DKK3於原發性⾼醛固酮症的表現和其可能之應⽤

Abstract

原發性高醛固酮症是最常見的次發性高血壓原因之一。病患體內的醛固酮因基因遺傳或不明原因體細胞突變而自主性地過度分泌，持續活化鹽皮質激素受體，造成水分滯留、氧化壓力增加、以及器官發炎和纖維化反應的惡化。高醛固酮對腎臟的早期傷害因血液容積和腎臟血流灌注的增加續所造成的高腎絲球過濾率不容易被察覺。 Wnt/β-catenin訊息傳遞路徑是上皮細胞間質化還有腎臟纖維化的過程中非常重要的路徑。Dickkopf (DKK) 蛋白質是腎小管上皮細胞承受壓力時的Wnt/β-catenin訊息傳遞路徑的調控因子，其中DKK3能促進Wnt/β-catenin的訊息傳遞。我們查詢腎臟精準醫學計畫(KPMP) 研究發現慢性腎臟病患的組織中DKK3會增加，且過去許多研究也表示急慢性腎臟病患的尿液中DKK3的增加跟病患日後的腎功能惡化有關。 高醛固酮對於腎臟持續的傷害會造成不可逆的後果，因此我們希望能在更早期的時候偵測到腎臟的傷害。我們比較原發性高醛固酮症和高血壓患者的尿液中的DKK3數值，並且觀察DKK3數值是否跟病患的長期預後有明顯的相關。同時，我們也執行動物研究來釐清DKK3在原發性高醛固酮症與腎功能惡化之間扮演的角色。 這篇研究共有163位高血壓病患參與，原發性高醛固酮症（primary aldosteronism, PA）的病患佔73.6%（n=120，年紀的中位數：53.8歲，男性：48.3%）而原發性高血壓（essential hypertension, EH）的患者佔了26.4%（n=43，年紀的中位數：51.9歲，男性：46.5%）。PA病患比EH病患使用更多的高血壓藥物（2.1±1.2 vs. 1.1±1.0，p<0.05）。儘管PA和EH病患的尿液中的DKK3並沒有明顯差別（2006 pg/ml [IQR: 909-5200 pg/ml] vs. 4255 pg/ml [IQR: 2494-5468 pg/ml]，p=0.64），我們發現治療和追蹤一年後的腎功能變的比較差的PA病患的尿液中有比較高的DKK3（3253 pg/ml [IQR: 1764-8715 pg/ml] vs. 1712 pg/ml [IQR: 845-3770 pg/ml]，p=0.04）。 動物研究共使用了十隻小鼠。所有的小鼠都在實驗的一開始接受單邊腎臟切除（unilateral nephrectomy）和植入皮下注射幫浦（subcutaneous osmotic minipump），並且每天飲用生理食鹽水。其中五隻小鼠會每天被注射醛固酮（aldosterone，600 μg/kg/day），另外五隻只接受不含醛固酮的酒精溶劑（5% EtOH）。轉錄組定序實驗顯示體內醛固酮增加的小鼠的腎臟會有更多的的DKK3基因表現 [log2(fold change) = 1.67，p<0.01]。腎臟組織的染色（包含一般化學和免疫組織化學染色法）顯示體內醛固酮增加的小鼠會有更多的DKK3（36.4±5.1% vs 16.4±4.4%，p<0.05），以及上皮間質轉化（纖維黏連蛋白fibronectin：14.4±2.6% vs 4.0±0.6%，p<0.05）和腎臟纖維化（膠原蛋白collagen：17.6±3.0% vs 2.6±0.5%，p<0.05）的蛋白表現。PA病患的腎臟組織也比EH病患有更嚴重的腎臟纖維化（膠原蛋白H-score：132±2.1 vs 148±2.3，p=0.005）以及更明顯的DKK3表現（DKK3 H-score：138±0.8 vs 158±0.3，p=0.04）。 我們發現尿液DKK3是亞臨床腎傷害指標，跟腎臟預後不佳的PA病患之間的關聯性。透過動物實驗和轉錄組定序實驗，我們檢測到醛固酮關係DKK3的表現以及跟後續腎臟惡化相關的蛋白的表現。

