*2.1. SH045 Treatment Does Not Affect Renal Function and Trpc Expression in UUO Model*

To investigate the impact of in vivo TRPC6 inhibition on renal function, target molecules and fibrosis, we performed UUO in the NZO mice. During the one week period, we administrated SH045 (TRPC6 inhibitor) or vehicle once daily (Figure 1A). After 7 days, urinary tract obstruction led to hydronephrosis (Figure S1). Consistent with our previous findings, *Trpc6* expression significantly increased in UUO kidneys. SH045 affected neither *Trpc6* mRNA expression nor the expression of other TRPC channels, including *Trpc1, Trpc2, Trpc3 and Trpc4* (Figure 1B and Figure S2A–D). SH045 had no impact on renal function. Serum creatinine (*p* = 0.1098; Figure 1B) and blood urea nitrogen (BUN) (*p* = 0.928; Figure 1C), serum cystatin C, urine albumin, and urine albumin-to-creatinine ratio in SH045-treated mice were unchanged (Figure 1D–F). In addition, we found no differences in serum levels of glucose, sodium, potassium, ionized calcium, total CO2, hemoglobin, hematocrit, and anion gap in SH045-treated animals compared to the vehicle group (Table S1). SH045-treated mice exhibited a slightly higher serum chloride concentration (*p* = 0.047), albeit within the normal physiological range.

**Figure 1.** Impact of SH045 administration on renal function and Trpc6 expression in UUO model. (**A**) Experimental design of unilateral ureteral obstruction (UUO) model. NZO mice were subjected to UUO and then injected with SH045 (*n* = 11) or vehicle (*n* = 11) once every 24 h between day 0 and day 7. All mice were euthanized on day 7 after UUO surgery. (**B**) Renal mRNA levels of *Trpc6* (control *n* = 10, UUO *n* = 11). Control group includes kidneys that were not subjected to the UUO. (**C**) Serum levels of creatinine, (**D**) blood urea nitrogen, and (**E**) cystatin C in the experimental UUO groups. (**F**) Urine albumin and (**G**) ratio of albumin to creatinine in the experimental UUO groups (UUO vehicle *n* = 10–11, UUO SH045 *n* = 8–11). Data expressed as means ± SD. Two-way ANOVA followed by Sidak's multiple comparisons post hoc test. \*\* *p* < 0.01 and \*\*\*\* *p* < 0.0001 defined as significant. ns, not statistically significant. AU, arbitrary units.
