机械敏感通道 Piezo 1 和 Piezo 2 促进幼鼠神经源性膀胱纤维化的初步研究

A preliminary study of mechanosensitive channels Piezo 1 and Piezo 2 promoting neurogenic bladder fibrosis in young rats

摘要

目的 探究机械敏感通道 Piezos（Piezo1 和 Piezo2）在神经源性膀胱（NB）幼鼠中的变化及其对膀胱纤维化的作用，为临床寻找治疗 NB 的新靶点提供参考。方法 将 30 只雌性幼年 SD 大鼠按随机数表法分为 5 组：假手术组（Sham 组）、神经离断 2 周组（NB-2W 组）、神经离断 6 周组（NB-6W 组）、神经离断+Piezos 抑制剂 2 周组（NB-P-2W 组）和神经离断+Piezos 抑制剂 6 周组（NB-P-6W 组），每组 6 只。通过离断幼鼠 L6 及 S1 脊神经构建 NB 动物模型，神经离断组造模后不进行干预，神经离断+Piezos 抑制剂组则在造模后每 2 天腹腔注射 Piezos 抑制剂 GsMTx4（10 mg/kg）。在离断后 2 周和 6 周进行幼鼠膀胱测压和超声检查，并取膀胱组织检测 Piezos 及纤维化相关指标Ⅰ型胶原蛋白（Collagen Ⅰ）及α-平滑肌肌动蛋白（α-SMA）的相对表达水平。结果 膀胱测压显示，NB-2W 组幼鼠基础膀胱压力明显升高，NB-6W 组略有下降但仍高于 Sham 组（P＜0.05）。NB-P-2W 组的基础膀胱压力低于 NB-2W 组但较 Sham 组高，NB-P-6W 组的基础膀胱压力低于 NB-6W 组但高于 Sham 组（P＜0.05）。与 Sham 组比较，NB-2W 组和 NB-6W 组最大膀胱测压容量（MCC）出现先升高后下降趋势（P＜0.05）。与 NB-2W 组比较，NB-P-2W 组的漏尿点压（BLPP）更低，MCC 和膀胱顺应性（BC）更高（P＜0.05）；与 NB-6W 组比较，NB-P-6W 组的 BLPP 更低，MCC 和 BC 更高（P＜0.05）。HE、MASSON 染色及超声结果显示，随着神经离断时间延长，膀胱纤维化逐渐加重，膀胱壁粗糙增厚，内可见结石，肾脏逐渐出现肾盂扩张积水，而 NB-P-2W 组和 NB-P-6W 组纤维化程度轻于 NB-2W 组和 NB-6W 组，未见上尿路扩张积水。此外，Weston blot 及免疫组化结果显示 NB-2W 组和 NB-6W 组的 Piezos、Collagen Ⅰ及α-SMA 的相对表达水平明显高于 Sham 组（P＜0.01）；NB-P-2W 组和 NB-P-6W 组的 Piezos、Collagen Ⅰ及α-SMA 的相对表达水平则分别低于 NB-2W 组和 NB-6W 组（P＜0.01）。结论 机械敏感通道 Piezos 在 NB 幼鼠中表达升高，可能参与膀胱纤维化进展，但其机制有待进一步研究。

