*2.5. SH045 Treatment Reduces Renal Expression of Fibrotic Markers*

Since progressive fibrosis is a typical lesion occurring after UUO [18], we examined the impact of SH045 administration on renal fibrosis. We measured the renal mRNA expression of pro-fibrotic markers, including collagen I (*Col1a2)*, collagen III (*Col3a1)*, collagen IV (*Col4a3)*, α-smooth muscle actin (*Acta2)*, connective tissue growth factor (*Ccn2*), and fibronectin (*Fn1*). All these fibrosis-associated genes were upregulated after UUO (Figure 5A–F). Notably, SH045 treatment significantly reduced *Col1a2*, *Col3a1*, *Col4a3*, *Acta2*, *Ccn2,* and *Fn1* expressions in the UUO kidney (Figure 5A–F).

**Figure 5.** SH045 impact on expression of renal fibrotic markers UUO. (**A**) Renal mRNA levels of collagen type I α 1 (*Col1α2*), (**B**) Collagen type III α 1 (*Col3α1*), (**C**) Collagen type IV α 1 (*Col4α1*), (**D**) α-Smooth muscle actin (*Acta2*), (**E**) Connective tissue growth factor (*Ccn2*), and (**F**) Fibronectin (*Fn1*) (Control *n* = 10, UUO *n* = 11). Data expressed as means ± SD. Two-way ANOVA followed by Sidak's multiple comparisons post hoc test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001 defined as significant. ns, not statistically significant. AU, arbitrary units.

To further confirm our qPCR data, Sirius red (SR) and fibronectin immunofluorescence staining was performed. Control kidneys exhibited small SR-positive (+) areas. In contrast, UUO kidneys displayed markedly increased SR+ areas compared to control kidneys, indicating that UUO caused considerable collagen deposition (Figure 6A,B). SH045 effectively decreased this collagen deposition (Figure 6A,B). Similarly, immunofluorescence staining revealed increased fibronectin deposition and chromogenic immunohistochemistry increased α-smooth muscle actin (α-SMA) expression in UUO kidneys in comparison to control kidneys, which were reduced by SH045 treatment (Figure 6C–F). Taken together, these data suggest that renal fibrosis and inflammatory reactions are ameliorated in response to in vivo TRPC6 inhibition by SH045.

**Figure 6.** *Cont*.

**Figure 6.** SH045 impact on renal fibrogenesis after UUO. (**A**) Representative images of control and UUO-injured kidneys stained with Sirius red (magnification: 400×). Scale bars are 50 μm. (**B**) Semiquantification in renal Sirius red+ area proportion (control *n* = 6, UUO *n* = 8). (**C**) Representative images of control and UUO-injured kidneys stained with fibronectin (magnification: 400×). Scale bars are 50 μm. (**D**) Quantification in fibronectin+ area (control *n* = 6, UUO *n* = 8). (**E**) Representative images of control and UUO-injured kidneys stained with α-SMA (magnification: 400×). Scale bars are 50 μm. (**F**) Quantification of α-SMA+ staining (control *n* = 6, UUO *n* = 8). Data expressed as means ± SD. Two-way ANOVA followed by Sidak's multiple comparisons post hoc test. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\*\* *p* < 0.0001 defined as significant. ns, not statistically significant.
