The role of nitric oxide (NO) during cyclosporin renal vasoconstriction was evaluated by glomerular hemodynamic and histological changes produced by chronic NO synthesis inhibition and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) NO synthases mRNA expression in renal cortex and medulla. Uninephrectomized rats treated during 7 days with vehicle (Veh), cyclosporin A (CsA) 30 mg/kg, CsA + nitro-l-arginine methyl ester (l-NAME), and Veh +l-NAME (10 mg/dl) in the drinking water were studied. Increase in arterial pressure and afferent and efferent resistances, as well as decrease in glomerular plasma flow, ultrafiltration coefficient, and single-nephron glomerular filtration rate were significantly greater with CsA +l-NAME than with CsA alone. The increase in afferent resistance was higher with CsA +l-NAME than with Veh +l-NAME. In addition, glomerular thrombosis, proximal tubular vacuolization, and arteriolar thickening were more prominent. In renal cortex, eNOS mRNA expression exhibited a 2.7-fold increase in CsA, whereas, in medulla, nNOS and iNOS expression were lower in CsA than in Veh, while eNOS tended to increase. Our results support the hypothesis that NO synthesis is enhanced at cortical level during CsA nephrotoxicity, counterbalancing predominantly preglomerular vasoconstriction. Higher NO production could be the result of increased eNOS mRNA expression.
- micropuncture studies
- preglomerular vasoconstriction
- renal histopathology
- reverse transcription-polymerase chain reaction
- nitric oxide
cyclosporin A (CsA) is a potent immunosuppressive agent with indubitable efficacy in the prevention of organ allograft rejection; however, nephrotoxicity is a serious complication of the therapy. CsA nephrotoxicity is characterized by renal vasoconstriction that often progresses to chronic injury with irreversible structural renal damage (20, 27). Renal vasoconstriction is attributed to an imbalance in the release of vasoactive substances: on the one hand, increased release of vasoconstricting factors, such as thromboxane (22), endothelin (15), and angiotensin II (21); and, on the other, a decrease in vasodilating factors such as prostacyclin (21) and nitric oxide (NO) (10, 30). Participation of NO, however, has not been well defined. Some studies suggest that cyclosporin impairs NO production. Zoja et al. (39) showed that endothelial cell cultures developed structural damage when the cells were exposed to cyclosporin. Diederich et al. (10) and Takenaka et al. (30) reported a deficient response to the endothelium-dependent agonist in vascular beds of cyclosporin-treated animals. This response, however, was attributed to an enhanced generation of free radicals, which inactivate NO in mesenteric cyclosporin-treated arteries (9). In contrast, more recent studies demonstrated that NO synthesis is well preserved during cyclosporin nephrotoxicity (6, 17, 29). We have shown that, in rats treated with cyclosporin, the acute renal hemodynamic responses to arginine or NO synthesis inhibition were similar to those obtained in vehicle-treated rats, suggesting that the ability of the renal endothelium to produce NO was maintained and appeared to attenuate the renal vasoconstriction induced by cyclosporin (6). In addition, in bovine aortic endothelial cells in culture, López-Ongil et al. (17) recently demonstrated enhanced NO production in the presence of cyclosporin, which correlated with an increase in endothelial nitric oxide synthase (NOS) mRNA, protein, and activity. Stroes et al. (29), studying forearm blood flow of healthy volunteers, found that CsA increases NO activity.
In the kidney, NO is a vasoactive factor that plays a key role in maintaining vascular tone. NO is produced froml-arginine by the action of NOS isoforms, of which at least three molecular-level isoforms have been identified. These isoforms are the products of three different genes: neuronal NOS (nNOS) that encodes for a calcium-calmodulin-dependent enzyme, which is markedly expressed in the brain; iNOS that encodes a second isoform expressed in macrophages after appropriate immunological and inflammatory stimuli; and eNOS, a third isoform, that is expressed mainly in endothelial cells (14).
The three NOS isoforms are present in the kidney. In renal cortex, nNOS exhibits a macula densa cell-specific expression, iNOS has been observed in mesangial and proximal tubule cells, and eNOS is expressed mainly in endothelial cells of the afferent and efferent arterioles and glomerular capillaries. In renal medulla, nNOS is expressed in inner medullary collecting duct, iNOS is detected in medullary thick ascending limb of Henle’s loop (MTAL), and eNOS is located in thick ascending limb and collecting duct (for review, see Ref. 16).
