Kidney-specific Clock Machinery Coupled With Disturbed Circadian Rhythm Of Urinary Sodium Excretion In Nephropathy Rats

BACKGROUND:Normal kidney is a rhythm organ with the circadian pattern and contribute to diurnal blood pressure and urinary sodium excretion. It has been found that some circadian physiological problems including nondipping blood pressure, diurnal urinary sodium excretion disturbance, sleep-wake disturbance, and restless leg syndrome in chronic kidney disease patients. Some of the disorders disappeared after kidney transplantation. Increasing evidences suggested that the endogenous molecular machinery of the circadian clock functional in peripheral organs contributes to the regulation of oscillation of the physiological ryhthm.Our earlier study provided the first evidence that the nephrotic syndrome model adriamycin rats presented with the disturbed circadian rhythm of blood pressure. We also charaterized the adriamycin rats lost the circadian rhythm of blood prssure coupled with the disturbance of urinary sodium excretion rhythm compared with the SD control rats. In this study we would continue to explore the underlying chronobiologic mechanisms revolved with the kidney-specific clock machinery in nephropathy rats.METHODS:Male Sprague-Dawley (SD) rats were intravenously injected with adriamycin as the nephropathy rats group. Others were injected equal dose of saline as the control group. The rats were raised adaptively for two weeks, lights on at 8:00 a.m noted as zeitgeber time (ZT) 00:00; and lights off at 8:00 p.m noted as ZT12:00.Time from ZT00:00 to ZT12:00 noted as Light period, and ZT12:00 to ZT24:00 as Dark period. The urines in Dark and Light period were collected synchronously for analysis the rate of urinary sodium excretion. Three adriamycin-induced nephrotic syndrome rats and three SD control rats were sacrificed every four hours throughout a 24-hour day-night cycle to determine the mRNA expression of clock genes CLOCK, BMAL1, Per1, Per2, Cry1, Cry2 mRNA and clock controlled genes NHE3, aENaC, NCC, Ptges, V1aR, V2R mRNA in the kidney. Meanwhile, plasma rennin activity level, plasma aldosterone concentration and angiotensin Ⅱ concentration were measured. Data were analysed by a partial Fourier analysis and a stepwise regression technique.RESULTS:1.The rhythm of the urine volume, urinary potassium and urinary sodium excretion rate displayed high level at Dark period and low level at Light period in SD rats.Clock gene CLOCK, BMAL1, Per1, Per2, Cry1, Cry2 mRNA (p<0.05)and kidney specific clock-controlled gene NHE3, aENaC, NCC, Ptges, V1aR, V2R mRNA (p<0.05) expression showed circadian pattern. The peak time of the genes were at Dark period, coupled with the urinary sodium excretion oscillating.2.Compared to control rats group, urine volume and urinary sodium and potassium excretion showed high level at Light period and low level at Dark period in nephrotic ADR rats (p<0.05). Renal clock gene BMAL1 mRNA expression displayed 24h and 12h circadian rhythm, Cryl mRNA 4.8h, clock controlled gene NCC, V2R mRNA both 12h, and the peak time of the genes mentioned above shifted from Dark period to Light period<p<0.05).But clock genes CLOCK, Per1, Per2, Cry2 and clock controlled genes aENaC, NCC, Ptges, V1aR displayed no rhythm (p>0.05) 3.Plasma rennin activity level displayed 12h rhythm (p<0.05), plasma aldosterone concentration showed 24h rhythm (p<0.05), and plasma angiotensin II concentration presented no rhythm (p>0.05) in control rats group, while the ADR nephropathy rats showed no significant circadian oscillating in plasms rennin, aldosterone, and angiotensin II concentration (p>0.05)CONCLUSIONS:We demonstrated the ADR nephropathy rats lost the rhythm of the clock genes expression in the kidney which was conservative and stable in SD control rats. The kidney specific clock-controlled genes for tubular sodium and water transporting of NHE3, aENaC, NCC, Ptges, V1aR, V2R expression pattern were also disturbed in ADR rats, while all these genes exhibit normal circadian patterns in SD control rats. To our knowledge, this is the first demonstration of the disturbance at molecular level of self-sustained circadian timing system in nephropathy rats. Kidney-specific clock machinery coupled with disturbed circadian rhythm of blood pressure and urinary sodium excretion in nephropathy rats. It is likely to prove useful in unraveling the role of clock system in renal diesease.