適量的使用紅血球生成刺激劑與延緩末期腎病變患者開始透析有關

Abstract

慢性腎臟病(chronic kidney disease, CKD)在全世界廣為盛行，而貧血(anemia)又是慢性腎臟病患者的常見共病症。在健康成人，正常的紅血球生成(erythropoiesis)是由紅血球生成素(erythropoietin, EPO)所促成；而紅血球生成素是接受第二型低氧誘導因子(hypoxia-inducible factor-2, HIF-2)調控，由腎臟內的血管周邊細胞(pericyte)與纖維母細胞(fibroblast)所製造。當腎臟發生纖維化(fibrosis)現象時，腎臟裡的血管周邊細胞會轉變為成肌纖維細胞(myofibroblast)，同時喪失製造紅血球生成素的能力。當紅血球生成素製造減少，合併鐵代謝異常(disturbance of iron homeostasis)與尿毒環境(uremic milieu)影響時，將會造成慢性腎臟病患者的貧血現象。紅血球生成刺激劑(erythropoiesis-stimulating agent, ESA)包括有基因重組人類紅血球生成素(recombinant human EPO)與紅血球生成類似物(EPO analogues)，是目前治療慢性腎臟病貧血的主要藥物。然而，近年來的大型臨床試驗證實了使用高劑量紅血球生成刺激劑治療會帶來死亡與心血管疾病的風險；觀察性研究也顯示高濃度的內生性紅血球生成素與糖尿病患者、心衰竭患者、腎臟移植病患的死亡有相關。但許多動物實驗卻顯示使用低劑量紅血球生成刺激劑治療對腎臟有保護效果。我們提出假說來解釋上述現象。我們認為紅血球生成刺激劑的劑量與體內紅血球生成素濃度是決定病患接受治療後預後(outcome)的關鍵因子。明確而言我們認為，在體內紅血球生成素濃度過低(absolute EPO deficiency)的病患使用低劑量紅血球生成刺激劑治療，可以改善腎臟預後(renal outcome)；而高劑量紅血球生成刺激劑治療與過度提升體內紅血球生成素濃度會導致不良預後。為了驗證這個假說，首先我們設計臨床實驗來分析紅血球生成刺激劑的劑量與腎臟預後的關係，接下來我們建立體內具有高濃度紅血球生成素的動物模型(animal models)來檢視紅血球生成素濃度對於預後的影響。在我們的臨床實驗中，總共有423位病患追蹤了約1.37年時間，有使用紅血球生成刺激劑的病患(ESA user)接受了平均每個月9,600 ± 5,500 U的劑量治療。有使用紅血球生成刺激劑的病患血色素(hemoglobin)的變化為每年下降0.29 ± 2.19 g/dL；而沒有使用紅血球生成刺激劑的病患則是每年下降0.99 ± 2.46 g/dL，兩者差距達統計意義(P = 0.038)。而平均估計腎絲球過濾率(estimated glomerular filtration rate, eGFR)的每年下降程度則無統計差異。為了檢視紅血球生成刺激劑與預後的關係，我們建置了時間相依共變數迴歸模型(time-dependent Cox regression model)，以校正每個月的平均估計腎絲球過濾率、每個月的血色素、以及其他重要變項。經過校正後，紅血球生成刺激劑的使用與透析延緩(deferred dialysis initiation)開始有關(hazard ratio 0.63, 95% confidence interval 0.42-0.93, P = 0.021)。當把每個月的紅血球生成刺激劑劑量也考慮在模型中，依然可以得到相同結論。在我們的動物實驗中，我們建立了可以在特定時間後(inducible)，於全身(non-selective)或特定於血管周邊細胞上(pericyte-specific)，進行低氧誘導因子的穩定表現(stabilization)的動物模型。在Tg(UBC-CreERT2);VhlFlox/Flox 與Tg(UBC-CreERT2);Phd2Flox/Flox這兩個全身低氧誘導因子穩定表現的小鼠品系，給予三苯甲氨(tamoxifen)後可以觀察到體內紅血球生成素濃度大量提升的現象。在Tg(UBC-CreERT2);Phd2Flox/Flox小鼠，腎臟內的血管周邊細胞與纖維母細胞可能是主要製造紅血球生成素的細胞；而在Tg(UBC-CreERT2);VhlFlox/Flox 小鼠可以觀察到死亡、體重減輕、與脾臟進行大量紅血球生成(severe splenic erythropoiesis)現象。在血管周邊細胞上低氧誘導因子穩定表現的小鼠品系，Gli1CreERT2/+;VhlFlox/Flox這個品系可以在給予三苯甲氨10週後，觀察到體重逐漸下降、脾臟大量進行紅血球生成、以及腎臟肝臟肺臟等多個器官充血(congestion)現象；而在Gli1CreERT2/+;Phd2Flox/Flox這個品系則可以觀察到類似Tg(UBC-CreERT2);VhlFlox/Flox 小鼠的紅血球增多(polycythemia)與脾臟紅血球生成現象。總結而言，我們的臨床分析顯示保守的低劑量使用紅血球生成刺激劑與延緩透析開始有關，而我們的動物實驗則顯示大量提升體內紅血球生成素濃度與死亡及體重減輕有關。然而，因為我們的臨床分析是回顧性研究，動物實驗結果無法完全推論適用於臨床，故本研究結論尚未完全確認不宜過度解釋。未來如果有臨床隨機對照試驗(randomized control trial)，可以設計來分析紅血球生成刺激劑的劑量與體內紅血球生成素濃度對於預後的影響，也許可以進一步提供慢性腎臟病貧血患者除了血色素外更好的治療標的(treatment targets)與指引。

