實驗性腎病中腎感覺功能失調的機制

Abstract

中文摘要 腎臟感覺功能主要是透過由快肽 (tachykinin)活化神經激肽受體（neurokinin receptors）所引發的訊息傳遞路徑，進而造成傳入性腎神經活性的增加，導致尿流速與尿中鈉離子排泄速率增加。依據本實驗室之前研究，在許多腎病變中，包括：嘌呤黴素核苷酸（puromycin aminonucleoside, PAN）、內毒素（lipopolysaccharide, LPS）、 以及環孢菌素A（cyclosporine A, CsA） 所引起的腎病變中，都觀察到腎臟感覺功能失調的現象，而且本實驗室之前的研究也發現低氧預處理能夠減緩這些疾病中腎感覺功能失調的現象。除了神經激肽受體之外，本實驗室最近也發現類鴉片受體在腎盂表現並且其中以delta亞型的類鴉片受體（delta-opioid receptor, DOR）為主，活化腎盂內DOR可夠劑量依存性地增加傳入性腎神經的活性，所以DOR可調控腎臟感覺功能。因此，我們假設快肽、神經激肽受體以及DOR可能在腎臟疾病所引發的腎感覺功能失調中扮演相當重要的角色；並且低氧預處理可能透過這些因子來達到其感覺功能的保護效果。 大鼠腎病變的誘發模式是參照本實驗室之前的研究方法。藥物處理前先將各組動物區分為海平面正常組 （sea level，SL）以及低氧預處理組（hypoxia-adapted，HA）。低氧預處理的進行方式是將大鼠置放於模擬海拔高度5,500公尺的低氧艙中，每天15小時，共放置4週，而後再進行與SL組相同劑量的藥物處理。腎病變的誘發模式如下：皮下注射60或150 mg/kg的PAN處理 4天、7天以及10 天；腹腔注射4 mg/kg的LPS處理8 小時以及48小時；每天腹腔注射15 mg/kg的CsA連續注射15 天。實驗動物被置於代謝箱中，收集24小時尿液以測定腎功能指標，包括：血漿肌酸酐 （creatinine，Cr）、和尿素氮（blood urea nitrogen，BUN），尿液排泄速率（UV）、鈉離子排泄速率（UNaV）、尿蛋白質排泄速率（UPROV）、尿中Cr與計算肌酸酐清除率（Cr clearance , Ccr）。最後，各組動物使用過量的麻醉藥犧牲，灌流後將腎臟取出並分離腎盂，以西方墨漬法進一步分析腎盂組織中神經激肽第一型受體（neurokinin-1 receptor, NK-1R）、第三型受體（neurokinin-3 receptor, NK-3R）以及 DOR的表現量；另外，我們也使用免疫定量法（EIA），來測量腎盂組織與尿液中物質P（substance P, SP）以及Neurokinin B（NKB）的含量，並計算尿液中SP和NKB的排泄速率 （USPV和UNKBV）。 經過低氧預處理的HA組與SL組相比，體重較低且血比容增加，腎功能指標在兩組並無明顯差異；此外，HA組的腎盂內NK-1R、NK-3R和DOR的表現量以及尿液中SP和NKB的排泄速率，和SL組無明顯差異。 以不同劑量的PAN引發腎病變後，在不同觀察的時間點血液中Cr和BUN與UPROV會顯著增加，而Ccr和UV顯著下降；但是在150 mg/kg的PAN處理後UNaV會下降。有趣的是，60 mg/kg的PAN處理後，在不同時間點SL與HA組腎盂內NK-1R、NK-3R和DOR的表現均會增加；而150 mg/kg的PAN處理後10天，在兩組腎盂內NK-1R和DOR的表現顯著減少，但對NK-3R的表現並無顯著影響。同時兩組USPV均明顯下降，而腎盂中SP的含量在SL組顯著增加。UNKBV在兩組PAN處理動物中卻無顯著變化，但是腎盂中NKB含量均增加。 LPS處理8小時後，兩組的體重明顯下降，血漿中Cr以及BUN顯著增加，但Ccr在HA組明顯下降，UV在SL組明顯下降，而兩組的UNaV均顯著上升。兩組動物的腎盂中NK-3R表現均顯著上升，但NK-1R以及DOR表現卻無顯著變化。此外，腎盂中SP以及NKB的的含量在兩組並無顯著變化，但USPV和UNKBV均大量增加。LPS處理48小時後，可觀察到SL組體重下降與HA組的血比容降低，但Ccr在兩組都明顯下降。腎盂中NK-1R的表現量在HA組顯著增加，但在SL組卻無改變。DOR的表現量在兩組均下降，此外，SL組的NK-3R表現量明顯下降，HA組下降但是不具統計意義，顯示HA組的NK-3R下降幅度較SL組小。腎盂中SP和NKB的含量在處理48小時的LPS-SL組較高（P<0.05），而USPV和UNKBV皆無顯著變化。 在CsA所引發的腎病變中，除了Ccr的顯著增加外，其他腎功能指標並無顯著改變；腎盂中包括NK-1R、NK-3R與DOR的表現量以及USPV以及組織中SP的含量也無明顯改變。 腎臟的感覺功能在體液平衡中扮演相當重要的角色。綜合以上結果，可知在高劑量PAN所造成的腎病變中，腎盂內NK-1R的表現量降低與腎盂中SP的釋放發生障礙，可能是PAN導致腎感覺與排泄功能失調的原因；而低氧預處理可經過減緩NK-1R的表現量下降，來達到腎感覺功能的保護。此外，在低劑量PAN處理，NK-3R與DOR表現量的變化程度相似，此意味著兩者可能一起參與PAN所誘導的腎感覺功能變化。在LPS所造成的腎病變中，處理8小時後可觀察到NK-3R的表現增加以及尿中SP和NKB排泄量的增加與利鈉反應平行，顯示出神經激肽受體功能的增加可能參與LPS早期調控腎排泄反應。而在48小時後，NK-3R與DOR表現量的下降可能為LPS所引起寡尿的原因。CsA所造成的腎病變中，無論是正常或低氧鼠其腎盂中任何受體的表現量以及神經激肽的排泄量均無明顯改變，所以CsA所造成的腎感覺功能失調可能是有別於腎盂內快肽系統。

