Department of Nephrology, West China Hospital of Sichuan University, Chengdu, China.
Department of Nephrology and Rheumatology, Yongchuan Hospital of Chongqing Medical University, Chongqing, China.
Am J Physiol Renal Physiol. 2020 Oct 1;319(4):F664-F673. doi: 10.1152/ajprenal.00292.2020. Epub 2020 Jul 27.
Tubular changes contribute to the development of renal pathologies in diabetic kidney disease (DKD), including interstitial fibrosis. It is unclear how tubular cells relay signals to interstitial fibroblasts. Recently, exosomes have been recognized as crucial mediators of intercellular communication. We hypothesized that exosomes secreted from tubular cells may stimulate fibroblasts for interstitial fibrosis in DKD. In this study, we isolated and purified exosomes from the renal cortex of DKD mice and high glucose-treated mouse proximal tubular cells. Compared with nondiabetic mice, exosome secretion in kidney tissues decreased in DKD mice. Likewise, high glucose incubation reduced exosome secretion in mouse kidney proximal tubular BUMPT cells. To study the effect of tubular cell exosomes on fibroblasts, exosomes from BUMPT cells were added to renal fibroblast NRK-49F cell cultures. Notably, exosomes from high glucose conditioned BUMPT cells induced higher proliferation, significant morphological change, and substantial production of fibronectin, α-smooth muscle actin, and collagen type Ι in NRK-49F fibroblasts. Proteomics analysis was further performed to profile the proteins within tubular cell exosomes. Interestingly, 22 proteins were found to be differentially expressed between tubular exosomes derived from high glucose conditioned cells and those from normal glucose conditioned cells. Cytoscape analysis suggested the existence of two protein-protein interaction networks in these exosomal differentially expressed proteins. While one of the protein-protein interaction networks comprised enolase 1 (Eno1), heat shock protein family A member 8 (Hspa8), thioredoxin 1 (Txn1), peptidylprolyl isomerase A (Ppia), phosphoglycerate kinase 1 (Pgk1), DNA topoisomerase II-β (Top2b), and β-actin (Actb), the other had the family proteins of human leucocyte antigen F (Ywhag), a component of the ND10 nuclear body (Ywhae), interferon regulatory factor-8 (Ywhaq), and human leucocyte antigen A (Ywhaz). Gene expression analysis via Nephroseq showed a correlation of Eno1 expression with DKD clinical manifestation. In conclusion, DKD is associated with a decrease in exosome secretion in renal tubular cells. Exosomes from high glucose conditioned tubular cells may regulate the proliferation and activation of fibroblasts, contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.
管状变化有助于糖尿病肾病 (DKD) 中的肾脏病变发展，包括间质纤维化。目前尚不清楚管状细胞如何将信号传递给间质成纤维细胞。最近，外泌体已被认为是细胞间通讯的重要介质。我们假设来自管状细胞的外泌体可能会刺激 DKD 中的成纤维细胞引起间质纤维化。在这项研究中，我们从 DKD 小鼠和高糖处理的小鼠近端肾小管细胞的肾皮质中分离和纯化了外泌体。与非糖尿病小鼠相比，DKD 小鼠肾脏组织中外泌体的分泌减少。同样，高糖孵育会降低小鼠肾脏近端肾小管 BUMPT 细胞中外泌体的分泌。为了研究管状细胞外泌体对成纤维细胞的影响，我们将 BUMPT 细胞的外泌体添加到肾成纤维细胞 NRK-49F 细胞培养物中。值得注意的是，高糖条件下 BUMPT 细胞的外泌体诱导 NRK-49F 成纤维细胞的增殖增加、形态显著改变、以及纤连蛋白、α-平滑肌肌动蛋白和胶原 I 型的大量产生。进一步进行蛋白质组学分析以描绘管状细胞外泌体中的蛋白质。有趣的是，在高糖条件下 BUMPT 细胞衍生的外泌体与正常葡萄糖条件下衍生的外泌体之间发现了 22 种差异表达蛋白。Cytoscape 分析表明，这些外泌体差异表达蛋白中存在两个蛋白质-蛋白质相互作用网络。其中一个蛋白质-蛋白质相互作用网络包含烯醇酶 1 (Eno1)、热休克蛋白家族 A 成员 8 (Hspa8)、硫氧还蛋白 1 (Txn1)、肽基脯氨酰异构酶 A (Ppia)、磷酸甘油酸激酶 1 (Pgk1)、DNA 拓扑异构酶 II-β (Top2b) 和 β-肌动蛋白 (Actb)，另一个网络则包含人类白细胞抗原 F (Ywhag)、核体 ND10 的组成部分 (Ywhae)、干扰素调节因子-8 (Ywhaq) 和人类白细胞抗原 A (Ywhaz) 的家族蛋白。通过 Nephroseq 进行的基因表达分析显示 Eno1 表达与 DKD 临床表现相关。总之，DKD 与肾小管细胞中外泌体分泌减少有关。高糖条件下肾小管细胞的外泌体可能调节成纤维细胞的增殖和激活，从而参与导致 DKD 肾间质纤维化的旁分泌信号机制。