Background: Primary aldosteronism (PA) is the leading cause of secondary hypertension, characterized by autonomous aldosterone secretion and persistent activation of aldosterone synthase. This activation leads to volume expansion, oxidative stress, and increased inflammatory and fibrotic responses. However, renal impairment is often overlooked in the early stages of PA due to glomerular hyperfiltration. The Wnt/β-catenin signaling pathway plays a crucial role in epithelial-mesenchymal transition and the progression of kidney fibrosis. Dickkopf 3 (DKK3) protein modulates Wnt/β-catenin signaling under renal tubular cell stress. The role of DKK3 in aldosterone-induced kidney injury, however, remains inconclusive. Hypothesis: We hypothesized that PA patients will have higher urinary levels of DKK3 compared to those with essential hypertension and similar renal function. Elevated urinary DKK3 levels were expected to correlate with adverse kidney events in PA patients. Furthermore, we aimed to elucidate the mechanism underlying DKK3 production and its role in mediating subclinical kidney damage in PA patients. Methods: In this cross-sectional study, data from patients with confirmed PA (n = 120) and essential hypertension (EH, n = 43) were analysed. Demographic data and relevant clinical parameters were collected and compared between PA and EH patients, as well as among PA patients with different levels of urinary DKK3. Hypertensive mice were generated by giving drinking water with sodium chloride, and kidney stress was induced by unilateral nephrectomy on the first day of experiment. Subcutaneous osmotic minipump was implanted to administer either aldosterone or vehicle (5% ethanol) to create animals with aldosteronism. Histology and immunohistochemistry staining were performed to visualize the presence of kidney fibrosis, epithelial mesenchymal transition as well as DKK3. Transcriptome analysis was also carried out to figure out the difference in gene expression between aldosterone-infused mice and controls. Results: PA patients (n=120, median age: 53.8 years, male: 48.3%) required a higher number of antihypertensive medications compared to those diagnosed with EH (n=43, median age: 51.9 years, male: 46.5%) (2.1±1.2 vs. 1.1±1.0, p<0.05). DKK3 were found to be similar between PA patients and EH patients (2006 pg/ml [IQR: 909-5200 pg/ml] vs. 4255 pg/ml [IQR: 2494-5468 pg/ml], p=0.64). However, within the PA group, urinary DKK3 levels were significantly elevated among patients with more than 20% of estimated glomerular filtration rate (eGFR) dip at one year after targeted treatment compared to those with less eGFR decline (3253 pg/ml [IQR: 1764-8715 pg/ml] vs. 1712 pg/ml [IQR: 845-3770 pg/ml], p=0.04). In the animal study, histology and immunohistochemistry of the kidney tissues revealed remarkable increment of the areas with epithelial-mesenchymal transition (fibronectin: 14.4±2.6% vs 4.0±0.6%, p<0.05), interstitial fibrosis (collagen: 17.6±3.0% vs 2.6±0.5%, p<0.05) and DKK3 (36.4±5.1% vs 16.4±4.4%, p<0.05) staining in the aldosterone-infused mice. Transcriptome analysis showed DKK3 gene expression increased after aldosterone treatment [log2(fold change) = 1.67, p < 0.01]. In addition, kidney tissues of the PA patients also had more severe interstitial fibrosis [collagen H-score: 132±2.1 vs 148±2.3, p=0.005] and DKK3 [H-score: 138±0.8 vs 158±0.3, p=0.04)] expression compared to patients with essential hypertension. Conclusion and Future Perspectives: Our study found comparable urinary DKK3 levels between PA and EH, but baseline urinary DKK3 was associated with post-treatment kidney function decline, suggesting it as a potential subclinical biomarker for monitoring PA-related kidney outcomes. Animal study and transcriptome analysis showed elevation of kidney specific DKK3 expression in concomitant with the kidney fibrosis and epithelial mesenchymal transition.