NO produced by the nNOS in macula densa cells modulates renal vascular tone by direct relaxation of the afferent arteriole, thus attenuating the vasoconstriction mediated by tubuloglomerular feedback activation (31, 35); in addition, NO produced by macula densa cells enhances renin secretion in yuxtaglomerular cells of the afferent arteriole (4, 26), whereas the NO produced by eNOS contributes to maintain the vascular tone and regulates the glomerular plasma flow through vasodilation of the glomerular vasculature (3, 37). In normal and during immunostimulatory conditions, NO produced by the iNOS in mesangial cells can also modulate the vascular tone by an indirect mechanism due to mesangial cell relaxation (7, 19). Thus the source of NO determines its effect on the glomerular function in normal and pathophysiological conditions.
The present study was undertaken to characterize the contribution of NO during renal vasoconstriction induced by cyclosporin administration. For this purpose, we evaluated 1) the renal hemodynamic and histological changes during chronic inhibition of NO synthesis and 2) the level of mRNA expression in the cortex and medulla of each NOS isoforms during cyclosporin administration.
Male Wistar rats weighing 300–350 g, with right nephrectomy, were used for the study. Fifteen days after surgery, animals received daily subcutaneous injections of either cyclosporin (30 mg/kg of body wt) or vehicle (0.1 ml olive oil). Rats receiving vehicle were pair fed and served as controls. Renal hemodynamic and histological studies were performed 7 days after cyclosporin or vehicle administration in four groups of six rats each. Group Iincluded rats that received vehicle, group II consisted of rats treated CsA, andgroup III was formed by rats that received CsA plus the inhibitor of the NO synthesis, nitro-l-arginine methyl ester (CsA +l-NAME), in the drinking water at 10 mg/dl, which provides a daily ingestion of ∼6 mg/kg for a period of 7 days. This dose ofl-NAME has been previously demonstrated to be enough to produce systemic and renal NO blockade (11). Group IV was composed of rats treated with vehicle and chronicl-NAME.
Micropuncture studies. For micropuncture studies, rats were anesthetized with pentobarbital sodium (30 mg/kg ip), and supplemental doses were instilled as required. The rats were placed on a thermoregulated table, and temperature was maintained at 37°C. Trachea and both jugular veins, femoral arteries, and the left urether were catheterized with polyethylene tubing PE-240, PE-50, and PE-10, respectively. The left kidney was exposed, placed in a Lucite holder, sealed with elastomer (Xantropen, Bayer), and covered with Ringer solution. Mean arterial pressure (MAP) was monitored with a pressure transducer (model P23db, Gould) and recorded on a polygraph (Grass Instruments, Quincy, MA). Blood samples were taken periodically and replaced with blood from a donor rat.
Rats were maintained under euvolemic conditions by infusion of 10 ml/kg body wt of isotonic rat plasma during surgery, followed by an infusion of 25% polyfructosan, at 2.2 ml/h (Inutest, Laevosan). After 60 min, five to six 3-min collection samples of proximal tubular fluid were obtained to determine flow rate and polyfructosan concentration. Intratubular pressure under free- and stop-flow conditions and peritubular capillary pressure were measured in other proximal tubules with a servo-null device (Servo Nulling Pressure System; Instrumentation for Physiology and Medicine, San Diego, CA), as previously described (13). Polyfructosan was measured in plasma samples. Glomerular colloid osmotic pressure was estimated in protein from blood of the femoral artery and surface efferent arterioles. Polyfructosan concentrations were determined by the Davidson et al. (8) technique. Tubular fluid volume was estimated as previously described (13). Concentration of tubular polyfructosan was measured by the method of Vurek and Pegram (34). Protein concentration in afferent and efferent samples was determined according to the Viets et al. (33) method.
MAP, glomerular filtration rate (GFR), single nephron glomerular filtration rate (SNGFR), glomerular capillary hydrostatic pressure (Pgc) glomerular capillary hydrostatic pressure gradient (ΔP), single-nephron filtration fraction, single-nephron plasma flow (QA), single-nephron blood flow, afferent and efferent resistances, ultrafiltration coefficient (K f), and oncotic pressure were calculated according to equations given elsewhere (2).
