**3. Discussion**

Renal fibrosis is the final common outcome of progressive CKD, which is often observed in metabolic syndrome [19]. To date, there are few clinical treatments that successfully target fibrosis in CKD. Thus, developing new drug treatments is the current focus. Increasing evidence indicates that TRPC6 could play a critical role in kidney fibrosis [20]. In our previous study using *Trpc6*–/– mice, we found that TRPC6 deficiency ameliorated renal fibrosis and immune cellular infiltration in the UUO model [7]. However, the results were difficult to interpret due to confounding genomic and non-genomic effects of other TRPC channels, e.g., TPRC1, TRPC3, TRPC4 and TRPC5. Previous studies identified SH045 (larixyl N-methylcarbamate) as a novel, highly potent, subtype-selective inhibitor of TRPC6 channels [15]. In our previous study, we found that the in vivo inhibition of TRPC6 by SH045 had no effects on acute kidney injury (AKI) [14]. However, there are no studies on the effects of SH045 in kidney fibrosis. In the present study, we tested the hypothesis that SH045 ameliorates UUO-accelerated renal fibrosis in NZO mice.

Our results show that SH045 ameliorates fibrotic processes in UUO kidneys. Expressions of all investigated fibrosis or fibrosis-related genes were ameliorated by SH045 treatment. The histological assessment of deposited collagen and extracellular matrix protein confirmed the expression data of the genes. Of note, renal fibrosis arises after an insult, whereas resident kidney fibroblasts and cells of hematopoietic origin differentiate

into myofibroblasts [21–23]. Myofibroblasts acquire a contractile/proliferative phenotype upon activation by profibrotic factors and become principal kidney collagen-producing cells [24]. Considerable evidence indicates that renal inflammation plays a central role in the initiation and progression of fibrosis [19]. Myofibroblasts are regulated by a variety of means, including paracrine signals derived from lymphocytes and macrophages. Critical chemokines recruiting macrophages and lymphocytes are CCL2/CCR2, CCL5, and CXCL1/2. ICAM-1 is an endothelial- and leukocyte-associated transmembrane protein in facilitating leukocyte endothelial transmigration [25]. Interestingly, our results show that SH045 inhibits the overexpression of these chemokines and the infiltration of numerous immune cells, suggesting that TRPC6 inhibition may antagonize renal fibrosis by affecting inflammatory processes. TRPC6 is expressed in a wide range of cell types, including neutrophils, lymphocytes, platelets and the endothelium, which might be a modulator of tissue susceptibility to inflammatory injuries [26,27]. Some studies suggested that TRPC6 channels may enhance chemotactic responses by increasing Ca2+ concentration, which promotes actin-based cytoskeleton remodeling [28,29]. Furthermore, Ca2+ currents within T-lymphocytes are influenced by TRPC6, which can affect the function of T-lymphocytes [30]. Novel myeloid cell subsets could be targeted to ameliorate injury or enhance repair, including an *Arg1+* monocyte subset present during injury and *Mmp12+* macrophages present during repair [31]. It is intriguing to speculate that TRPC6 inhibition might ameliorate fibrotic processes in UUO kidneys by modulating the function(s) of theses cell types.

On the other hand, TRPC6 was also reported to contribute to fibroblast transdifferentiation and healing in vivo [32]. Thus, the beneficial effects of TRPC6 inhibition seen in the UUO model might also involve fibroblasts. A TPRC6 blockade may decrease Ca2+ dependent activation of MEK/ERK signaling pathway [33]. Of note, this pathway was implemented in the detrimental differentiation and expansion of kidney fibroblasts [34]. The inhibition of the ERK1/2 pathway by trametinib ameliorated UUO-induced fibrosis through the mammalian target of rapamycin complex 1 (mTORC1) and its downstream targets.

In the present study, SH045 did not affect renal function parameters in 7-day-UUO mice, which is not surprising. In this short-term UUO model, the kidney function of contralateral undamaged kidney remained preserved and compensated for the loss of the obstructed kidney at the early stage [35]. We used the NZO inbred obese mouse strain, which carries susceptibility genes for diabetes and hypertension, conditions similar to metabolic syndrome and CKD in humans [36]. Our data observed in UUO induced fibrosis in NZO mice, and thus might be of importance in mimicking human CKD pathophysiology.

Renal fibrosis involves complex interactions among multiple cells and cytokine signaling pathways. Further studies of the TRPC6 modulation of renal fibrosis using single-cell RNA sequencing could help to better understand the exact mechanism(s) of action in the different cell types. Single-cell RNA sequencing enables the precise discrimination of specific cell type(s) or cell state(s) enriched in certain conditions (e.g., UUO) [31]. Thus, selecting cellular labels based on gene expression markers could represent a novel approach to determine cell type(s) or cell state(s) predominantly influenced by the inhibition of TRPC6 (by SH045) in the UUO model. Understanding the mechanisms behind TRPC6-induced fibrogenesis is essential for developing novel therapies to slow the progression of CKD.

Our study demonstrates that the in vivo administration of SH045 ameliorates immune cell infiltration and fibrosis in NZO mice subjected to UUO, which makes SH045 a promising therapeutic drug strategy in CKD treatment for metabolic syndrome.
