*2.3. SH045 Treatment Ameliorates Renal Expression of Inflammatory Markers*

Next, we measured the renal mRNA expression of inflammatory cytokines and chemokines using qRT-PCR. The expression of inflammatory molecules was markedly increased in kidneys subjected to UUO compared to control groups (Figure 3). The mRNA expression of chemokine (C-X-C motif) ligand 1 (*Cxcl1*), chemokine (C-C motif) ligand 5 (*Ccl5*), and chemokine (C-C motif) receptor 2 (*Ccr2*) was significantly lower in UUO kidneys of SH045-treated mice (SH045 UUO kidneys) compared to UUO kidneys of vehicletreated mice (vehicle UUO kidneys) (Figure 3A–C). The expressions of chemokine (C-C

motif) ligand 2 (*Ccl2*), chemokine (C-X-C motif) ligand 2 (*Cxcl2*), and intercellular adhesion molecule 1 (*Icam1*) were increased in both SH045 UUO and vehicle UUO kidneys compared to control kidneys, although there were no differences between SH045 UUO and vehicle UUO kidneys (*p* = 0.056, *p* = 0.068 and *p* = 0.076, respectively) (Figure 3D–F). Furthermore, immunofluorescence staining of ICAM-1 markedly increased in UUO kidneys in comparison to control kidneys (Figure S3A–C). Additionally, the pharmacological inhibition of TRPC6 by SH045 decreased ICAM-1 expression after UUO in comparison to vehicle-treated kidneys (Figure S3A–C). Whereas ICAM1 expression was similar in the vessels of UUO kidneys, vehicle-treated kidneys had a much higher expression in SH045-treated UUO kidneys due to more ICAM-1-positive immune cell infiltration (Figure S3A).

**Figure 3.** SH045 impact on renal expression of inflammatory markers. (**A**) Renal mRNA levels of chemokine (C-X-C motif) ligand 1 (*Cxcl1*), (**B**) chemokine (C-C motif) ligand 5 (*Ccl5*), (**C**) chemokine (C-C motif) receptor 2 (*Ccr2*), (**D**) chemokine (C-C motif) ligand 2 (*Ccl2*), (**E**) chemokine (C-X-C motif) ligand 2 (*Cxcl2*), and (**F**) intercellular adhesion molecule-1 (*Icam1*) (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, and \*\*\*\* *p* < 0.0001 defined as significant. ns, not statistically significant. AU, arbitrary units.
