Abstract
KCl Cotransport (KCC) is highly expressed in sickle red blood cells (SS RBC) and recent data have demonstrated its abnormal response to cell swelling and acid pH. We showed that the final MCHC achieved by SS reticulocytes upon activation of KCC by these stimuli was higher than that of normal (AA) reticulocytes (Joiner et al, Blood, in Press). Here we report studies examining the sensitivity of KCC to activation by urea at concentrations present in the kidney and the effect of urea stimulation of KCC on reticulocyte MCHC. KCC fluxes were assayed as Rb uptake over 20 min in isotonic saline solutions buffered with HEPES to pH 7.4 (37°C) containing 0.1 mM ouabain, and 0.01 mM bumetanide, with 27 mM RbCl replacing equimolar NaCl. Under these conditions > 95 % of Rb uptake was Cl-dependent (assessed by sulfamate replacement of Cl). The maximal volume-stimulated KCC flux (VSmax KCC) was measured for each sample in cells swollen isotonically to MCHC < 27 gm/dl (nystatin method). Urea (100 to 1000 mM) increased osmolality of buffers, but did not alter initial MCHC. MCHC of reticulocytes (detected by flow cytometry) was tracked by measuring density changes on calibrated OPTIprep® gradients. Cl-dependent, ouabain- and bumetanide-insensitive Rb influx in both AA and SS RBC was increased by urea, reaching a plateau at 1000 mM urea that was similar to VSmax KCC. SS RBC were more sensitive to urea stimulation than AA RBC: 50% VSSmax KCC was achieved at 330 mM urea in SS RBC vs 550 mM in AA RBC. This effect was sulfhydryl dependent: exposure to the reducing agent dithiothreitol (preincubation for 30 min at 37°C with 10 mM DTT, then 1 mM in flux media) normalized the response to urea in SS RBC, with no effect in AA RBC. When swollen to MCHC 30 gm/dl, SS and AA retics exhibited Regulatory Volume Decrease (RVD) which increased MCHC. RVD in both AA and SS retics was abolished by incubation in sulfamate media, indicating mediation by KCC. As previously reported, final MCHC achieved after two hours incubation by SS retics was greater than AA retics (see Table, Control).
Final Reticulocyte MCHC, gm/dl [mean (SD), n = 3]
AA SS p (AA vs SS) Control 31.9 (0.7) 34.7 (1.2 0.03 Urea 35.3 (0.5) 37.8 (0.3) 0.002 p (Control vs Urea) 0.005 0.03
Urea (600 mM) enhanced RVD in both AA retics and SS retics. Sulfhydryl reduction with DTT had no effect on urea-stimulated RVD in SS reticulocytes. RVD stimulated by urea was complete within 60 minutes, and was irreversible: additional incubation without urea did not lower MCHC. The partially dehydrating effect of brief (10 min) exposure to urea was also irreversible, and was cumulative: cells exposed to two 10 min exposures to high urea (600 mM), separated by 10 min at a low, non-stimulating concentration of urea (100 mM), yielded the same MCHC as a continuous 20 min exposure. These data demonstrate that urea, at concentrations found in the renal medulla, is a powerful stimulant of KCC and intiates a striking RVD in reticulocytes. To the extent that intermittent stimulation of KCC by urea in the kidney occurs in vivo, this could contribute an exaggerated RVD resulting in dehydration of SS reticulocytes.
Pathology of Infestation of the Rat With Nippostrongylus Muris (Yokogawa) III. Jejunal Fluxes in Vivo of Water, Sodium, and Chloride