Histological studies. After micropuncture studies, the kidney was perfused through the femoral catheter with phosphate buffer, conserving the MAP of each animal. The kidney was excised, fixed in alcoholic Bouin’s solution, and processed for light microscopy. After appropriate dehydration, kidney slices were embedded in paraffin, sectioned at 4 μm, and stained with routine methods: hematoxylin/eosin, periodic acid-Schiff, Jones’ methenamine silver, Masson trichromic, and Weigert’s resorcine-fuchsin with a Van Gieson counterstain for elastic fibers demonstration. The whole colored slides containing at least 100 glomeruli each were analyzed. Morphological analyses were performed using a semiquantitative scale with values from 0 to 3 for each of the following alterations: for tubular vacuolization, 1 = scant, 2 = abundant and focal, and 3 = abundant and diffuse. For arteriolar lesions, such as wall thickening, lumen narrowing, and elastic fibers folding, 0 = none, 1 = slight, 2 = moderate, and 3 = severe. In addition, the percents of glomerular thrombosis and focal fibrinoid necrosis were evaluated. Histological samples were analyzed blindly.
RNA isolation. Kidneys were obtained from five rats of either CsA- or vehicle-treated groups. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium, and their left kidneys were excised, macroscopically divided into cortex and medulla, frozen in liquid nitrogen, and kept at −80°C until used. Total RNA was isolated from individual cortex or medulla, following the guanidine isothyocianate-cesium chloride method (24). Integrity of isolated total RNA was examined by 1% agarose gel electrophoresis, and RNA concentration was determined by the ultraviolet (UV) light absorbance at 260 nm (Beckman DU640; Beckman, Brea CA).
Relative quantitation of NOS mRNA. The relative level of NOS mRNA expression was assessed at renal cortex and medulla by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), as previously described (5). Briefly, Table1 shows the primers sequence used to amplify a fragment of nNOS, iNOS, and eNOS. The NOS primers were custom obtained from Life Technologies. The nNOS primers were designed from a region of low identity between the three NOS isoforms (14). The iNOS and eNOS primers have been previously reported (19, 32).
The specificity of the primers was demonstrated by sequencing PCR products in both directions by the dideoxy chain termination method (25), using the Sequenase V 2.0 DNA sequencing kit (U.S. Biochemical). To monitor nonspecific effects of the experimental treatment and to semiquantitate NOS isoform expression, we amplified a fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using primers previously described (23), which yield a PCR product of 515 bp. Genomic DNA contamination was checked by carrying samples through PCR procedure without adding reverse transcriptase.
RT was carried out using 10 μg of total RNA from renal cortex or medulla of each rat. Prior to RT reaction, the RNA was heated at 65°C for 10 min. RT was performed at 37°C for 60 min in a total volume of 20 μl, using 200 U of the Moloney murine leukemia virus reverse transcriptase (Life Technologies), 100 pmol of random hexamers (Life Technologies), 0.5 mM of each dNTP (Sigma), and 1× RT buffer (75 mM KCl, 50 mM Tris ⋅ HCl, 3 mM MgCl2, 10 mM dithiothreitol, pH 8.3). Samples were heated at 95°C for 5 min to inactivate the reverse transcriptase and diluted to 40 μl with PCR grade water. One-tenth of the RT individual samples of each group was used for each NOS isoform or GAPDH amplification in 20 μl final volume reactions containing 1× PCR buffer (10 mM Tris ⋅ HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), 0.1 mM of each dNTP, 0.2 μCi of [α-32P]dCTP (∼3,000 Ci/mmol, 9.25 MBq, 250 μCi), 10 μM of each primer, and one unit of Taq DNA polymerase (Biotecnologı́as Universitarias, Mexico City, Mexico). The samples were overlaid with 30 μl mineral oil, and PCR cycles were performed in a DNA thermal cycler (M.J. Research, Watertown, MA) with the following profile: denaturation 1 min at 94°C, annealing 1 min at 55°C for nNOS primers and 60 °C for iNOS and eNOS primers, and 1 min extension step at 72°C. The last cycle was followed by a final extension step of 5 min at 72°C. The control gene was coamplified simultaneously in each reaction.
Preliminary studies were performed to determine the optimum number of cycles for quantitation. To analyze the PCR products, one-half of each reaction was electrophoresed in a 5% acrylamide gel. Bands were ethidium bromide stained and visualized under UV light, cut out, suspended in 1 ml of scintillation cocktail (Ecolume, ICN), and counted by liquid scintillation (Beckman LS6500). The amount of radioactivity recovered from the excised bands was plotted in a log scale against the number of cycles. Figure 1 shows amplification kinetics of the three NOS isoforms and housekeeping gene in renal cortex and medulla total RNA that was pooled from vehicle group. From these curves, we chose the optimal number of cycles for each primers pair as follows: in renal cortex, 28 for nNOS, 30 for iNOS, 32 for eNOS, and 20 for GAPDH; in renal medulla, we chose 24, 31, 23, and 18, respectively. To semiquantitate each NOS isoform, all reactions were performed individually from each cortex or medulla total RNA from vehicle- and CsA-treated rats in quadruplicate.
