Salamanca, Spain Communications. Renal Physiology

Transcription

1 Salamanca, Spain Communications Renal Physiology

2 86S Serotonergic effects in the in situ autoperfused rat kidney A. Moran, C. Velasco, P. Prieto, M.L. Martin and L. San Roman Lab. Farmacognosia y Farmacodinamia, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain The in situ autoperfused rat kidney technique allows continuous measurement of renal blood flow in the rat and evaluates rapid changes in renal blood flow induced by direct intrarenal arterial drug administration to the kidney (Fink & Brody, 1978). In this work in anaesthetized rats, we have analysed both the direct local renal action of 5-HT and the possible indirect action induced by liberation of the vasoconstrictor or vasodilator humoral agents, as it has been proposed with this agent in other animal species (Shoji et al. 1989; Blackshear et al. 1991). The local injection of 5-HT and m-cpp increased the perfusion pressure in the in situ autoperfused rat renal vascular beds. This local vascular renal effect was completely antagonized by both ritanserin and enalapril, and was not modified by propranolol, prazosin or indomethacin pretreatment. These agonists do not modify the perfusion responses to electrical stimulation of the sympathetic renal nerves. Our results suggest a vasoconstrictor renal serotonergic effect mediated by local 5-HT2 activation and by an increase of angiotensin II. Blackshear, J.L., Orlandi, C. & Hollenberg, N.K. (1991). J Cardiovasc. Pharmacol. 17, 68. Fink, G.D. & Brody, M.J. (1978). Am. J Physiol. 234 (2), H219. Shoji, T., Tamaki, T., Fukni, K., Iwao, H. & Abe, Y. (1989). Eur. J Pharmacol. 171, 219. Gentamicin induces rat mesangial cell activation. A role for calcium C. Martinez-Salgado, A. Rodriguez-Barbero, M. MacLaughlin and J.M. L6pez-Novoa Medicina, Universidad de Salamanca, Salamanca, Spain Journal of Physiology (1996) 493.P Gentamicin-induced decreases in glomerular filtration rate have been associated with a marked decline in the glomerular capillary ultrafiltration coefficient, which could be mediated by mesangial cell contraction or release of vasoactive hormones. Gentamicin induces mesangial cell contraction, proliferation and calcium mobilization in rat mesangial cells. To assess whether calcium could be a mediator in mesangial cell activation, we have studied the effect of verapamil and TMB-8 on gentamicn-induced contraction and proliferation. Reduction of planar mesangial cell surface area induced by 10-5 M gentamicin, after 60 min of incubation, was blunted by a calcium-channel blocker, 10-5 M verapamil (-10 ± 1P6 vs % in gentamicin-treated cells) and by an endoplasmic reticulum calcium release blocker, 10`5 M TMB-8 ( P6 vs % in gentamicin-treated cells). Gentamicin stimulated [3H] thymidine incorporation into DNA and cell proliferation, measured by the amount of tetrazolium salt converted to blue formazan (Cell Titer 96TM), indicating the number of viable cells in the well. Both effects were also reduced by verapamil ( vs c.p.m. well-'; vs c.p.m. well-' in gentamicin-treated cells) and TMB-8 (222 ± 4 vs c.p.m. well-'; vs cells well-' in gentamicin-treated cells). Gentamicin induced an increase in cytosolic free calcium (from 100 to 230 nm). The present studies demonstrate that gentamicin-induced contraction and proliferation in mesangial cells is mediated, at least in part, by an increase in cytosolic free calcium both extracellular and intracellular origin. Role of nitric oxide in gentamicin-induced nephrotoxicity in rats L. Rivas-Cabaniero, J.M. Valdivielso, J.R. Alonso, M. Arevalo and J.M. L'pez-Novoa Instituto Reina Sofia de Investigaciones Nefrol6gicas, Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Espaiia The aim of this study was to asses the role of nitric oxide (NO) synthesis on gentamicin (G)-induced nephrotoxicity. Some female Wistar rats were given G (100 mg (kg body wt)-' day-') for 6 days (n = 27), and others were used as controls (n = 9) (C). G-treated rats were divided into three groups, receiving G alone (n = 9), G and the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME), 4 mg (kg body wt)-' day-' in the drinking water (n = 9), and G and the NO synthesis precursor L-arginine (1 %) in the drinking water. On the last day of treatment, renal function was measured. Rats receiving G + L-NAME, showed higher plasma creatinine levels (37-14 ± 3*5 vs ± 1-7 mg l1'), as well as significantly lower creatinine clearance (0-17 ± 0-02 vs ml min-') than rats receiving G alone. However, rats receiving G + L-arginine showed lower

