Potential Ototoxicity of Aluminum in Hemodialysis Patients

Objectives/Hypothesis. Aluminum (Al) is a neurotoxin in both human and animal models. Al accumulation is usually observed in patients with end- stage renal disease (ESRD). To clarify whether Al also exhibits toxic effects on the specified neural organ of inner ear, we recruited hemodialysis (HD) patients to investigate the effect of serum Al level on the auditory physiology. Study Design: Forty patients in maintenance HD as well as 40 age-matched healthy subjects without hearing complaints were enrolled. The auditory function tests, including pure-tone audiometry( PTA ), distortion-product otoacoustic emissions (DPOAEs), and auditory brainstem response (ABR) were performed in all subjects. The serum Al levels determined within 3 months of auditory tests were used for analysis . Results: High- frequency hearing impairment was the predominant auditory dysfunction in HD patients who showed worse high-tone hearing level on PTA and diminished amplitudes of DPOAEs at 3 K and 4 K as compared with the controls (P <.001). Age was a significant factor determining the auditory dysfunction in both HD patients and control subjects. After age correction, serum. Al level correlated reversely with the amplitude of DPOAEs-2 K (P =.002), but not with amplitudes of DPOAEs-3 K, -4 K, hearing levels on PTA, or wave latencies on ABR. Conclusion. High-frequency hearing impairment is a common presentation in HD patients. Serum Al level correlates reversely with the amplitude of DPOAEs-2 K but not those of DPOAEs-3 K -4 K, hearing levels on PTA, and wave latencies on ABR. Possibly, the correlation between the Al level and the high-frequency OAE results was obscured by the significantly diminished amplitudes of DPOAEs- 3 K, -4 K in ESRD patients. These results implicate that the effect of Al is mainly of cochlear origin rather than of retrocochlear origin

Clinical Characteristics of Patients with Segmental Renal Infarction

Background: Renal infarction is usually an underestimated disease due to its rare and non-specific presentations; the renal survival of these patients is not well studied. The aim of the present analysis is to study the clinical features and outcome in patients who had documented renal infarction. Methods: Twenty-two patients (12 men and 10 women, mean age of 57.7 ± 3.44 years (28.4–83.3 years)) with image-confirmed segmental renal infarction in the past 15 years were enrolled. All patients were followed up at outpatient department with a median of 4 years (1–14 years ) . Initial and follow-up clinical characteristics and laboratory results were recorded. Results: The most common underlying disease was cardiovascular disease. Renal infarction often presented with non-specific symptoms, including flank pain (55%), vague abdominal pain (50%), nausea/ vomiting (46%) and fever (27%). The levels of leucocytes, lactate dehydrogenase, blood urea nitrogen and serum creatinine were all elevated at admission. The early diagnosis group (12/22) had more obvious flank pain, nausea/ vomiting(P < 0.001) and higher alanine transaminase (P = 0. 02). It also predisposed to undergo antiplatelet or anticoagulant therapy (all P < 0.04). During follow up, there was no recurrence in the whole study group, and a trend of better recovery of renal function was noted in the early diagnosis group. Conclusion: The serum creatinine level correlates with longer hospitalization length (P < 0. 05). As regards long -term prognosis, no definite factor or treatment was found to have significant effect in segmental renal infarction patients. However, early diagnosis and early initiation of treatment seems to have a positive effect on future renal outcome

The Impact and Mechanism of Angiotensin II Type 2 Receptor（AT2） on Aldosterone Secretion and Adrenocortical Cell Apoptosis