Chronic kidney disease (CKD) incurs a heavy disease burden globally and anemia is highly prevalent in patients with CKD. In the healthy adult, normal erythropoiesis is stimulated by erythropoietin (EPO). The EPO is produced by renal pericytes and fibroblasts under the control of hypoxia-inducible factor (HIF)-2. In the fibrotic kidney, renal pericytes may transform into myofibroblasts and lose the ability of EPO production. Decreased EPO production, together with disturbance of iron homeostasis and uremic milieu, contribute to the development of anemia in CKD. Erythropoiesis-stimulating agents (ESAs), including recombinant human EPO and EPO analogues, are the mainstay of treatment for anemia in CKD. However, recent clinical trials revealed the risks for death and adverse cardiovascular outcomes with high dose ESA treatment. On the contrary, plenty animal studies suggested the renoprotective effects with low dose ESA treatment. In addition, observational studies showed association between elevated endogenous EPO level and mortality in diabetes, heart failure, and renal transplant patients. We hypothesized that the dose of ESA and the level of serum EPO may be critical factors determining outcomes. Specifically, low dose ESA treatment may provide benefits for renal outcomes in CKD patients with absolute EPO deficiency. High dose ESA treatment and inappropriately elevated serum EPO level may result in adverse outcomes. To test the hypothesis, we first designed a clinical study to analyze the association between the doses of ESA and renal outcomes. Second, we created animal models with highly elevated serum EPO levels to exam the outcomes. In the clinical study, totally 423 patients were followed up for a median of 1.37 year. The standardized monthly ESA dose (per person) in ESA users was 9,600 ± 5,500 U of epoetin beta. Annual changes of hemoglobin were –0.29 ± 2.19 and –0.99 ± 2.46 g/dL in ESA users and ESA non-users, respectively (P = 0.038). However, annual estimated glomerular filtration rate (eGFR) decline rates were not different between ESA users and non-users. A time-dependent Cox regression model, adjusted for monthly levels of eGFR and hemoglobin, was constructed to investigate the association between ESA and outcome. After adjustment, ESA use was associated with deferred dialysis initiation (hazard ratio 0.63, 95% confidence interval 0.42-0.93, P = 0.021). The protective effect remained when the monthly ESA doses were incorporated. In the animal study, we created animal models of inducible non-selective HIF stabilization or inducible pericyte-specific HIF stabilization. We confirmed that in non-selective HIF stabilization mice strains, Tg(UBC-CreERT2);VhlFlox/Flox and Tg(UBC-CreERT2);Phd2Flox/Flox, highly elevated serum EPO levels developed after tamoxifen induction. Renal pericyes or fibroblasts might be the major source of EPO producing cells in Tg(UBC-CreERT2);Phd2Flox/Flox mice. Mortality developed in Tg(UBC-CreERT2);VhlFlox/Flox mice, along with progressive body weight loss and severe splenic erythropoiesis. In pericyte-specific HIF stabilization mice strains, Gli1CreERT2/+;VhlFlox/Flox mice developed progressive body weight loss, severe splenic erythropoiesis, and congestion in the kidney, liver, and lung 10 weeks after tamoxifen induction. Gli1CreERT2/+;Phd2Flox/Flox mice developed polycythemia and splenic erythropoiesis similar to Tg(UBC-CreERT2);VhlFlox/Flox mice. In conclusion, restricted use of ESA was shown to be associated with deferred dialysis initiation in our clinical study, while highly elevated serum EPO level was associated with mortality and body weight loss in our animal study. However, retrospective design of the clinical study and the difference between human and mice weakened the robustness of the conclusion. Future randomized control trials designed to test the effects of ESA dose and serum EPO level on outcomes may be considered to find better treatment targets other than hemoglobin level for CKD patients with anemia.