Changes Of Large Conductance Ca2+-actirated K+Channels On Vascular Smooth Muscle Cells Derived From Uremic Rats

Background:At present,Uremia has been in high incidence in most parts of the world. Macroangiopathy has become one of the main complications which could cause disability or death. But the mechanism has not been clear until now, one of the possibilities is that it could be involved with abnormality of signal transduction of ion channel. Large conductance of calcium-activated potassium(BK) channel is an important ion channel mainly exclusively expressed in vascular muscle cells, which acts as regulators of VSMC membrane potential and has a vital role in regulating arterial resting tone and VSMC relaxation. Impaired BK channel activities in smooth muscle cells may contribute to the development of vascular reconstruction in uremic.OBJECTIVE:The present study was therefore to investigate whether BK activity and expression has altered in VSMCs isolated from aortic vascular tissues of rats with uremic using approaches of electrophysiology and molecular biology to elucidate the molecular mechanisms of vascular dysfunctions due to uremia.METHODS:(1) Adult SD rats underwent 5/6 nephrectomy(5/6 Nx) or a sham operation(Sham). The following parameters were measured respectively pre-operation and postoperation at week 2, 8, 16, 24, 30, 36: creatini ne concentration in serum(Scr) and blood urea nitrogen(BUN), which was measured in the blood taken from the retro-orbital plexus under anesthesia. The values of systolic pressures were measured by the tail-cuff method using an RTBP apparatus in conscious animals. After uremic animal model was established successfully by 5/6th nephrectomy, Kidney and aorta of both groups from paraffin-embedded were processed with hematoxylin-eosin(HE) and/or Masson’s trichrome staining for later analysis.(2) Vascular smooth muscle cells(VSMCs) were isolated by enzyme digestion. The BK currents of both control group and model group were recorded by patch clamp technique in whole cell configuration.(3) m RNA and proteins of the α-subunit and the β1-subunit of BK channels were detected using real-time reverse transcription polymerase chain reaction and Western blot analysis in rat aorta tissues.RESULTS :(1) 5/6 nephrectomy-induced rat uremic animal model was established successfully: ①Renal function:Compared with control group, creatinine concentration and blood urea nitrogen in serum of model group increased significantly since the second week after surgery. Scr was(102.67±3.28) μmol/L and BUN reached(24.78±5.14) mmol/l at week 36 after surgery. ②Morphologic findings in kidneys: The size of random-shaped renal stump in uremic group was increased. Histological features include tubulo-interstitial damage reflected by inflammation, glomeruli enlargement and sclerosis, concomitant expansion and atrophy of renal tubular, tubulointerstitial fibrosis. ③ Morphologic findings in aorta: The characteristics of the arterial lesions were necrosis of medial smooth muscle cells and, in some cases, calcification of the media.④SBP: The values of systolic pressure showed rising tendency in subtotally nephrectomized rats, the significant difference was found at least 2 weeks after surgery(the model group(128.7±5.4) mm Hg vs.(113.5±3.6) mm Hg in control group). SBP in the model group at the 8, 16, 24, 30 and 36 week was respectively(141.2±4.8) mm Hg、(153.9±7.4) mm Hg、(163.5±6.7) mm Hg、(174.2±6.6) mm Hg、(181.3±7.7) mm Hg, which was significantly different from the control group(P<0.05);(2) Macroscopic Current of BK Channels in VSMCs from arota: Compared with control group, the current densities in uremic group were significantly decreased when test potentials >10 m V(P<0.05).BK current densities were(199±17) p A/p F in control group and(85±13) p A/p F in uremic group at 70 m V test potentials(n=6);(3) m RNA of BK α- and β1-Subunits: The results were normalized by the 2-ΔΔCt, the sham group as the control(2-ΔΔCt=1). In m RNA study, a dramatic decrease in the m RNA expression of α subunits and β1-subunit in model group was found(P<0.05).(4)Proteins of BK α- and β1-Subunits: Remarkable significance of difference in BK α-subunit protein expression was found between the two groups(P<0.05),β1-subunit protein expression was also significantly reduced in uremic group than in control group(P<0.05).CONCLUSION:Downregulation of BK channel and decrease of BK channel current density in uremic aortic arteries may be associated with aortic dysfunction.