Krawczyk Krzysztof M, Hansson Jennifer, Nilsson Helén, Krawczyk Katarzyna K, Swärd Karl, Johansson Martin E
Department of Translational Medicine, Clinical Pathology, Lund University, SUS Malmö, Jan Waldenströms gata 59, SE-20502, Malmö, Sweden.
Department of Laboratory Medicine, Lund University, Lund, Sweden.
BMC Nephrol. 2017 Oct 24;18(1):320. doi: 10.1186/s12882-017-0738-8.
Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined.
Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or HO. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed.
In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (HO), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules.
Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.
小窝是一种膜内陷结构，大小为50 - 100纳米。这些细胞器由小窝蛋白和窖蛋白相关蛋白组成，对细胞信号传导和存活至关重要。小窝在人类肾脏中发挥的作用尚未完全明确。此前已有关于病变肾小管中诱导小窝蛋白-1/CAV1的描述，但其相关机制仍有待确定。
对健康和萎缩的人类肾脏进行小窝蛋白（小窝蛋白1 - 3和窖蛋白相关蛋白1 - 4）染色，并通过电子显微镜检查。在分离的近端肾小管和原代肾上皮细胞中研究小窝蛋白的诱导情况。这些细胞分别用缺氧或过氧化氢进行刺激。原代肾小管细胞还进行了巨核母细胞白血病1（MKL1）的病毒过表达以及用MKL1抑制剂CCG - 1423抑制MKL1。通过蛋白质免疫印迹法检测癌细胞侵袭抑制因子（SCAI）和细丝蛋白A（FLNA），以研究MKL1活性的假定共调节因子。最后，对小窝蛋白和MKL1的mRNA表达进行相关生物信息学研究。
在健康肾脏中，小窝蛋白由鲍曼囊的壁层上皮细胞（PEC）、内皮细胞和血管平滑肌表达。电子显微镜证实了PEC中有小窝。近端肾小管中未见表达。相比之下，萎缩的近端肾小管中表达小窝蛋白。原代肾小管上皮细胞培养物中也诱导出小窝蛋白。缺氧或自由基应激（过氧化氢）并未增强其表达，但对MKL1的抑制敏感。MKL1的病毒过表达诱导了小窝蛋白-1/CAV1、小窝蛋白-2/CAV2和SDPR/窖蛋白相关蛋白2。在肾脏组织中，MKL1的mRNA水平与小窝蛋白-1/CAV1、小窝蛋白-2/CAV2以及典型的MKL1靶标腱生蛋白C（TNC）的mRNA水平相关，MKL1共激活因子FLNA的情况也是如此。以TNC作为MKL1活性的读数进行共染色显示，在PEC以及近端肾小管的萎缩节段中，TNC与小窝蛋白-1/CAV1的表达有重叠。
我们的研究结果支持以下观点，即MKL1有助于健康肾脏中小窝蛋白的表达，并在肾损伤时协调肾小管小窝蛋白的诱导。
