This study explored the mechanism of Piezo1 regulation related macrophage-to-myofibroblast transition (MMT) in neurogenic bladder fibrosis (NBF) by using rat spinal cord half-transection model and in vitro cell experiments (Figure 1).

Neurogenic bladder (NB) refers to the dysfunction of lower urinary tract storage and urination due to damage to the central or peripheral nervous system, resulting in a series of lower urinary tract symptoms (LUTS) and complications [1]. It is necessary to further understand the mechanism of NB bladder fibrosis in order to effectively delay or block it. Recently, the role of MMT in promoting multiple organ fibrosis has attracted attention. Studies have showed that MMT can be found in progression of renal fibrosis, myocardial fibrosis et al and play a key role in fibrosis [2-3]. However, there is no reports on the MMT changes in bladder, especially in severe NBF. Therefore, this study explores the mechanism of Piezo1 regulation related MMT in NBF. Forty 1-week-old SD rats were randomly divided into sham-operated group, sham+GsMTx4 (10 mg/kg), NBF and NBF + GsMTx4 (10 mg/kg, Intraperitoneal Injections) groups. After cystometry, bladder and bladder tissue samples were collected for Single-Cell RNA Sequencing(scRNA-seq), Transcriptome Sequencing, Hematoxylin and Eosin (HE), Masson staining, and WB and qPCR detection. Primary rat bone marrow mononuclear cells were isolated, extracted, and cultured. After co-stimulation with TGF-β1 (TGFB, 10 ng/mL) for 24h, cells were collected. Cell marker was detected using WB, immunofluorescence, and annexin/PI assays.

Bladder tissue can detect the following cell types, including immune cells, myeloid cells, fibroblasts, epithelial cells, NK cells and endothelial cells et al by using scRNA-seq. It presented M2 macrophages (Mø) found more expression of myofibroblast markers like ACTA2 in NBF group. Meanwhile, drug treatment can alleviate M2 Mø ACTA2 expression in sham+GsMTx4 and NBF + GsMTx4 group. This preliminarily speculated that Piezo1/Piezo1 inhibitor GsMTx4 influence the MMT in NBF. Transcriptome Sequencing GO results showed that differentially expressed genes were enriched in the integrin related pathways like HYPOXIA and INTEGRIN. Compared with the different groups, HE/Masson staining/WB results showed differences in the expression of PKM2/ITGB6/TGFB signaling pathway and fibrosis markers(Figure 2). Immunofluorescence/WB experiments suggest that co-cultivation of Mø and TGFB affects the expression of ACTA2 in Mø(Figure 3).

Piezo1 is highly expressed in spinal cord injury-induced neurogenic bladder, correlating with bladder pressure and fibrosis severity. Animal experimental results using Piezo1 inhibitors in animal models with bladder hypertension demonstrate that Piezo1 is a key initiating marker in the MMT and PKM2/ITGB6/TGFB pathways. TGFB stimulation of Mø can induce MMT conversion. It provides new molecular therapy intervention targets which will help develop drugs to alleviate fibrosis.

Upregulation of Piezo1 through PKM2/ITGB6/TGFB pathway can exacerbate bladder fibrosis progression and influence the MMT.

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Zhuang T, Chen MH, Wu RX, et al. ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice. Nat Commun. 2024 Mar 5;15(1):1995.