腎臟草酸鈣結石的大鼠模式中，kappa類鴉片受器對腎功能變化的影響。

行政院國家科學委員會專題研究計畫 成果報告

腎臟草酸鈣結石的大鼠模式中，kappa 類鴉片受器對腎功能

變化的影響。

中 華 民 國 94 年 10 月 31 日

行政院國家科學委員會補助專題研究計畫

■ 成 果 報 告

□期中進度報告

Effects of kappa opioid receptor on the renal functional responses in the

kidney with calcium oxalate formation of rats

腎臟草酸鈣結石的大鼠模式中，kappa 類鴉片受器對腎功能變化的影響

計畫類別：個別型計畫

□ 整合型計畫

計畫編號：NSC 93-2314-B-002-151

執行期間：93 年 08 月 01 日至

94 年 07 月 31 日

計畫主持人：

共同主持人：

計畫參與人員：

成果報告類型(依經費核定清單規定繳交)：精簡報告 □完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

涉及專利或其他智慧財產權，□一年二年後可公開查詢

執行單位：臺大醫學院泌尿部

中

華

民

國

94 年

10 月

20

日

(一)、中文摘要及關鍵詞 根據文獻上也有證據顯示，內生的類鴉片也會作用在活體內的周邊系統來調節心臟血 管系統和腎臟功能。但是，目前文獻上所報告的有關類鴉片對腎功能的影響，研究的結果 似乎都指出類鴉片是借由腎臟以外的機制來調控腎功能，而非腎臟內的類鴉片受體的作用 而來。但是本計劃初步結果卻顯示腎臟內亦有不同類鴉片受體的表現；而且腎臟內的鴉片 受體的表現在乙烯甘醇餵食大鼠所誘發草酸鈣腎結石的過程中會改變。 內生的鴉片產生生理作用是經由活化三種不同的鴉片受體，也就是 mu (MOR), delta (DOR) 和 kappa (KOR) 等三種鴉片受體。本實驗發現在腎臟內確實有這三種鴉片受體存 在，但是在比較正常大鼠和腎臟有草酸鈣結石的實驗組大鼠的腎臟標本後，我們發現 DOR 在結石的腎臟中的皮層顯著的增加而髓質中的表現則無影響；而 MOR 在結石的腎臟中的 表現則無顯著變化。KOR 在結石的腎臟中的表現只有在初期會增加，有趣的是其 mRNA 與蛋白量表現的變化一致，顯示出大鼠餵食乙烯甘醇早期之高草酸尿症與 KOR 之變化有 重要的關聯。活化 kappa 類鴉片受體會產生顯著的利尿效果，但是對尿中的電解質卻沒有 影響。而由我們先前的實驗已發現，使用乙烯甘醇餵食大鼠以誘發草酸鈣腎結石的過程中， 尿流量和尿鈉的排泄在餵食乙烯甘醇 42 天後會有顯著的下降現象。這些結果顯示在結石的 大鼠，腎臟的排泄功能可能有受損，而鴉片受體表現的增加可以代償腎功能的改變。此外， 鴉片受體在大腦中有神經保護的功能，是否腎臟 DOR 與 KOR 表現的增加可以保護腎臟細 胞對高草酸血症的傷害，需進一步的試驗。 關鍵字：κ 類鴉片受體，腎功能，草酸鈣，腎結石，高草酸尿症。 (二)、英文摘要及關鍵詞 (keywords)

Based on previously reports, endogenous opioid receptors in peripheral system have been suggested to be involved in regulation of both cardiovascular and renal function. However, the effect of opioid receptors on renal function was suggested as an extrarenal event. In our preliminary study, we found that one of opioid receptor subtype was increased in the rat kidney of experimental nephrolithiasis induced by fed with ethylene glycol. Therefore, we are interested to know whether this change has a significant role on regulation of crystal formation.

Endogenous opiates act on its receptor subtypes, classified as mu (MOR), delta (DOR) and kappa (KOR) opioid receptor, to elicit a physiological effect such as addition and so on. In the present study, we found that three subtypes of opioid receptors were existed in rat kidney. Compared to the normal kidney, the DOR expression in the kidney with calcium oxalate was significantly increased in renal cortex but not in the medulla. However, the MOR expression was unchanged in any part of renal tissues. The KOR expression was increase during the initial phase of crystal formation. Most interesting is that the expressed patterns of KOR mRNA and protein were similar, this indicated that the changes in KOR were participated in EG-induced hyperoxaluria. As previous studies, urinary excretion of electrolytes won’t be affected by diuresis elicited by intrarenal KOR activation. Our previous results also showed that urinary output and

sodium excretion were significantly lowered in EG-treated 42 day rats. Together these, we concluded that increased expression of opioid receptor may have an effect on compensation of impaired renal excretory response observed in the nephrolithiatic kidney. In addition, opioid receptor expressed in neurons of brain cortex offered a neuroprotection in respond to detrimental damage. Whether renal opioid receptor show the similar effect on renal cell protection is our further interest.