Statistical analysis. Statistical significance is defined as two-tailedP < 0.05, and the results are presented as means ± SE. For the hemodynamics data, the significance of the differences between groups were tested by two-way ANOVA with multiple comparison, using Student-Neumann-Kuels correction. The hystopathological data were analyzed using one-way ANOVA with multiple comparison using Bonferroni’s correction. NOS level of expression is shown as the ratio between NOS/GAPDH PCR product (means ± SE of 5 rats/group) and was analyzed using Student’s unpaired two-tailed t-test or Mann-WhitneyU-test, as needed.
Hemodynamic studies. Table 2 summarizes the results obtained in glomerular hemodynamics studies. Although chronic CsA administration did not change MAP, marked renal hemodynamic effects were observed. In the CsA group, the decrease in total GFR was associated with glomerular vasoconstriction. Glomerular plasma flow was reduced due to a sharp rise in afferent and efferent resistances. Because MAP was not increased, Pgc remained unchanged, and a significant reduction was observed inK f: sinceK f is determined in part by mesangial cells tone, its decrease suggests that CsA induces mesangial cell contraction. The fall in two of the determinants of glomerular filtration rate, that is, QA andK f, was responsible for the decrease in SNGFR to almost half of the values obtained in control animals. Thus the CsA group exhibited the characteristic renal vasoconstriction observed during chronic cyclosporin nephrotoxicity.
The simultaneous administration ofl-NAME with CsA enhanced the effects observed with CsA alone. In the CsA +l-NAME group, the marked elevation of MAP demonstrated the effect of systemic NO inhibition. Total kidney GFR was further reduced, although the change did not reach statistical significance. Constriction of glomerular vasculature induced by CsA was further increased byl-NAME administration, as shown by the marked increase in afferent and efferent resistances, producing a fall in glomerular plasma flow to ∼50% of the flow observed in rats treated with CsA alone. It is noteworthy that the vasoconstriction induced by l-NAME was significantly different in afferent and efferent arterioles, whereas efferent resistance rose 1.8-fold, and afferent resistance rose as much as 3.15-fold. The higher preglomerular vasoconstriction prevented transmission of elevated systemic blood pressure to glomerular capillaries, as disclosed by the unchanged values of Pgc. The decrease in QA andK f values resulted in a further decrease in SNGFR compared with that observed with CsA alone.
The vehicle + l-NAME group was designed to demonstrate that the dose ofl-NAME used was enough to inhibit NO synthesis and to induce systemic and renal vasoconstriction, as previously reported (11). Indeed, we found that this was the case. Table 2 shows that the dose ofl-NAME used in vehicle-treated rats induced a rise in MAP and significant renal vasoconstriction, as demonstrated by a decrease in QA. The pattern of glomerular vasoconstriction induced by NAME in vehicle-treated rats was, however, different from that observed in CsA-treated rats. In the vehicle group, afferent and efferent resistances rose in the same proportion (2-fold), whereas, in CsA-treated rats, l-NAME produced a predominant afferent vasoconstriction. A rise in intraglomerular capillary pressure secondary to the elevation in MAP was observed in response to a proportional elevation of afferent and efferent resistances. However, despite the rise in Pgc, SNGFR fell as a result of the proportionally greater reduction in QA andK f.
Histological studies. Table3 summarizes the light microscopy findings observed in the four groups studied. CsA administration produced glomerular thrombosis in 1.0% of the glomeruli, abundant and focal proximal tubular vacuolization, and moderate arteriolar thickening. Addition of the NO synthesis inhibitor to CsA resulted in greater structural changes. Glomerular thrombosis was present in 11% of the glomeruli, proximal tubular vacuolization was abundant and diffuse, and arteriolar thickening was severe. Figure2 shows a representative light microscopy image of a glomerulus and arterioles of a CsA +l-NAME-treated rat showing glomerular thrombosis and arteriolar thickening. These lesions were not observed in vehicle-treated rats and appeared in much lesser proportion in CsA alone and vehicle +l-NAME groups.
