Division of Hematology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, USA.
Blood Cells Mol Dis. 2011 Aug 15;47(2):95-9. doi: 10.1016/j.bcmd.2011.04.007. Epub 2011 May 14.
The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes - KCC1, KCC3, and KCC4 - are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20-30min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5-29g/dl in SS reticulocytes compared to 25-26.5g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3mmolK/kgHb/min in SS reticulocytes, compared to 2.9-7.2mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.
钾氯协同转运蛋白（KCC）是电中性阳离子氯离子家族的成员之一，存在于多种组织中，参与跨上皮离子转运和细胞内离子含量和细胞体积的调节。我们之前已经表明，四个 KCC 基因中的三个 - KCC1、KCC3 和 KCC4 - 在红细胞（RBC）中表达（Exp. Hem. 33:624, 2005）。在功能上，KCC 介导网织红细胞的体积减少，从而建立成熟 RBC 更高的细胞血红蛋白浓度（CHC）。KCC 在网织红细胞中的活性较高，并且随着年龄的增长而降低。与正常（AA RBC）相比，镰状血红蛋白（SS RBC）中的 RBC 中的 KCC 活性升高，部分原因是 SS 血液中的网织红细胞增多。然而，我们已经证明，SS 网织红细胞的 KCC 活性异常调节，导致与 AA 网织红细胞相比，KCC 激活后 CHC 增加（Blood 104:2954, 2004; Blood 109:1734, 2007）。这些发现表明 KCC 是 SS RBC 脱水的一个因素，导致 Hb S 浓度升高，并增强 Hb S 聚合和溶血。由于 KCC 活性与细胞年龄相关，因此对具有不同数量的网织红细胞或年轻的非网织红细胞的血液样本进行标准通量测量是不可比的。Advia 自动化细胞计数器测量网织红细胞的细胞体积（MCV）和细胞血红蛋白浓度（CHC），这是一个年龄定义的细胞群体，因此避免了 SS 和 AA 血液样本中网织红细胞计数的差异。在这项研究中，新鲜血液中的网织红细胞 CHC 测量结果清楚地区分了 AA 和 SS 细胞之间的差异，反映了 SS 网织红细胞的体内脱水，尽管存在显著的个体间变异，并且两组的 CHC 分布重叠。体外通过使用制霉菌素方法使细胞肿胀来激活 KCC 后，最初的网织红细胞 MCV 和 CHC 随时间的变化用于估计 KCC 介导的通量率，假设变化与等渗 KCl 运动有关。在 20-30 分钟后，达到并维持最终的稳态 MCV/CHC（设定点），反映了转运蛋白的失活。SS 网织红细胞的 CHC 设定点为 26.5-29g/dl，而 AA 网织红细胞的 CHC 设定点为 25-26.5g/dl，反映了 SS 细胞的异常调节。这些结果在同一个体中随时间是可重复的。从 CHC 中得出的 KCC 通量在 SS 网织红细胞中为 5-10.3mmolK/kgHb/min，而在 AA 网织红细胞中为 2.9-7.2mmolK/kgHb/min。这种控制细胞年龄的 RBC 群体中 KCC 活性的测量方法将有助于进一步研究 KCC 活性与镰状细胞病中的表型或遗传变异性的相关性。