Key Words: Opioid receptor, calcium oxalate, kidney crystal

(三)、前言 我們研究團隊曾於民國 89 年提出國科會研究計畫:”以乙烯甘醇誘發大白鼠產生腎結石的 模式中一氧化氮所扮演的角色 (NSC89-2314-B-002-298)”，發現經由腎動脈導管直接給予一 氧化氮合成酵素的受質灌注腎臟 — 即 L-arginine 的灌注，我們發現在腎結石的實驗組大鼠其 腎臟利尿和利鈉的反應與正常組的大鼠相比會顯著的降低。利用 volume expansion 的方法來測 試腎臟內 NOS 的活性，結果一樣發現在腎結石的大鼠腎臟內，利鈉和利尿的反應與正常大鼠 相比顯著的降低。腎臟內一氧化氮系統是一很重要調控利鈉利尿反應的物質，相對於腎臟內 鴉片受體系統，也是一很重要調控排泄反應的物質。所以本計劃探討是否在結石的腎臟，其 鴉片系統的變化會參與在結石形成的過程中。 (四)、研究目的 本計畫首先觀察在結石的腎臟內鴉片受體 mRNA 和蛋白表現的時間性的變化，我們檢 測: 1. 三種不同鴉片受體 MOR、DOR 和 KOR 在實驗性腎臟草酸鈣腎結石的大鼠中的變化情 形。 2. 利用 RT-PCR 偵測鴉片受體 mRNA 量的變化。 3. 利用 immunoblot 偵測鴉片受體蛋白量的變化。 (五)、重要文獻

1. Baylis C and Bloch J: Nitric oxide (NO) in renal physiology and pathophysiology. Nephrol Dial Transplant, 11:1955, 1996

2. Chen C, Mitchell KD, Navar LG: Role of endothelial-derived nitric oxide in the renal hemodynamic response to amino acid infusion. Am J Physiol, 263:R510, 1992.

3. Peters H, Border WA, Noble NA: L-arginine supplementation increases mesangial cell injury and subsequent tissue fibrosis in experimental glomerulonephritis. Kidney Int,

55:2264, 1999.

4. Huang HS, Chen CF, Chien CT, Chen J: Changes in the oxidant-antioxidant balance in the kidney of rats with nephrolithiasis induced by ethylene glycol. J Urol, 167:2584, 2002 5. Reyes AA, Purkerson ML, Karl I, Klahr S: Dietary supp;ementation with L-arginine

Dis 20:168-176, 1992.

6. Reyes AA, Karl I, Kissane J, Klahr S: L-arginine administration prevents glomerular hyperfiltration, and decreases proteinuria in diabetic rats. J Am Soc Nephrol 4:1039-1045, 1993.

7. De Nicola L, Thomson SC, Wead LM, Brown MR, Gabbai FB: Arginine feeding modifies cyclosporine nephrotoxicity in rats. J Clin Invest 92:1859-1865, 1993.

8. Reyes AA, Porras BH, Chasalow FI, Klahr S”L-arginine decreases the infiltration of the kidney by macrophages in obstructive nephropathy and puromycin-induced nephrosis. Kidney Int 45:1346-1354, 1994.

9. Ashab I, peer G, Blum M, et al: Oral administration of L-arginine and captopril in rats prevents chronic renal failure by nitric oxide production. Kidney Int 47:1515-1521, 1995, 10. Ingram A, Parbtani A, Thai K, et al: Dietary supplementation with L-arginine limits cell

proliferation in the remnant glomerulus. Kidney Int 48:1857-1865, 1995.

11. Peters H, Border WA, Noble NA: L-arginine supplementation increases mesangial cell injury and subsequent tissue fibrosis in experimental glomerulonephritis. Kidney Int 55:2264-2273, 1999.

12. Nicola LD, Minutolo R, Bellizzi V, et al: Enhancement of nitric oxide synthesis by L-arginine supplementation in renal disease: Is it good or bad? Miner Electrolyte Metab 23:144-150, 1997.

13. Zhang C, Walker LM, Mayeux PR: Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney. Biochemical Pharmacology 59:203-209, 2000.

14. White R, Brock T, Chang LY, et al: Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci USA 91:1044-1048, 1994.

15. Malinsky T, Bailey F, Zhang Z, et al: NO measured by a porphyrinic microsensor in rat brain after transient middle cerebral occlusion. J Cereb Blood Flow Metab 13:355-358, 1993.