Expression of NOS mRNA. The level of gene expression of each NOS isoform was individually determined by semiquantitative RT-PCR analysis from renal cortex and medulla total RNA of each vehicle- and CsA-treated rats. Results obtained on kinetic amplification experiments (Fig. 1) show that the three NOS isoforms in renal medulla reached plateau phase earlier than in renal cortex, suggesting that renal medulla has greater capacity to generate NO than renal cortex, as was recently proposed by Kone and Baylis (16). Therefore, we used more cycles to amplify each NOS from renal cortex than medulla.
Figure 3 shows that in renal cortex, mRNA levels of nNOS and iNOS were similar in vehicle and CsA groups. In contrast, eNOS expression was 2.7-fold higher in CsA-treated rats than in the control group. The difference was statistically significant (P < 0.05). In the renal medulla, however, mRNA levels of nNOS and iNOS were 47 and 75% lower in CsA than in vehicle group, respectively. The differences were statistically significant (P < 0.05). In contrast, eNOS expression was twofold higher in CsA group, but this difference did not reach statistical significance (P = 0.09).
The mechanisms involved in CsA-induced renal vasoconstriction have not been completely elucidated. Previous studies demonstrated that administration of the drug stimulates the production of vasoconstrictor factors such as endothelin, thromboxane A2, and angiotensin II (15, 21,22). In addition, CsA inhibits the release of the vasodilator prostacyclin (21). However, participation of NO, an important renal vasodilator that maintains the low vascular resistances in the kidney, is not well defined. Studies on endothelial cell cultures showed that exposure of cells to cyclosporin results in structural damage (39), and several in vitro studies reported that acetylcholine-induced vasodilation is impaired in vascular beds of cyclosporin-treated animals, suggesting a deficient endothelial NO synthesis (10,30), although these findings can also be explained by enhanced generation of free radicals that inactivate NO (9).
Previous studies from our laboratory evaluating the acute hemodynamic renal response to NO synthesis inhibition withl-NAME or stimulation with arginine infusion in cyclosporin-treated rats showed that the responses to both l-NAME and arginine were similar to that observed in controls, suggesting that the ability of the renal endothelium to produce NO is preserved (6). Moreover, Amore et al. (1) and López-Ongil et al. (17) recently found in kidney homogenates and in bovine aortic endothelial cells, respectively, that NOS activity is increased in the presence of cylcosporin. Thus, as a whole, these studies suggest that during cyclosporin nephrotoxicity NO release is normal or even increased.
To characterize the contribution of NO during renal vasoconstriction induced by cyclosporin administration in the rat, we evaluated the effect of chronic NO inhibition on the renal hemodynamics and histology in uninephrectomized rats treated with CsA, a model with functional and hypertrophic compensatory changes similar to those observed in renal transplant recipients (12, 36). In addition, we determined the renal expression level of each NOS isoforms mRNA in cyclosporin-treated rats.
To determine whether renal vasoconstriction induced by cyclosporin was mediated by NO synthesis deficiency or by other mechanisms, the effects of chronic inhibition on NO production induced byl-NAME was evaluated in CsA-treated rats. Accordingly, if NO synthesis is completely suppressed by cyclosporin, l-NAME would not enhance existing renal vasoconstriction; if NO is only partially suppressed, the vasoconstrictive effect ofl-NAME would be mild or moderate, but, if NO synthesis is preserved or increased,l-NAME will markedly accentuate renal vasoconstriction.
In CsA-treated rats, NO synthesis inhibition produced elevation of arterial pressure and further increased renal vasoconstriction, which was disproportionally enhanced in preglomerular vessels. The rise in afferent resistance was significantly greater than the increment in efferent resistance and prevented the transmission of systemic pressure to glomerular capillaries. These findings contrast with the effect observed in control animals, in which there was a proportional rise in pre- and postglomerular resistances and elevation in glomerular capillary pressure. This unique effect ofl-NAME suggests that, in cyclosporin nephrotoxicity, NO plays a proportionally greater role in maintaining vascular tone of preglomerular vessels. Supporting this suggestion, light microscopy analysis demonstrated that vascular structural changes and glomerular thrombosis were enhanced by NO inhibition in cyclosporin-treated rats.
Mesangial cells tone can be indirectly evaluated in vivo by changes in the K f, which is the product of glomerular permeability and filtration area that is determined by mesangial cells tone (2). Cyclosporin nephrotoxicity was associated with mild reduction ofK f, which was further reduced by NO synthesis inhibition.l-NAME produced similar changes in K f values of control animals, suggesting that NO participates in maintaining mesangial tone to the same extent in control and CsA groups. Taken all together, these findings support the statement that NO synthesis is preserved and plays an important role in maintaining the vascular tone and renal function during cyclosporin nephrotoxicity.