Abstract

Objective To explore the changes of mechanosensitive channels Piezos (Piezo 1 and Piezo 2) in neurogenic bladder (NB) of young rats and their effects, so as to provide reference for clinical search of new therapeutic targets. Methods A total of 30 female young SD rats were divided into 5 groups based on random number table method: sham operation group (sham), 2-week nerve transection group (NB-2W), 6-week nerve transection group (NB-6W), 2-week nerve transection + Piezos inhibitor group (NB-P-2W) and 6-week nerve transection + Piezos inhibitor group (NB-P-6W), with 6 rats in each group. The NB models were constructed by transecting the L6 and S1 spinal nerves of young rats. The NB-2W and NB-6W groups were not intervened after modeling, while the NB-P-2W and NB-P-6W groups were intraperitoneally injected with Piezos inhibitor GsMTx4 (10 mg/kg) every 2 days after modeling. Bladder cystometry and ultrasound were performed after 2 and 6 weeks of transection. The expressions of Piezos and fibrosis-related indexes (Collagen Ⅰ and α-smooth muscle actin) were detected in bladder tissues. Results The results of bladder cystometry showed that the basal bladder pressure in NB-2W group was significantly increased, while it was slightly decreased but was still higher in NB-6W group than in the sham group (P<0.05). Basal bladder pressure was lower in NB-P-2W group than in NB-2W group, but was higher than that in the sham group; basal bladder pressure was lower in NB-P-6W group than in NB-6W group, but higher than that in the sham group (P<0.05). Compared with the sham group, the NB-2W and NB-6W groups had firstly increased and then decreased maximum cystometric capacity (MCC) (P<0.05). Compared with NB-2W group, NB-P-2W group had lower bladder leakage point pressure (BLPP), but higher MCC and bladder compliance (BC) (P<0.05). Compared with NB-6W group, NB-P-6W group had significantly lower BLPP but higher MCC and BC (P<0.05). HE and MASSON staining and ultrasound results showed that, with the extension of nerve transection time, bladder fibrosis gradually worsened, the bladder wall became rough and thickened, calculi were visible inside, and hydronephrosis gradually appeared; the degree of fibrosis in NB-P-2W and NB-P-6W groups was less than that in NB-2W and NB-6W groups, and no hydronephrosis was observed in the upper urinary tract. In addition, Western blotting and immunohistochemical results showed that NB-2W and NB-6W groups had significantly higher relative expression levels of Piezos, Collagen Ⅰ and α-SMA than the sham group (P<0.01), while NB-P-2W and NB-P-6W groups had lower relative expression levels of Piezos, Collagen Ⅰ and α-SMA than NB-2W and NB-6W groups (P<0.01). Conclusion The increased expressions of mechanosensitive channels Piezos in NB young rats may be involved in the progression of bladder fibrosis, but its mechanism needs further study.

关键词

Piezo 1 / Piezo2 / 神经源性膀胱 / 膀胱纤维化 / 幼鼠 / 机械敏感通道

Key words

Piezo 1 / Piezo 2 / neurogenic bladder / bladder fibrosis / young rat / mechanosensitive channel

参考文献

[1] PARK JS, LEE YS, LEE CN, et al. Efficacy and safety of mirabegron, a β3-adrenoceptor agonist, for treating neurogenic bladder in pediatric patients with spina bifida: a retrospective pilot study [J]. World J Urol, 2019, 37(8): 1665-1670.

[2] 汲凤平, 张艳平, 马源, 等. 儿童神经源性膀胱纤维化研究进展 [J]. 中华实用儿科临床杂志, 2021, 36(13): 1037-1040.

[3] SAGER C, BARROSO U JR, BASTOS JMN, et al. Management of neurogenic bladder dysfunction in children update and recommendations on medical treatment [J]. Int Braz J Urol, 2022, 48(1): 31-51.

[4] STEIN R, BOGAERT G, DOGAN HS, et al. EAU/ESPU guidelines on the management of neurogenic bladder in children and adolescent part Ⅱ operative management [J]. Neurourol Urodyn, 2020, 39(2): 498-506.

[5] STEIN R, BOGAERT G, DOGAN HS, et al. EAU/ESPU guidelines on the management of neurogenic bladder in children and adolescent part Ⅰ diagnostics and conservative treatment [J]. Neurourol Urodyn, 2020, 39(1): 45-57.

[6] YADAV P, ALSABBAN A, DELOS REYES T, et al. A systematic review of paediatric neurogenic lower urinary tract dysfunction guidelines using the Appraisal of Guidelines and Research Evaluation (AGREE) Ⅱ instrument [J]. BJU Int, 2023, 131(5): 520-529.

[7] WIENER JS, CHAUDHRY R. Neurogenic lower urinary tract dysfunction [J]. Urol Clin North Am, 2023, 50(3): 415-432.

[8] MARSHALL KL, SAADE D, GHITANI N, et al. Piezo2 in sensory neurons and urothelial cells coordinates urination [J]. Nature, 2020, 588(7837): 290-295.

[9] THOMAS T. Piezo2 in urinary function [J]. Nat Rev Urol, 2020, 17(12): 658.

[10] DALGHI MG, RUIZ WG, CLAYTON DR, et al. Functional roles for Piezo 1 and Piezo 2 in urothelial mechanotransduction and lower urinary tract interoception [J]. JCI Insight, 2021, 6(19): e152984.

[11] XU Y, HUANG Y, CHENG X, et al. Mechanotransductive receptor Piezo 1 as a promising target in the treatment of fibrosis diseases [J]. Front Mol Biosci, 2023, 10: 1270979.