16. Dinerman J, Lowenstein C, Snyder S: Molecular mechanisms of nitric oxide regulation. Potential relevance to cardiovascular disease. Circ Res 73:217-222, 1993.

17. Backman JS, Ischiropoulos H, Zhu L, et al: Kinetics of superoxide dismutase and iiron-catalyzed nitration of phenolics by peroxynitrite Arch Biochem Biophys 298:438-445, 1992.

18. Mundel P, Backman S, Bader M, et al: Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int 42:1017-1019, 1992.

19. Goligorsky MS, Eisei N: Duality of nitric oxide in acute renal injury. Seminars in Nephrology 19(3):263-271, 1999.

20. Patel VC, Yellon DM, Singh KJ, et al: Inhibition of nitric oxide limits infarcts. Biochem Biophy Res Commun 194:234-238, 1993.

(六)、研究方法

Induction of Experimental Nephrolithiasis and Animal Grouping.

Male Wistar rats (200–250 g) were housed under a constant temperature and a light/dark cycle (light from 07:00 to 18:00). The animal care and experimental protocol were in accord with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The rats were divided into control group and group rats received 0.75% ethylene glycol (EG) in drinking water. There were six rats in each group, fed for 7, 14, 21, and 42 days. During the experimental period, all groups had free access to ground regular rat chow.

Semi-quantitative RT-PCR to Detect opioid receptor mRNA Expression.

Total cellular RNA in the cultured neurons was extracted by using the TRIzol reagent (Gibco) as the manufacturer’sprotocol. The RNA pellet was washed twice with 75% ethanol, dried, and resuspended in the diechypyrocarbonate-treated water. Complementary DNA (cDNA) was synthesized from 5g of the total RNA sample at 42oC for 47 min using 0.5g of poly dT15 oligonucleotide primer (Invitrogen, Carlsbad, CA) and 200 units of reverse transcriptase (M-MLV, Promega, Madison, WI) in a final volume of 20 l. Opioid receptor and glyceraldehyde 3-phosphate dehydrogenase cDNA were amplified using specific primers designed as previously described (55). The sequences for GAPDH were5’-TTA GCA CCC CTG GCC AAG G-3’ and 5’-CTT ACT CCT TGG AGG CCA TG-3’ (535 bases). The reverse transcription product (2.5 l) was amplified with a mixture of primers and 2.5 units of REDTag DNA polymerase (Sigma) for 30 cycles under the following conditions: 1 min at 95oC for denaturation, 50 sec at 58oC for annealing, and 1 min at 72oC for extension. Reaction products were electrophoresed through a 2% agarose gel and visualized by ethidium bromide staining. The density of appreciate band was determined using an image analytic system (Alpha Innotech, San Leandro, CA). Expression of opioid receptor mRNA was calculated as a ratio of opioid receptor and glyceraldehyde 3-phosphate dehydrogenase PCR products.

Immunoblotting of opioid receptor.

The abundance of KOR, MOR, DOR and actin were examined by Western blots as described previously (Ma et al., 2005). Kidneys (divided into cortex and medulla) were sampled to prepare total proteins, a part of hepatic tissue was served as a positive control. The same amount of protein from each preparation (150 g for opioid receptor) was separated on a 7% SDS-PAGE under denaturing, and electrophoretically transferred to nitrocellulose (Amersham, Buckingham, England, UK). Membrane was incubated with monoclonal anti-MOR antibody (1:500; BD Biosciences), anti-DOR (1:250; BD Biosciences), anti-KOR (1:500; BD Biosciences), and anti-actin (1:1000, Sigma), respectively, overnight at 4℃, washed, and then incubated with goat biotinylated anti-mouse IgG conjugated to horseradish peroxidase (Leinco Technologies, Saint Louis, Missouri, USA) for one hour at room temperature. The samples were washed and the bound antibody was visualized using a commercial DAB (3, 3’-diaminobenzidine) substrate kit (Vector, Burlingame, CA, USA) for peroxidase, following the manufacturer’sprotocol.The density of the acquired band in each lane was semi-quantitatively determined by densitometer with an image analytic system (Alpha Innotech, San Leandro, CA, USA). The data in each group was presented as the mean standard error for the integrated digital value (IDV) divided by corresponding actin of each lane.

Figure 1. Representative Western blot of DOR, KOR, and MOR in the specimen of renal cortex

and medulla from the controls and ethylene glycol (EG)-treated rats with different time-points.

Figure 2. Results of immunoblot and PCR of KOR in the specimen of renal cortex and medulla

from the controls and ethylene glycol (EG)-treated rats with different time-points. Insects showed the representative pictures.

(八)、計畫成果自評部份

本計劃進行相當順利，預計在本年度下半年中即可將所得之結果彙理並投稿於腎臟與 泌尿學相關學門之國際雜誌。