As an alternative approach, to discern the pathway of NO production during vasoconstriction induced by cyclosporin, the gene expression levels of NOS in cortex and medulla were determined by RT-PCR. We chose the semiquantitative RT-PCR approach because this strategy offers the possibility to detect changes in the expression level of low abundant mRNAs, as in the case of NO synthases in the kidney (28).
In the renal cortex, synthesis of NO due to nNOS activity is mainly produced at macula densa cells, which is directly involved in the local control of vascular tone and renin secretion (4, 26, 31, 35). Changes in NaCl concentration sensed by the macula densa cells determine nNOS expression: low-NaCl delivery to the distal nephron stimulates nNOS gene expression, whereas high-NaCl delivery suppresses it (28). In CsA-treated rats, we previously found that, although SNGFR was markedly reduced, proximal fractional sodium reabsorption was also decreased (6). Therefore, we reasoned that nNOS expression should not change because the delivery of NaCl to macula densa cells did not fall as much as SNGFR. In the present study, nNOS mRNA levels in the renal cortex were no different in CsA treated, compared with control group, suggesting that NO production in macula densa cells was not suppressed by CsA. In addition, iNOS mRNA levels in renal cortex were not affected by CsA administration.
NO release by endothelial cells is responsible for the low vascular resistances that characterize renal circulation and contributes to the regulation of glomerular plasma flow. Shear stress on the luminal surface of the endothelium by the streaming blood is considered to be the most important physiological stimulus for the release of NO from endothelial cells (7, 14). In this study, we found that renal cortex expression of eNOS was 2.7-fold higher in CsA-treated rats, suggesting increased NO synthesis in endothelial cells, which agrees with our hemodynamic and histological results.
The increase in eNOS mRNA can be due to either an indirect or direct effect. Indirect stimulation can result from renal vasoconstriction induced by CsA, which decreases arteriolar diameter at constant flow rate, increasing shear stress to which the endothelial cell layer is exposed, thereby eliciting an increase in eNOS expression. In this regard, in cerebral blood vessels, eNOS was markedly upregulated during cerebral ischemia (38), suggesting that NOS expression can be indirectly enhanced by shear stress resulting from vasoconstriction. Direct effect of CsA can be at transcriptional level and/or the stabilization of eNOS transcript. In bovine aortic endothelial cells, López-Ongil et al. (17) recently showed that cyclosporin enhances NO production that correlates with an increase in endothelial NOS mRNA, protein, and activity, suggesting that cyclosporin itself can stimulate eNOS gene expression.
In this study, we found that CsA administration induced differential changes in the expression pattern of each NOS isoforms between cortex and medulla. At cortical level, nNOS and iNOS mRNA levels did not change, whereas eNOS increased significantly. In contrast, at medullary level, nNOS and iNOS expression were diminished by CsA, whereas eNOS tended to increase. In support of our observation of iNOS mRNA reduction in renal medulla, Marumo et al. (18) reported that CsA inhibits iNOS mRNA expression and nitrite products in vascular smooth muscle cells.
This different expression of NOS isoforms pattern observed in CsA-treated rats could result from several factors, such as vascular changes and/or tubular damage induced by CsA; however, more studies are necessary to investigate the mechanisms involved in the different regulation of NOS isoforms by CsA.
In summary, the results obtained in our hemodynamic and histological studies, as well as the change in renal cortex eNOS expression, support the notion that NO synthesis is enhanced at cortical level during CsA nephrotoxicity, counterbalancing predominantly preglomerular vasoconstriction induced by CsA. The increase in NO production could be secondary to an increase in the renal cortex expression of eNOS mRNA. In addition, CsA did not suppress the nNOS and iNOS expression in the renal cortex, which represents another source of NO.
We thank Juan A. Alvarado for technical assistance and the members of the Molecular Physiology Unit for their suggestions and stimulating discussion.
Address for reprint requests: N. A. Bobadilla, Dept. of Nephrology, Instituto Nacional de Cardiologı́a I.Ch., Juan Badiano No 1, Col Sección XVI, Mexico City, CP 14080, Mexico.
This work was supported by research grant no. 0356PM (to J. Herrera-Acosta) and nos. 2036 and 3900 (to G. Gamba) and a scholarship grant to N. A. Bobadilla from the Mexican Council of Science and Technology.
Parts of this work were presented at the 27th meeting of the American Society of Nephrology, Orlando, FL, 1994, and at the XIth Inter-American Society of Hypertension Meeting in Mexico City, Mexico, in 1997.
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