The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport

1Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, UFC, Fortaleza, Ceará, Brasil, 2Instituto Superior de Ciências Biomédicas, Universidade Estadual do Ceará, UECE, Fortaleza, CE, Brazil, 3BioMolLab, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, UFC, Fortaleza, CE, Brazil, 4Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, CE, Brazil

Despite the structural similarities, Diocleinae lectins exhibit distinct biological activities, varying in potency and efficacy in comparison with CFL: lower mitogenic activity of Canavalia ensiformis lectin (ConA) in human blood mononuclear cells (Barral-Netto et al., 1992); higher histamine secretory activity of many Diocleinae lectins in rat peritoneal mast cells (Gomes et al., 1994).In addition, the higher affinity of CFL for glucose-mannose binding sites on the eye and mouth mucosal surfaces in relation with other glucose-mannose lectins (Banchonglikitkul et al., 2000) and its intermediate inhibitory activity in relation to the lectins of Dioclea violacea and Dioclea guianensis (DguiL) on the rat peritonitis and paw edema (Assreuy et al., 1997).
In respect to renal effects of Diocleinae lectins, our group demonstrated that the lectin of Canavalia brasiliensis (ConBr) (Teixeira et al., 2001), DguiL and ConA (Havt et al., 2003) distinctly alter physiological parameters in ex vivo rat kidneys.Considering that kidneys are responsible for the maintenance and excretion of electrolytes in the human body and the structure-activity relationship among Diocleinae lectins, the direct renal effects promoted by the seed lectin of Cratylia floribunda in isolated perfused kidneys and mesenteric bed of rats were investigated.

Wistar rats
Animals, maintained with free access to water and fasted for 24 h before experiments, were anesthetized with sodium pentobarbital (50 mg/kg body weight; i.p.).Experimental protocols were approved by the Animal Care and Use Committee of the Federal University of Ceara (UFC n° 107/07), according to international guidelines (NIH publication n°85-23, 1985).''Guide to the Care and Use of Experimental Animal Care" (Canadian Council on Animal Care guidelines, 1984).
Renal perfusion pressure (RPP) was measured at the tip of the stainless steel cannula in the renal artery.Renal vascular resistance (RVR) was calculated by dividing the RPP by the flow, measured in a flowmeter.Urinary flow (UF) was measured directly and glomerular filtration rate (GFR) by the clearance of inulin (Walser, Davidson, Orloff, 1955, Fonteles et al. 1983).Osmolality was evaluated by means of a vapor pressure osmometer (Wescor 5100C, USA).Samples of urine and perfusate were collected at 10min intervals.Electrolytes (Na + , K + and Cl -) were measured using ion-selective electrodes (Rapid Chem 744 ® , Bayer Diagnostic, UK) and sodium (%TNa + ), potassium (%TK + ) and chloride (%TCl) tubular transport were calculated (Martinez-Maldonado, Opava-Stitzer, 1978).CFL (10 µg/ mL) was added to the organ bath 30 min after the perfusion was initiated.In another set of experiments, amiloride (10 -4 M), a distal tubular sodium channel blocker, was added at 0 time (immediately after a 20-min equilibration period), 30 min before the CFL challenge.All experiments lasted 120 min and results were compared to the internal control groups (inside the group) or to time-matched controls (between groups).

Mesenteric bed perfusion
The abdomen of rats (280-350 g) was opened for tightening of pancreatic-duodenal, ileum-colic and colic branches of the superior mesenteric artery.The superior mesenteric artery was cleaned of surrounding tissue and cannulated with a polyethylene tube (PE20).The mesenteric bed (n=4) was perfused with MKHS with addition of 3.60 mM glucose at 37°C at a flow rate of 4 mL/min (MCGREGOR 1965).Direct vascular effects of CFL (3 and 10 µg/mL/min) or phenylephrine (1 µM/ mL/min) after 10 min of infusion were compared to the vehicle.Endothelium integrity was evaluated by addition of acetylcholine (1 µM) in the plateau-phase of phenylephrine-induced contraction.Perfusion pressure was measured using a pressure transducer (Statham P23, Gould, Oxnard, CA, USA) and recorded on a four-channel physiograph (Narco BioSystems, Houston, TX, USA).

