elsevier; 1991.p.199206.
34. Faraji B, Kang HK, Valentine JL. Methods compared for determining glutathione peroxidase activity in blood. Clin Chem 1987;33:539544.
35. Ohkawa H, Orishi N, Yagi K.Assay for lipid peroxidation in animals and tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351357.
36. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:165169.
37. Mezzano, S.A., Droguett, M., Burgos, M.E. Overexpression of chemokines, fibrogenic cytokines, and myofibroblasts in human membranous nephropathy. Kidney Int 2000; 57(1):147151.
38. Hansch, G.M.,Wagner, C., Burger, A. Matrix protein synthesis by glomerular mesangial cells in culture:effects of tranforming growth factor beta (TGF beta) and platelet-derived growth factor (PDGF) on collagen type IV mRNA. J Cell Physiol 1995;163(3):451456.
39. Ito, Y., Aten, J., Bende, R.J. Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998;53(4):853858.
40. Fine, L.G., Orphanides, C., Norman, J.T. Progressive renal disease: The chronic hipoxia hypothesis, Kidney Int 1998;53 (Suppl.65):S7477.
41. León, O.S., Menéndez, S., Merino, N., López, R., Castillo, R., Sam, S., Pérez, L., Cruz, E., Jouseph, F., Fernández, A. Influencia del precondicionamiento oxidativo con ozono sobre los niveles de calcio. Revista CENIC Ciencias Biológicas 1998;29(3):134-136.
 Atrás
Regresa a Indice-Back to Index topics |
Para
consultas y comentarios