From the Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, LMU München, Germany (Y.Y., D.J., E.T.A.P., L.S.M., H.S., F.B., M.R., T.A.W.).
Endocrine Division, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
Hypertension. 2019 Oct;74(4):809-816. doi: 10.1161/HYPERTENSIONAHA.119.13476. Epub 2019 Aug 26.
Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (=0.435, =0.007), a negative correlation was noted in adenomas with no mutation detected (=-0.548, =0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference <0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (<0.0001), G151E (=0.008), and L168R (<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel.
醛固酮瘤中 KCNJ5 G 蛋白偶联内向整流钾通道的体细胞突变是原发性醛固酮增多症的一个原因。这些突变导致醛固酮过多，但它们在细胞生长中的作用尚不清楚。我们的目的是确定 KCNJ5 突变在肾上腺细胞增殖和凋亡中的作用。Ki67 增殖指数与 KCNJ5 突变的醛固酮瘤直径呈正相关（=0.435，=0.007），与未检测到突变的腺瘤呈负相关（=-0.548，=0.023）。用 cumate 诱导的野生型或突变型 KCNJ5 稳定表达建立人肾上腺皮质细胞系。与对照细胞相比，低水平诱导 KCNJ5-T158A 表达可诱导细胞增殖增加（=0.009），但增加诱导可消除这种差异。KCNJ5-G151R 没有明显的增殖作用，但 KCNJ5-G151E 和 L168R 突变均导致细胞增殖减少（与对照细胞相比差异<0.0001，两种比较）。在测试的条件下，T158A 对细胞凋亡没有影响，但 G151R 表达增加时凋亡增加（<0.0001），G151E（=0.008）和 L168R（<0.0001）。我们生成了一种特异性的 KCNJ5 单克隆抗体，用于免疫组化，以证明在没有突变的腺瘤和腺瘤旁的肾小球带中均有强烈的 KCNJ5 表达，无论基因型如何，以及在产生醛固酮的细胞簇中也是如此。KCNJ5 和 CYP11B2（醛固酮合酶）的双重免疫荧光染色显示，在 KCNJ5 突变的醛固酮瘤中，与 CYP11B2 阴性细胞相比，CYP11B2 阳性细胞中的 KCNJ5 免疫染色明显减少。综上所述，这些发现支持这样一种观点，即 KCNJ5 突变的细胞生长效应取决于突变通道的表达水平。
