Repetti Robert, Majumder Nomrota, De Oliveira Karin Carneiro, Meth Jennifer, Yangchen Tenzin, Sharma Mukut, Srivastava Tarak, Rohatgi Rajeev
Northport VA Medical Center, Northport, NY, United States.
School of Medicine, Stony Brook University, Stony Brook, NY, United States.
Front Physiol. 2021 Feb 3;12:583453. doi: 10.3389/fphys.2021.583453. eCollection 2021.
Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, > 6 per group, < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive in static mpkCCD cells was 25.3 ± 1.7 μA/cm ( = 21), but after exposure to 24 h of FSS the increased to 41.4 ± 2.8 μA/cm ( = 22; < 0.01) and returned to 19.1 ± 2.1 μA/cm ( = 18, < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.
肾单位丢失会引发对剩余肾单位的代偿性血流动力学和细胞效应。单个肾单位肾小球滤过率和肾小管流速的增加会对肾小管施加更高的流体剪切力（FSS）。在主细胞（PC）培养模型中，FSS可诱导细胞外信号调节激酶（ERK），并且ERK参与跨上皮钠（Na）转运的调节以及细胞增殖。因此，我们推测高肾小管流速和FSS介导孤肾皮质集合管（CCD）中ERK的激活，这调节了氨氯地平敏感的Na转运并影响CCD细胞数量。对全肾蛋白裂解物进行免疫印迹以确定磷酸化ERK（pERK）的表达。接下来，用抗pERK抗体和双花扁豆凝集素（DBA）对假手术和单侧肾切除的小鼠进行染色，以鉴定具有pERK的主细胞。将小鼠主细胞（mpkCCD）在静态、FSS以及添加U0126（一种MEK1/2抑制剂）的FSS条件下培养于半透膜支架上，以测量FSS和ERK抑制对氨氯地平敏感的Na短路电流（）的影响。单侧肾切除组肾脏裂解物中pERK的丰度高于假手术组。肾切除小鼠的CCD中细胞总数和pERK阳性主细胞增加（分别为每CCD肾切除组9.3±0.4个细胞与假手术组6.1±0.2个细胞，以及5.1±0.5个细胞与3.6±0.3个细胞，每组n>6，P<0.05）。然而，增殖标志物Ki67在肾切除术后1个月的样本中通过免疫印迹或免疫组织化学检测与假手术组相比并无差异。接下来，静态mpkCCD细胞中氨氯地平敏感的为25.3±1.7μA/cm²（n=21），但在暴露于FSS 24小时后增加至41.4±2.8μA/cm²（n=22；P<0.01），在用U0126处理后恢复至19.1±2.1μA/cm²（n=18，P<0.01）。虽然FSS未改变mpkCCD细胞中α-或γ-ENaC的表达，但γ-ENaC在U0126处理的细胞中减少。总之，肾切除术后全肾尤其是CCD细胞中的pERK增加，但肾切除术后1个月pERK与活跃增殖无关。研究表明高肾小管流速诱导ERK依赖的ENaC钠吸收，可能在肾切除术后的钠平衡中起关键作用。