Abstract Intrapelvic neurokinin system is important on regulating renal sensory function via an activation of afferent renal nerve activity (ARNA) to reflexly inhibit efferent renal sympathetic nerve activity and induce a diuresis/natriuresis. Previously we found that ARNA response was impaired in rat nephropathies of puromycin aminonucleoside (PAN), lipopolysaccharide (LPS), or cyclosporine A (CsA), and these sensory defects were ameliorated in rats pretreated with hypoxia preconditioning (HPC). However, the underlying mechanisms are not clear. Besides neurokinins, recently we found that the dominant opioid receptor, delta subtype (DOR), was existed in renal pelvis and intrapelvic activation of DOR resulted dose-dependent increases in ANRA, suggested that DOR plays a role in regulating renal sensory response. In this study, we therefore hypothesized that changes in intrapelvic neurokinin system and DOR detrimental to renal sensory response in nephropathies and HPC may affect above molecules to prevent the loss of ARNA response. Nephropathic models were induced by the subcutaneous injection of PAN 60 or 150 mg/kg for 4, 7 and 10 days, intraperitoneal injection of LPS 4 mg/kg for 8 and 48 h, or intraperitoneal injection of CsA 15 mg/kg/day for 15 days in rats. Before nephrotoxic treatment, rats kept at room air pressure as controls were termed sea level (SL) group and been hypoxia-adapted (HA) by exposing to an altitude chamber (5,500 m) for 15 h/day for 4 weeks for HPC. Rats were placed in metabolic cages to assess renal function by determining plasma levels of creatinine (Cr) and blood urea nitrogen (BUN). Urine was collected to analyze the changes in urinary sodium (UNaV) and protein (UPROV) excretion, and determine urinary Cr for Cr clearance (Ccr). After functional study, animals were sacrificed and renal pelvis was sampled after transcardiac perfusion. The expression of neurokinin-1 receptor (NK-1R), neurokinin-3 receptor (NK-3R), and DOR were analyzed by Western Blot. The amount of substance P (SP) and neurokinin B (NKB) in renal pelvis and in urine were measured by EIA to calculate urinary excretion of SP (USPV) and NKB (UNKBV). Except a lower body weight and a higher hematocrit observed in HA group, there is no obvious differences of any functional indices between SL and HA groups. Also, the renal pelvic expressions of NK-1R, NK-3R, and DOR as well as USPV and UNKBV in SL rats were similar as those of HA rats. Significant increases in plasma Cr and BUN and UPROV were observed at various time-points after treated with different doses of PAN, and these were associated with decreases in Ccr and UV. Impaired UNaV was noticed after treatment of 150 mg/kg PAN but not for 60 mg/kg. Interestingly, renal pelvic expressions of NK-1R, NK-3R, and DOR of both groups were increased at various time-points after treated with 60 mg/kg of PAN. In the rats treated with 150 mg/kg of PAN for 10 days, the expressions of NK-1R and DOR were decreased, but not for NK-3R. The USPV of both groups treated with 150 mg/kg of PAN significantly lower than those of corresponding controls, but tissue amount of SP in renal pelvis was increased in SL group. There were no changes in UNKBV in both groups, but tissue amount of NKB in renal pelvis was increased in both groups. 8-h after LPS treatment, body weights of both groups were lower than controls, associated with increases in plasma Cr and BUN of both groups and a decreased Ccr was found in the HA group，a decreased UV was found in the SL group, and an increased UNaV was found in both groups. The expression of NK-1R and DOR were not altered, but a significant increase in NK-3R expression was observed in both SL and HA groups after 8-h LPS treatment. Besides, the amount of SP and NKB in the tissue of renal pelvis were no change, but the secretion of SP and NKB were both increased after 8-h of LPS treatment。 48-h after LPS treatment, a lower body weight was observed in SL group and a lower hematocrit was observed in HA group, and these were associated with decreases in Ccr in both groups. The expression of NK-1R was increased in HA group, but not in SL group. Decrement of both NK-3R and DOR abundances were observed in SL group. However, the reduction of both receptor expression were attenuated in HA. Besides, the amount of SP and NKB in renal pelvis tissue in SL group were higher, but the secretions of SP and NKB were not changed after 48-h of LPS treatment。 There were no changes of renal functional indices of both groups after CsA treatment except increased Ccr. CsA showed no effect on renal pelvic receptor expressions as well as the tissue amount of SP and USPV. Together above, the decrement of NK-1R expression and impaired SP release after a high dose of PAN treatment were paralleled with oliguria, suggested that the defective in NK-1R and SP resulted renal excretory impairment. However, HPC prevented loss of sensory function by restoring the levels of NK-1R which lowered by PAN. In addition, changes in renal pelvic NK-3R and DOR after a low dose of PAN treatment were also observed, implied that both receptor types participated in PAN-mediated renal sensory response. Increases in secretion of SP and NKB were paralleled with increased NK-3R expression and natriuresis after 8 h, suggested that neurokinin system was participated in LPS-induced early response. After 48 h, decreases in NK-3R and DOR might cause oliguria in response to LPS. Finally, intrapelvic receptor expression and the releases of neurokinin showed no obvious differences among groups, suggested that CsA-induced sensory impairment was independent of intrapelvic neurokinin system.