3 Journal of Physiology (1996) 493.P plasma creatinine ( vs mg F-) and higher creatinine clearance levels ( X14 vs X14 ml min-') than rats receiving G alone. At the end of the study, glomeruli were obtained and glomerular NO synthesis was tested as nitrite production by a modification of the Griess reaction. After 24 h of incubation, glomerular nitrite production was significantly higher in G-treated groups compared with controls, and in G + L-arginine compared with all the other groups (G: , G+ L-NAME: ; G + L-arginine: vs. control: pmol per 1000 glomeruli). NO synthase activity was evaluated as positive NADPH diaphorase histochemistry in renal cortex slices. Glomeruli from G-treated animals showed more NADPH diaphorase-positive cells than those from control rats. These data showed that NO inhibition aggravates and NO stimulation improves G-induced renal failure, and together with the increased glomerular NO production in G-treated animals suggest that NO plays a protective role in G-induced nephrotoxicity. Glomerular nitric oxide synthesis is increased in rats with ischaemic acute renal failure J.M. Valdivielso, L. Rivas-Cabaniero, J.R. Alonso and J.M. Lopez-Novoa Medicina, Universidad de Salamanca, Salamanca, Spain Ischaemic acute renal failure (ARF) is characterized by changes in renal haemodynamics, tubular obstruction, backleak of filtrate and changes in vascular reactivity. The aim of this study was to determine the role of nitric oxide in ARF induced by 1 h of renal ischaemia. Ischaemia was induced by clamping both renal arteries in female Wistar rats. The rats were divided into groups as follows: group 1-24, ischaemic rats with 24 h recirculation; group I-2, ischaemic rats with 2 h recirculation. Each ischaemic group had a corresponding sham-operated group as control. Blood samples were taken from the animals just before obtaining the glomeruli. Plasma creatinine concentration was significantly higher in rats with 24 h recirculation compared with all the other groups (sham-2, ; 1-2, ; sham-24, ; I-24, mg Fl). On the last day, glomeruli were obtained by mechanical sieving and incubated in sterile conditions for 24 h at 37 'C. Nitric oxide synthesis and release (nitrite production) were determined in the supernatant by a modification of Griess reaction. Nitrite production was significantly higher in I-24 than in all the other groups (sham-2, ; I-2, ; sham-24, ; I-24, pmol per 1000 glomeruli). NO synthase activity was evaluated as positive NADPH diaphorase histochemistry in renal cortex slices from I-24 rats. Glomeruli from I-24 animals showed a significantly higher NADPH diaphorase positive reaction compared with sham-24 rats. This increased glomerular NO synthesis after renal ischaemia agrees with previous studies that suggest a protective role of NO on ischaemic ARF; this increased glomerular NO production in ischaemic ARF after 24 h recirculation could play a role counteracting the vasoconstriction asociated with renal ischaemia. Nitric oxide production by rat glomeruli M. Reverte, J.M. Valdivielso and J.M. Lopez-Novoa Departamento de Fisiologia y Farmacologia, Edificio Departamental, Av. Campo Charro s/n, Salamanca, Spain There is increasing evidence that L-arginine NO pathway is active in the glomerulus (Bachmann & Mundel, 1994). NO formation has usually been assessed by the measurement of nitrite plus nitrate production. Recently, a NO-sensitive electrode has been developed, thus allowing the measurement of the actual NO formation. In glomeruli isolated from Wistar rats, we investigated the concentration of NO gas in aqueous solution by using a NO-specific electrode (ISO-NO, World Precision Instruments, Sarasota, Florida, USA) and the nitrite formation by the Griess reaction. Following isolation by sieving, glomeruli were suspended in Krebs buffer with 0 4 mm L-arginine, and subsequently incubated with 0 5 x 10-2 M NG-nitro- L-arginine methyl ester (L-NAME, 2 h), 1 jug ml-' lipopolysaccharide (LPS, 5 h) or L-NAME plus LPS. Results were as follows (mean + S.E.M.): Control L-NAME LPS L-NAME + LPS NO nm, n= nM, n= nm, n= nm, n=4 N02_ nm, n= nm, n = nm, n = nM, n=3 L-NAME inhibited basal and LPS-stimulated NO formation, but it stimulated nitrate accumulation. These results suggest that a pathway not involving NO synthase would contribute to nitrite formation in rat glomeruli. REFERENCE Bachmann & Mundel (1994). Am. J Kidney Dis. 24, S