[12] LI Z, LIN D, LUO C, et al. The expression and function of Piezo channels in bladder [J]. Bladder, 2023, 10: e21200008.

[13] OCHIAI K, MOCHIDA Y, NAGASE T, et al. Upregulation of Piezo 2 in the mesangial, renin, and perivascular mesenchymal cells of the kidney of Dahl salt-sensitive hypertensive rats and its reversal by esaxerenone [J]. Hypertens Res, 2023, 46(5): 1234-1246.

[14] BRAIDOTTI N, CHEN SN, LONG CS, et al. Piezo1 channel as a potential target for hindering cardiac fibrotic remodeling [J]. Int J Mol Sci, 2022, 23(15): 8065.

[15] ZHAO X, KONG Y, LIANG B, et al. Mechanosensitive Piezo 1 channels mediate renal fibrosis [J]. JCI Insight, 2022, 7(7): e152330.

[16] 蒲青崧, 杨兴欢, 王俊魁, 等. 大鼠腰骶神经离断后不同时间膀胱形态功能变化及其对上尿路扩张的影响 [J]. 中华泌尿外科杂志, 2023, 44(5): 369-375.

[17] CHEN Y, MA Y, HE Y, et al. The TGF-beta1 pathway is early involved in neurogenic bladder fibrosis of juvenile rats [J]. Pediatr Res, 2021, 90(4): 759-767.

[18] WYNDAELE JJ, WYNDAELE M, ROSIER P. Sensations reported during urodynamic bladder filling in spinal cord injury patients give additional important information [J]. Int Neurourol J, 2022, 26(Suppl 1): S30-S37.

[19] LYU L, YAO YX, LIU EP, et al. A Study of uodynamic parameters at different bladder filling stages for predicting upper urinary tract dilatation [J]. Int Neurourol J, 2022, 26(1): 52-59.

[20] AUSTIN PF, BAUER SB, BOWER W, et al. The standardization of terminology of lower urinary tract function in children and adolescents: update report from the standardization committee of the International Children's Continence Society [J]. Neurourol Urodyn, 2016, 35(4): 471-481.

[21] SONG S, ZHANG H, WANG X, et al. The role of mechanosensitive Piezo1 channel in diseases [J]. Prog Biophys Mol Biol, 2022, 172: 39-49.

[22] MIYAMOTO T, MOCHIZUKI T, NAKAGOMI H, et al. Functional role for Piezo1 in stretch-evoked Ca²⁺ influx and ATP release in urothelial cell cultures [J]. J Biol Chem, 2014, 289(23): 16565-16575.

[23] LI X, HU J, ZHAO X, et al. Piezo channels in the urinary system [J]. Exp Mol Med, 2022, 54(6): 697-710.

[24] SZCZOT M, NICKOLLS AR, LAM RM, et al. The form and function of Piezo2 [J]. Annu Rev Biochem, 2021, 90: 507-534.

[25] GAILLY P, DEVUYST O. Piezo 2, a mechanosensor in the urinary bladder [J]. Kidney Int, 2021, 100(1): 9-11.

[26] XU H, HE Y, HONG T, et al. Piezo 1 in vascular remodeling of atherosclerosis and pulmonary arterial hypertension: a potential therapeutic target [J]. Front Cardiovasc Med, 2022, 9: 1021540.

[27] CHEN J, MIAO J, ZHOU D, et al. Upregulation of mechanosensitive channel Piezo 1 involved in high shear stress-induced pulmonary hypertension [J]. Thromb Res, 2022, 218: 52-63.

[28] SHIMIZU N, SAITO T, WADA N, et al. Molecular mechanisms of neurogenic lower urinary tract dysfunction after spinal cord injury [J]. Int J Mol Sci, 2023, 24(9): 7885.

[29] SAITO T, GOTOH D, WADA N, et al. Time-dependent progression of neurogenic lower urinary tract dysfunction after spinal cord injury in the mouse model [J]. Am J Physiol Renal Physiol, 2021, 321(1): F26-F32.

[30] HOBBS SK, KRISCHAK M, TEJWANI R, et al. The importance of early diagnosis and management of pediatric neurogenic bladder dysfunction [J]. Res Rep Urol, 2021, 13: 647-657.

基金资助

国家自然科学基金项目(U1904208)

国家自然科学基金项目(82470807)