A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42‐mediated GSK‐3β/β‐catenin Signaling

Abstract Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay‐guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti‐renal fibrotic lead. DA shows significant anti‐kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down‐regulates its downstream phospho‐protein kinase Cζ(p‐PKCζ)/phospho‐glycogen synthase kinase‐3β (p‐GSK‐3β), thereby promoting β‐catenin Ser33/37/Thr41 phosphorylation and ubiquitin‐dependent proteolysis to block classical pro‐fibrotic β‐catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Table S2.Possible protein targets of DA.
Table S3.Antibodies used for immunoblotting.Table S4.Primes used for q-PCR experiments.Table S5.si-RNAs used for transfection experiments.Table S6.Clinical characteristics for patient samples included in this study.Supplementary Figure 2 The antifibrotic efficacy of DA was superior to RAAS blockers .Statistical analysis was performed using one-way ANOVA followed by a Bonferroni's multiple comparisons test.### P < 0.001, #### P < 0.0001 compared with CTL group.* P < 0.05, ** P < 0.01, **** P < 0.0001 compared with Veh group or groups under the lines; ns, not significant..

Figure S1 .
Figure S1.The effects of diterpene compounds on cell viability.

Figure S2 .
Figure S2.DA inhibited the activation of fibroblasts.

Figure S6 .
Figure S6.ZCL278 exhibited systemic and hepatic toxicity compared to DA.

Figure S8 .
Figure S8.DA targets Cdc42 to regulate macrophages and T cell functions.

Figure S1 .
Figure S1.The effects of diterpene compounds on cell viability.A) MTS assay of the influence of diverse diterpene compounds (Compd) (10 M) on cell proliferation of NRK-49F cells for 2 days (n=6 per group); B) Cell viability curves of NRK-49F cell line or pMRF treated with DA of indicated concentrations for 48 h, measured by MTS assay (n=6 per group).pMRF, primary murine renal fibroblast.

Table S2 .
Possible protein targets of DA.

Table S3 .
Antibodies used for immunoblotting.

Table S4 .
Primes used for q-PCR experiments.

Table S6 .
Clinical characteristics for patient samples included in this study.