背景與目的：Renin-angiotensin-aldosterone system (RAA system)是體內調控血流動力、體液及電解質平衡非常重要的系統。腎素（renin）使血管張力素原（angiotensinogen）轉換成第一型血管張力素（angiotensin I），再經過血管張力素轉換酵素 (angiotensin converting enzyme，ACE)將第一型血管張力素轉成第二型血管張力素(angiotensin II， ANGII)。ANGII已有不同的受器亞型被發現，主要有第一型及第二型(AT1 & AT2)。當ANGII作用在AT1時，會促使血管收縮使血壓上升，並促進腎上腺增加醛固酮（aldosterone）的產生和分泌。目前已知AT2在胚胎發育時期有廣泛表現,但在出生後卻迅速退化或消失除了在一些特定的組織外。目前對AT2的一般生理功能瞭解包括，抑制細胞生長、促進細胞分化、促進細胞凋亡、促進神經細胞再生、抑制血管新生、促進血壓下降及情緒穩定等，這些大部份似乎都在拮抗AT1的生理功能。在人類腎上腺，有40%的AT2表現在zona glomerulosa中，但這些AT2的生理功能不若AT1研究的透徹，尤其是在aldosterone的分泌及對細胞凋亡的影響，甚少被提出。因此吾人希望能針對此部分作進一步的探討，以瞭解AT2在人類腎上腺對aldosterone分泌及細胞凋亡所扮演的角色。 方法：首先，我們建立了穩定的H295R細胞株，並經由反轉錄聚合酵素連鎖反應（Reverse transcriptional polymerase chain reaction,RT-PCR）及西方墨點（Western blot）證實H295R細胞上確實有AT2的表現。並利用免疫放射分析法（radioimmunoassay,RIA）分析H295R細胞aldosterone基礎分泌量、ANGII對aldosterone分泌影響之時間與濃度效應、AT阻斷劑和AT2刺激劑對H295R細胞aldosterone分泌之影響。RT-PCR測定aldosterone synthase （CYP11B2）mRNA、西方墨點測定AT2之蛋白質在不同條件下之表現。最後並以流式細胞儀（Flow cytometry）進行細胞凋亡（cell apoptosis）分析。 結果：在加入10-6M ANGII後24小時測得aldosterone分泌明顯的增加4.8倍（p=0.03）。加入valsartan時，ANGII刺激的aldosterone分泌量會降低61%；而PD123319則對ANGII刺激的aldosterone分泌沒有影響，但若與valsartan合併使用，則可以使ANGII刺激aldosterone分泌的作用完全抑制，且分泌量下降達80%。利用RT-PCR則可發現在經過1μM ANGII處理後，在第2個小時偵測到CYP11B2 mRNA之表現開始增加，並於第8個小時達到最大量，約增加25倍。Valsartan可以減低CYP11B2 mRNA的表現量達35%，而同時加入valsartan及PD123319則減低CYP11B2 mRNA的表現量59.8%。雖然CGP42112A也會增加CYP11B2 mRNA的表現，但並不影響aldosterone的分泌。而在H295R細胞株上則並未看到刺激AT2所引起的細胞凋亡。 結論：ANGII增加aldosterone分泌主要是經由AT1的作用，且是藉由CYP11B2 mRNA的表現增加來達成，這個作用並無法以AT2的阻斷劑抑制。吾人進一步發現，同時阻斷AT1和AT2可以完全抑制ANGII刺激細胞分泌aldosterone，這個情形在CYP11B2的轉譯層次也有相同的結果。因此，AT1和AT2在腎上腺細胞也有“相互對話“的現象。這個相互對話的訊息傳遞有待進一步研究。而AT2的表現，目前認為並不影響腎上腺細胞之細胞凋亡。Renin-Angiotensin-Aldosterone system (RAA system) controls the hemodynamic status, body fluid and electrolyte balance in human body. In the system, ANGII is a key hormone which has 2 main receptor subtypes, AT1 and AT2. Most physiological effects of ANGII were mediated by AT1. It constricts arteries, elevates blood pressure and also stimulates adrenal gland to secret aldosterone. However, the role of AT2 in adrenal gland is not well studied as AT1. AT2, a G-protein coupled receptor has 7 transmembrane domains, is widely expressed during fetal development. It regresses or even disappears rapidly in early postnatal period except some limited tissue like limbic system, several thalamic nucleus, adrenal medulla, zona glomerulosa and uterine myometrium etc. The general understanding of its functions are inhibition of cell growth, promotion of cell differentiation or cell apoptosis, mediates neural cell remodeling, inhibition of angiogenesis and counteracts AT1’s effects. In adult adrenal gland, 40% of AT2 is presented in zona glomerulosa; however, the significance of its persistent expression is unknown. We established stable H295R cell line and aldosterone concentration was detected by radioimmunoassay (RIA) which was corrected by total protein amount. CYP11B2 mRNA and AT2 protein expression were detected by RT-PCR and Western blot later. Cell apoptosis was analyzed by flow cytometry. After ANGII stimulation, aldosterone was detectable since 24th hour and lasted up to 72 hours. The ANGII induced aldosterone secretion was 61% suppressed by AT1 blocker, valsartan; but AT2 blocker, PD123319 has no effect on it. However, co-treatment with valsartan and PD123319 could suppress aldosterone secretion almost totally. CYP11B2 mRNA expression could be detected since 2nd hour and maxially expressed at 8th hour after 1μM ANGII treatment. Valsartan suppressed CYP11B2 mRNA expression, whereas PD123319 alone has no impact on it. If pretreated with valsartan and PD123319 simultaneously, the expression of CYP11B2 mRNA was suppressed to basal level as control group.The mRNA change seems to correlate with the aldosterone secretion change. Although CGP42112A up-regulates CYP11B2 mRNA expression, it does not interfer aldosterone secretion as PD123319 does. By Western blot, AT2 expression was not altered by every tested condition and the flow cytometry showed no obvious apoptosis. In conclusion, AT1, is the main receptor for aldosterone secretion through CYP11B2；AT2 is involved too, but there might be a “cross talk”between AT1 and AT2. It does not function via CYP11B2 totally or even with post-transcription, translation modification by AT1 or other pathways. So far, AT2 mediated cell apoptosis was not observed in H295R cell.目錄： 頁次 一､中文摘要 3 二､縮寫表 5 三、緒論 6 （一）背景 6 （二）實驗假說 9 （三）目的 10 四､研究方法與材料 11 （一）人類腎上腺皮質腫瘤H295R細胞株的取得和培養 15 （二）H295R細胞之實驗前處理 15 （三）Aldostrone濃度的測定 16 （四）ANGII對H295R細胞的aldosterone分泌影響-劑量與時間的效應 16 （五）AT1與AT2拮抗劑對aldosterone分泌的影響 17 （六）AT2刺激劑對aldosterone分泌的影響 17 （七）以反轉錄聚合酵素連鎖反應（RT-PCR）測定醛固酮合成酵素傳訊RNA的表現 18 （八）以西方墨點法（Western blot）測定AT2蛋白質表現 19 （九）流式細胞儀（Flow cytometry）之細胞凋亡分析 20 （十）統計方法 21 五､結果 22 （一）人類腎上腺皮質腫瘤H295R細胞株AT2之表現 22 （二）ANGII對aldosterone分泌的影響-劑量與時間的效應 22 （三）AT2刺激劑對aldosterone分泌的影響 23 （四）AT1與AT2拮抗劑對aldosterone分泌的影響 23 （五）ANGII對CYP11B2 mRNA表現之影響 23 （六）AT2刺激劑對CYP11B2 mRNA表現之影響 24 （七）AT1與AT2拮抗劑對CYP11B2 mRNA表現之影響 24 （八）AT1與AT2拮抗劑及AT2刺激劑對AT2蛋白質表現之影響 24 （九）流式細胞儀之細胞凋亡分析 24 六､討論 26 七､展望 30 八､論文英文簡述（Summary） 32 九､參考文獻 35 十､圖表 4

