自體顯性多囊腎病的基因異常分析及其和臨床表現型的相關

自體顯性多囊腎病中85﹪為PKD1 基因異常，15﹪為PKD2 基因異常。PKD1基因很大，有46 個exon，且在其染色體上有此基因exon1-33 的repeats（似偽基因），益增分析的困難度。近年來變性高效能液相色層分析已被應用在一些複雜基因的篩選分析。本計畫目的乃探討自體顯性多囊腎病的PKD1 基因異常及其和臨床表現型的相關。 本計畫所用的方法為（1）收集自體顯性多囊腎病患者並整理臨床資料（2）抽血並萃取DNA（3）作long range PCR 以克服有repeats 的困難（4）製備DHPLCamplicon（5）製備gradient 並跑DHPLC（6）片段溶解溫度分析及改善DHPLC條件（7）DNA 定序找出基因異常部位。 結果顯示106 位病人中80% 有高氮血症，診斷時平均Cr 為3.3mg/dL，進入末期腎病需透析時平均為55 歲。基因檢測的部份已完成前幾個步驟，即將有完整的結果。此後病人基因診斷後，可及早偵測及預防腎功能惡化，延緩病人進入透析。基因診斷自體顯性多囊腎病方法建立後，以後可能用於產前篩檢，減少此種病人；或運用於基因治療上。Eighty five percent autosomal dominant polycystic kidney disease （ADPKD）patients have PKD1 gene defect；the remaining 15﹪has PKD2 gene defect. The PKD1 transcript is large, encoded by 46 exons, and embedded in a complex duplicated genomic area. The exon 1 to exon 33 of PKD1 is reiterated at least five times on the same chromosome. Recently, a new semi-automated method has been introduced for mutation analysis in human gene, denaturing high performance liquid chromatography（DHPLC）. The purpose of this study is mutation analysis of ADPKD and its correlation to phenotype. The methods included（1）recruit ADPKD patients and collect clinical data,（2）draw blood and extract DNA,（3）do long range PCR,（4）prepare DHPLC amplicon,（5）prepare gradient and run DHPLC ,（6）Fragment melting profile analysis and optimization of DHPLC conditions,（7）DNA sequencing. The results showed that 80% of 106 ADPKD patients had azotemia and the mean Cr was 3.3mg/dL. The mean age in patients entering end stage renal disease was 55 years old. For gene analysis, several steps have been finished. We will complete the work and gain entire results soon. If we find the correlation of genotype and phenotype, we may early diagnose the ADPKD patient and use some methods to delay the progression of renal failure. If we establish the genetic diagnosis of ADPKD, it maybe used in prenatal diagnosis, even gene therapy in future

副甲狀腺素對腎小管鈉氫交換器及重碳酸鈉共同運送器的調節

副甲狀腺素（parathyroid hormone）抑制近端腎小管對磷、鈉、重碳酸根的再吸收，近端腎小管經由鈉氫交換器再吸收鈉及重碳酸根，而近端腎小管腔側膜鈉氫交換器為NHE-3，負鼠腎（OKP）細胞是腎小管細胞，可表現NHE-3。目前已知有多種因子可調節NHE-3，如酸可增加NHE-3 活性、NHE-3 mRNA 表達及NHE-3 蛋白質表現量。在腎小管細胞急性給予副甲狀腺素可抑制蛋白質激脢A 及C 而抑制NHE-3。本計畫的研究目的是探討慢性給予副甲狀腺素對腎小管NHE-3 活性、NHE-3 蛋白質量的調節。 我們的方法是以負鼠腎（OKP）細胞為研究模式。細胞內pH 測定利用螢光物質BCECF 在不同pH 值下，其螢光強度不同，而以光譜螢光儀來測定，鈉氫交換器活性的測定乃給予細胞酸負荷後，再加鈉後看其pH 恢復速率而求得。以各種濃度之副甲狀腺素慢性給予後觀察對腎小管鈉氫交換器活性的作用。此外，亦以西方轉漬法來看副甲狀腺素對腎小管細胞NHE-3 蛋白質表現量的調節。 結果發現在OKP 細胞給予副甲狀腺素後其鈉氫交換器活性並無顯著變化。同樣地，在OKP 細胞給予副甲狀腺素後其NHE3 蛋白質表現量也無顯著變化。由此推論副甲狀腺素的作用主要在鈣、磷，而對鈉及酸的調節可能較無作用。Parathyroid hormone（PTH）decreases phosphate, sodium and bicarbonate reabsorption by the proximal tubule. Proximal tubule transcellular NaCl and NaHCO3 reabsorption is mediated by an apical membrane Na/H exchanger,（NHE-3）and a basolateral Na/HCO3（NBC-1）.OKP cells, a cell line of renal proximal tubule, express NHE-3 . The regulating factors of NHE-3 are multiple. Acid incubation increases NHE-3 activity, NHE-3 mRNA abundance, NHE-3 protein abundance. PTH acutely inhibits NHE-3 in OKP cells via a dual signaling cascade involving protein kinase A and C. The purpose of this project is to study the chronic effect of PTH on NHE-3 activity, NHE-3 protein abundance. We used OKP cell as the experiment model. Continuous measurement of intracellular pH was accomplished in cells using pH-sensitive dye BCECF. Na/H exchanger activity was assayed as the initial rate of Na-dependent pHi recovery from an acid load. We checked chronic effect of PTH in different concentration on Na/H exchange activity in renal tubular cell. We checked the effect of PTH on NHE-3 protein abundance in OKP cell. The results showed that there was no significant change of NHE-3 activity or NHE-3 protein abundance by PTH in OKP cells. We think the major effect of PTH is for Ca & P, not for Na & acid

