Ras-Related C3 Botulinum Toxin Substrate 1 Combining With the Mixed Lineage Kinase 3- Mitogen-Activated Protein Kinase 7- c-Jun N-Terminal Kinase Signaling Module Accelerates Diabetic Nephropathy

Abstract

Ras-related C3 botulinum toxin substrate 1 (RAC1) activation plays a vital role in diabetic nephropathy (DN), but the exact mechanism remains unclear. In this study, we attempted to elucidate the precise mechanism of how RAC1 aggravates DN through cellular and animal experiments. In this study, DN was induced in mice by intraperitoneal injection of streptozotocin (STZ, 150mg/kg), and the RAC1 inhibitor NSC23766 was administered by tail vein injection. Biochemical indicators, cell proliferation and apoptosis, and morphological changes in the kidney were detected. The expression of phosphorylated c-Jun N-terminal kinase (p-JNK), nuclear factor-κB (NF-κB), and cleaved caspase-3 and the interaction between RAC1 and the mixed lineage kinase 3 (MLK3)-mitogen-activated protein kinase 7 (MKK7)-JNK signaling module were determined. Furthermore, the colocalization and direct co-interaction of RAC1 and MLK3 were confirmed. Our results showed that RAC1 accelerates renal damage and increases the expression of p-JNK, NF-κB, and cleaved caspase-3. However, inhibition of RAC1 ameliorated DN by downregulating p-JNK, NF-κB, and cleaved caspase-3. Also, RAC1 promoted the assembly of MLK3-MKK7-JNK, and NSC23766 blocked the interaction between RAC1 and MLK3-MKK7-JNK and inhibited the assembly of the MLK3-MKK7-JNK signaling module. Furthermore, RAC1 was combined with MLK3 directly, but the RAC1 Y40C mutant inhibited the interaction between RAC1 and MLK3. We demonstrated that RAC1 combining with MLK3 activates the MLK3-MKK7-JNK signaling module, accelerating DN occurrence and development, and RAC1 Y40 is an important site for binding of RAC1 to MLK3. This study illustrates the cellular and molecular mechanisms of how RAC1 accelerates DN and provides evidence of DN-targeted therapy.

Receptor for advanced glycation end products aggravates cognitive deficits in type 2 diabetes through binding of C‐terminal AAs 2‐5 to mitogen‐activated protein kinase kinase 3 (MKK3) and facilitation of MEKK3‐MKK3‐p38 module assembly

“…HEK‐293T and HT22 cell lines were cultured and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) at 37°C with 20% O 2 and 5% CO 2 , and 0.05% trypsin/EDTA was used for cell passaging. As in our previous study (Ying et al, 2021 ), 25 and 80 mM glucose were used for the normal‐ and high‐glucose conditions in HEK293T cells experiments. After 12 h in serum‐free culture for synchronization, HEK‐293T cells were randomly divided into five groups: a normal group (NG; 25 mM glucose), a high‐glucose group (HG: 80 mM glucose), a high‐glucose plus RAGE inhibitor group (FPS; 2.5 μM FPS‐ZM1 dissolved in DMSO), a high‐glucose plus solvent control group (DMSO), and an osmotic pressure control (mannitol) group (MG; 25 mM glucose plus 55 mM mannitol).…”

Effects of acteoside on the expressions of MCP-1 and TGF-β1 in the diabetic nephropathy mice

“…Urine was collected and the ratio of urinary albumin to urinary creatinine (≥30 mg/g) in urine was detected for three consecutive times, when the result ≥30 mg/g, it means the DN model is successful. 12 Model mice were randomly divided into two groups as follows (n = 5 per group): in the acteoside treatment group (ACT-DN group), the mice were given a gavage of acteoside (Jintaihe pharmaceutical CoLtd, Chengdu, China) 20 mg/kg/ day, diluted in 0.9% (w/v) normal saline, acteoside was administered for 8 weeks after the onset of diabetes, and in the diabetic nephropathy group (DN group), the mice were left untreated. DN group was given the same amount of normal saline for 8 consecutive weeks.…”