Effects of Low- and High-Flux Dialyzers on Oxidative Stress and Insulin Resistance

Background: Cardiovascular disease (CVD) is the leading cause of mortality in patients with end-stage renal disease( ESRD). The cornerstone of high CVD incidence in ESRD patients is endothelial dysfunction which results from inflammation, oxidative stress and insulin resistance. Although various modalities of hemodialysis (HD) have been presumed to exert different effects on oxidative stress and insulin resistance, solid evidence is still lacking. Methods : 40 ESRD patients undergoing HD were prospectively enrolled and divided randomly into two groups. Patients in each group received either F8 HPS (low-flux) (Group A) or FX80 ( high-flux ) (Group B) as HD dialyzers for 2 consecutive months. Diet pattern and medications were kept as usual in both groups to avoid considerable blood glucose change during study period. Blood samples were taken at the start and end of the study. Results: A total of 38 patients (18 and 20 for Groups A and B, respectively) completed the study . Within each group, there was no change in adiponectin, plasma 8-iso-prostaglandin F-2 alpha, high-sensitivity C- reactive protein, blood glucose and insulin after 2 months of treatment except a significant change of HOMA(IR) (p = 0. 02) in high-flux group. The significant change of HOMA(IR) between the two groups (p = 0.017) mainly results from the parallel change of insulin between the two groups (p = 0.03) . Conclusion: For patients receiving HD, the high-flux dialyzer with synthetic polysulfone membranes fails to provide a better anti-inflammatory or antioxidative effect than the low- flux dialyzer; however, the high-flux dialyzer does significantly improve insulin resistance in this short- term study. This result implies that the high-flux dialyzer might provide better cardiovascular protection than the low - flux dialyzer. Therefore, the low-flux dialyzer might be considered for patients who only need short-term HD therapy. Regarding patients under long-term maintenance HD therapy, a high-flux dialyzer might be the choice of dialyzer