Statistical analysis
Results were presented as mean±S.E.M. and compared by the Student test.Statistical differences were adopted for p values < 0.05.
These results are suggestive of an indirect effect of CFL on renal hemodynamics.It is known that the increase in renal vascular resistance can lead to the increase in glomerular filtration and urinary flow.However, the present results showed that these effects could follow independent mechanisms (Koeppen, Stanton, 1997).Previous studies had demonstrated important vasodilator effects of lectins belonging to the genus Canavalia, that varied in efficacy and mechanisms either in vivo (paw edema) or in vitro (isolated aorta), and involved the nitric oxide pathway (Assreuy et al., 2009).Thus, changes in renal hemodynamics and tubular physiology may be a consequence of the release of inflammatory mediators from kidney cells (Koeppen;Stanton, 1997).Moreover, the increased release of renin elicited by amiloride can interfer in its mild natriuretic and diuretic effects (Endemann et al. 2002).In fact, amiloride intensified the vascular effects (RPP and RVR) elicited by CFL.
CFL did not alter the tubular transport of sodium or chloride, but reduced the tubular transport of potassium at 60 min (% TK + = 7.19), 90 min (% TK + = 20.79) and 120 min (% TK + = 22.53).As expected, amiloride reduced the transport of sodium at 60 min (% TNa + = 8.16), 90 min (% TNa + = 10.14) and 120 min (% TNa + = 7.25), but not that of chloride or potassium, whereas its addition to the perfusion solution before CFL did not alter none of these ion transports (Table II).In respect to renal tubular transport, CFL decreased %TK + , but did not alter %TNa + or %TCl -.Since amiloride, recognized as a potent potassium sparing drug, increases sodium levels in the distal segments, its effect opposes potassium reabsorption by CFL in this renal segment.The tubular alteration in  The effect of Cratylia floribunda lectin on renal hemodynamics and ion transport 759 %TK + elicited by CFL could be a consequence of an indirect mechanism.
Renal effects elicited by different Diocleinae lectins are quite diverse.For instance, CFL and ConBr greatly increased RPP and RVR in the perfused rat kidney, but the increase caused by CFL was twice longer than that induced by ConBr (Teixeira et al., 2001.On the other hand, ConA and DguiL showed minor alterations in RPP and RVR (Havt et al., 2003).ConA increased these parameters at 60 min, whereas DguiL increased from 90 to 120 min.In addition, only ConBr, but not DguiL, ConA (Teixeira et al., 2001, Havt et al., 2003) or CFL increased UF and GFR.With respect to tubular ion transport, ConBr reduced all transports (potassium, sodium and chloride) (Teixeira et al., 2001), while ConA only potassium (Havt et al., 2003).However, DguiL did not influence any of these parameters (Havt et al., 2003).Remarkably, CFL decreased potassium but not sodium or chloride transport.
The presence of specific amino acids in the structure of Diocleinae lectins (Del Sol et al., 2007) may explain such differences in pharmacological activities.The substitution of the amino acids Gln155 and Glu155, present in all Diocleinae by His155 solely on ConBr (Calvete et al., 1999) could also explain the better efficacy of ConBr, either in its antinociceptive property as well as in its renal hemodynamic changes compared to other lectins.Furthermore, the different activities and potencies observed between CFL and ConBr could be explained by the presence of specific amino acids in CFL, which are not present in ConBr due to the evolutionary processes in ancestors (Del Sol et al., 2007).
The knowledge of structural aspects and phylogenetic proximity among Diocleinae lectins is essential for the understanding of its relation structure-activity.Besides, this study opens avenues for the use of CFL as an important pharmacological tool to elucidate mechanisms implied in cardiovascular disorders.

CONCLUSION
The seed lectin of Cratylia floribunda alters renal hemodynamic parameters showing a kaliuretic effect, that could be of tubular origin, rather than a result from haemodynamic alterations.

FIGURE 1 -
FIGURE 1 -Effect of Cratylia floribunda lectin (CFL; 3 or 10 µg/mL/min) on mesenteric vascular bed.Panel (A) shows its effect in the basal mesenteric vascular tonus and (B) shows the effects of CFL in vascular bed contracted with phenylephrine (PHE; 5 µM).Mesenteric bed was perfused with Krebs Henseleit solution at 37 °C.Data were expressed as mean ± S.E.M. (n=4) and compared by using Student t test.*p< 0.05 compared to control (Krebs-Henseleit).

TABLE I -
Renal effects of the lectin from Cratylia floribunda seeds