4 88S Journal of Physiology (1996) 493.P Glomerular nitric oxide (NO) production in rats with bile duct ligation 0. Flores, M. Criado-Jimenez, L. Rivas-Cabafiero, F. Hidalgo, N. Eleno, L.M. Diez-Panero, J. Garcia Estaf* and J.M. L'pez-Novoa Instituto Reina Sofia de Investigacion Nefrologica, Medicina, Universidad de Salamanca, Salamanca, Spain, and *Departamento de Fisiologia, Facultad de Medicina, Universidad de Murcia, Murcia, Spain Recent indirect studies suggest that increased intrarenal NO synthase (NOSi) activity may contribute to the renal alterations of liver cirrhosis. We have evaluated the glomerular NO production in rats 21 days after inducing primary biliary cirrhosis by bile duct ligation (BDL), and in sham-operated (SO) animals. At that time, BDL rats showed a moderate hypotension ( vs mmhg in SO rats). Glomeruli were isolated according to sieving techniques and incubated for 24 h in RPMI 1640 (Gibco). NO production was measured as nitrite accumulation in culture medium. Plasma nitrite was gm in SO animals and /M in BDL rats (P < 0-01). Glomerular nitrite production was higher in BDL than in SO rats ( vs nmol (24 h)-1 (7000 glomeruli)f1). Either interferon-y (IFN, 100 U ml-') or lipopolysaccharide (LPS, 1,ug ml-') induced a marked increase in nitrite production in glomeruli from SO rats (IFN, ; LPS, nmol (24 h)- (7000 glomeruli)-') but not in glomeruli from BDL rats, in which only a mixture of IFN and LPS induced a small (30%) increase in nitrite production. Nitrite production from mononuclearlymphocyte cells was higher in BDL than in SO rats. NOSi was expresed in monocytes of cirrhotic rats, but not in SO rats, confirming that NO production is due to inducible NO syntase activity. When cells from SO rats were incubated with IFN, LPS or both, a marked increase in nitrite production was observed. However, cells from BDL rats respond to LPS or LPS and IFN, but not to IFN alone. Nitrite production by cultured mesangial cells was significantly stimulated (by 35%) by plasma from BDL rats, but not by plasma from SO rats. These results suggest that BDL rats show increased glomerular NO production, that is induced, at least in part, by factors present in plasma. Renal effects of antihypertensive therapy in uni-nephrectomized spontaneously hypertensive rats M.B. Gallego-Oviedo, C. Martinez-Salgado, J.M. Valdivielso, A. Rodriguez-Barbero, J.L. Garcia- Bastos, M. Arevalo* and J.M. Lopez-Novoa Departamento de Fisiologia y Farmacologia and *Departamento de Histologia, Universidad de Salamanca, Salamanca, Spain The aim of this study has been to assess the renal and arterial effect of 6 months of treatment with verapamil (V, 20 mg kg-' day-1) and the combination verapamiltrandolapril (VT, mg kg-1 day-1), and glomerular nitrite (NO) production in uni-nephrectomized spontaneously hypertensive rats (SHR). Treated rats showed, from the first month, a significant decrease in arterial pressure, measured by tail plethysmography (V, ; VT, vs mmhg in control rats at the sixth month). Both, verapamil and combination induced a decrease in urinary flow rate (V, ; VT, 4' #l min-' in control rats at the sixth month) vs. and in protein excretion (V, ; VT, vs mg day-' in control rats at the sixth month). No differences in creatinine clearance and natriuresis were observed after 6 months of treatment. At the end of the treatment, glomeruli were isolated and, after 24 h, glomerular nitrite production was measured. Verapamil induced an important decrease in glomerular NO production (V, 4f ; VT, vs nmol per 1000 glomeruli in control glomeruli). In summary, the combination verapamil-trandolapril reduces arterial pressure and proteinuria more effectively than verapamil alone in SHR. In addition glomeruli of rats treated with verapamil, but not with the combination, showed a decrease in glomerular NO production. Differential expression of fll and /?2 sodium pump subunits during nephrectomy. Correlation with enzymatic activity L.M. Diez-Panero, N. Eleno, A. Rodriguez-Lopez, M. MacLaughlin, F. Perez-Barriocanal and J.M. Lopez-Novoa Departamento de Fisiologia y Farmacologia, Universidad de Salamanca, Salamanca, Spain Male Wistar rats ( g) with unilateral nephrectomy develop a compensatory hypertrophy in order to maintain renal homeostatic function. One of the most

