Marković-Lipkovski Jasmina, Životić Maja, Müller Claudia A, Tampe Björn, Ćirović Sanja, Vještica Jelena, Tomanović Nada, Zeisberg Michael, Müller Gerhard A
Institute of Pathology, Medical Faculty, University of Belgrade, Belgrade, Serbia.
Section for Transplantation Immunology and Immunohematology, ZMF, University Medical Clinic, Eberhard Karls University, Tübingen, Germany; Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg-August University, Göttingen, Germany.
PLoS One. 2015 Sep 1;10(9):e0137028. doi: 10.1371/journal.pone.0137028. eCollection 2015.
Rare neural cell adhesion molecule (NCAM) positive cells have been previously described within the normal human adult kidney interstitium, speculating that they could increase in the interstitium with incipient interstitial renal fibrosis (IRF). In the present study, among 93 biopsy samples of various kidney diseases, NCAM+ interstitial cells were detected in 62.4% cases. An increased number of NCAM+ cells was significantly observed only in incipient IRF compared to normal renal tissues and advanced IRF stages (p<0.001), independently of underlying diseases (p = 0.657). All three major NCAM isoforms' RT-PCR bands were visible either in normal or in kidneys with incipient IRF, albeit their mRNA expression levels measured by qRT-PCR were different. Applying qRT-PCR on pure NCAM+ cells population, obtained by laser capture microdissection, significant mRNA over-expression of NCAM140kD isoform was found in NCAM+ cells within incipient IRF (p = 0.004), while NCAM120kD and NCAM180kD isoforms were not changed significantly (p = 0.750; p = 0.704; respectively). Simultaneously, qRT-PCR also showed significant αSMA (p = 0.014) and SLUG (p = 0.004) mRNAs up-regulation within the NCAM+ cells of incipient IRF, as well as highly decreased matrix metalloproteinases (MMP) -2 and -9 mRNAs (p = 0.028; p = 0.036; respectively). However, using double immunofluorescence MMP-9 could still be detectable on the protein level in rare NCAM+ cells within the incipient IRF. Further characterization of NCAM+ cells by double immunofluorescent labeling revealed their association with molecules involved in fibrosis. Fibroblast growth factor receptor 1 (FGFR1) and α5β1 integrin were extensively expressed on NCAM+ cells within the incipient IRF areas, whereas human epididymis protein-4 (HE4) was found to be present in few NCAM+ cells of both normal and interstitium with incipient fibrosis. Heterogeneity of NCAM+ interstitial cells in normal and incipient IRF, concerning molecules related to fibrosis and variable expression of NCAM isoforms, could suggest diverse role of NCAM+ cells in homeostasis and in regulation of renal fibrosis in diseased kidneys.
先前已在正常成人肾脏间质中发现罕见的神经细胞黏附分子（NCAM）阳性细胞，推测随着早期肾间质纤维化（IRF）的发生，它们在间质中的数量可能会增加。在本研究中，在93份各种肾脏疾病的活检样本中，62.4%的病例检测到NCAM+间质细胞。与正常肾组织和晚期IRF阶段相比，仅在早期IRF中观察到NCAM+细胞数量显著增加（p<0.001），与潜在疾病无关（p = 0.657）。正常肾脏或早期IRF肾脏中均可看到所有三种主要NCAM异构体的RT-PCR条带，尽管通过qRT-PCR测量的它们的mRNA表达水平有所不同。对通过激光捕获显微切割获得的纯NCAM+细胞群体进行qRT-PCR分析，发现在早期IRF中的NCAM+细胞中NCAM140kD异构体的mRNA有显著过表达（p = 0.004），而NCAM120kD和NCAM180kD异构体没有显著变化（分别为p = 0.750；p = 0.704）。同时，qRT-PCR还显示早期IRF的NCAM+细胞中αSMA（p = 0.014）和SLUG（p = 0.004）的mRNA上调，以及基质金属蛋白酶（MMP）-2和-9的mRNA高度降低（分别为p = 0.028；p = 0.036）。然而，使用双重免疫荧光法仍可在早期IRF中罕见的NCAM+细胞的蛋白质水平上检测到MMP-9。通过双重免疫荧光标记对NCAM+细胞进行进一步表征，揭示了它们与参与纤维化的分子之间的关联。在早期IRF区域的NCAM+细胞上广泛表达成纤维细胞生长因子受体1（FGFR1）和α5β1整合素，而在正常和早期纤维化间质的少数NCAM+细胞中发现了人附睾蛋白4（HE4）。正常和早期IRF中NCAM+间质细胞在与纤维化相关的分子以及NCAM异构体的可变表达方面的异质性，可能表明NCAM+细胞在患病肾脏的稳态和肾纤维化调节中具有不同作用。