血管舒張劑抑負鼠近端賢小管細胞增生

Renal tubule cell hyperplasia and hypertrophy have been regarded as antecedents of tubulointerstitial fibrosis. Dipyridamole and pentoxifylline inhibit rat glomerular mesangial cell growth. The purpose of this study was to examine the effects of pentoxifylline and other vasodilators on opossum kidney proximal tubular (OKP) cell growth. A modified 3-(4, 5-dimethylthiazil-2-yl)-2, 5- diphenyltetrazolium bromide assay was used to evaluate cell proliferation in OKP cells. Pentoxifylline caused a dose- dependent inhibition of serum-stimulated proliferation of OKP cells: inhibition was 17%, 25%, and 37% at 0.03 mg/mL, 0 .1 mg/mL, and 0.3 mg/mL, respectively. Dipyridamole 1.7 mg/L and 6 mg/L resulted in 34% and 46% inhibition, respectively . Verapamil 10^(-6) M, 10^(-5) M, and 10 ^(-4) M resulted in 34%, 46%, and 67% inhibitory, respectively. These inhibitory effects may contribute to renal protection or retardation of renal disease progression

The Outcome of Patients With 2 Different Protocols of Do-Not-Resuscitate Orders An Observational Cohort Study

Lack of clarity about the exact clinical implications of do-not-resuscitate (DNR) has caused confusion that has been addressed repeatedly in the literature. To provide improved understanding about the portability of DNR and the medical care provided to DNR patients, the state of Ohio passed a Do-Not-Resuscitate Law in 1998, which clearly pointed out 2 different protocols of do-not-resuscitate: DNR comfort care (DNRCC) and DNR comfort care arrest (DNRCC-Arrest). The objective of this study was to examine the outcome of patients with the 2 different protocols of DNR orders. This is a retrospective observational study conducted in a medical intensive care unit (MICU) in a hospital located in Northeast Ohio. The medical records of the initial admissions to the MICU during data collection period were concurrently and retrospectively reviewed. The association between 2 variables was examined using Chi-squared test or Student's t-test. The outcome of DNRCC, DNRCC-Arrest, and No-DNR patients were compared using multivariate logistic regression analysis. The total of 188 DNRCC-Arrest, 88 DNRCC, and 2051 No-DNR patients were included in this study. Compared with the No-DNR patients, the DNRCC (odds ratio =20.77, P<0.01) and DNRCC-Arrest (odds ratio =3.69, P<0.01) patients were more likely to die in the MICU. Furthermore, the odds of dying during MICU stay for DNRCC patients were 7.85 times significantly higher than that for DNRCC-Arrest patients (odds ratio =7.85, P<0.01). Given Do-Not-Resuscitate Law in Ohio, we examined the outcome of the 2 different protocols of DNR orders, and to compare with the conventional DNR orders. Similar to conventional DNR, DNDCC and DNRCC-Arrest were both associated with the increased risk of death. Patients with DNRCC were more likely to be associated with increased risk of death than those with DNRCC-Arrest