5 Journal of Physiology (1996) 493.P widespread enzymatic activities in kidney is that of Na+, K+-ATPase. The aim of the present work was to assay differential expression of /31 and /2 subunits of sodium pump (Western blotting) in the remaining right kidney over 13 days after left nephrectomy. Also, Na+, K+-ATPase was assessed in microsomal fraction of total homogenate by the Fiske-Subbarrow method. Na+, K+-ATPase specific activity (mmol Pi (mg protein)-' h-v) shows an initial increase achieving a maximum on the third day ( vs in controls), and then decreases, but stabilizing at a value higher than controls ( ); however, statistical analysis by one-way ANOVA does not show any significant difference. Also, the expression of /1 and,b2 subunits remained constant. The total amount of protein in the right kidney increases until the sixth day after nephrectomy ( vs f53 in controls), achieving a value that remains constant after the tenth day (62f32 + 3f26, day 10; 69' , day 13). If the proportion of #I1 and /B2 proteins remained constant as the total protein content varies as previously explained, then the expression of these subunits per cell must have increased until sixth day and remained constant afterwards. Total Na+, K+-ATPase activity varies in a similar way. Lecuona, E. et al. Cellular and Developmental Distribution Patterns. (en prensa). Norman, J.T. & Hardy, J. (1994). Exp. Nephrol. 2, activity in these regions, while the predominant Ca2+dependent NOS activity shows regional differences in distribution. However, cofactor requirements were identical in all regions. NOS activity was inhibited by and 25-30% in the absence of NADPH and BH4, respectively, while remaining unaffected in the absence of calmodulin or in the presence of W-7, a calmodulin antagonist. Both NOS activity in bladder and urethra, and relaxation responses to electrical field stimulation (EFS) in urethral preparations in vitro, were affected by NOS inhibitors with the same rank order of potency (N G-nitro-L-arginine > N G -nitro-l-arginine methyl ester> NG-methyl-L-arginine > 7-nitroindazole > L-cannavanine). These results suggest that the Ca2+dependent NOS activity found in the soluble fraction of ureteral, vesical and urethral homogenates represents a constitutive isoform present in neurons, which is mediating the NANC control of urinary tract function. NOS activity, NADPH-diaphorase positive nerves and responses to EFS (Triguero et al. 1993) are closely correlated in all regions. 898 Garcia-Pascual, A. & Triguero, D. (1994). J Physiol. 476, Triguero, D. et al. (1993). Neurosci. Lett. 163, 93. Characterization of nitric oxide synthase in the lower urinary tract A. Garcia-Pascual, A. Labadia, G. Costa and D. Triguero Departamento de Fisiologia, Facultad Veterinaria, Universidad Complutense Madrid, Madrid, Spain Nitric oxide (NO) mediates the NANC neural control of lower urinary tract motor function (Garcia-Pascual & Triguero, 1994). In the present study we have characterized the NO synthase (NOS) activity present within the muscular layer of the ureter, bladder and urethra in sheep. NOS activity was determined in both particulate and soluble fractions isolated from tissue homogenates by conversion of ["4C] arginine to ["4C] citrulline. No NOS activity was found in the particulate fraction. Soluble fractions showed a marked gradient in specific activity along the smooth muscle segments of the urinary tract, ranging (expressed as pmol (mg protein)' min-') from 1P in the ureter, in the bladder, to *5 in the urethra. In the absence of CaP, NOS activity in ureter, bladder and urethra was reduced to 65, 91 and 96%, respectively, suggesting the presence of a small and steady Ca2+-independent enzymic