Abstract: FR-PO754
Mechanism of TPPP3-Affecting Podocyte Cytoskeleton Stability by Regulating the Acetylation Level of α-Tubulin
Session Information
- Glomerular Diseases: Mechanisms and Podocyte Biology
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Chen, Qilin, Children's Hospital of Chongqing Medical University National Clinical Research Center for Child Health and Disorders, Chongqing, Chongqing, China
- Qiu, Li, Children's Hospital of Chongqing Medical University National Clinical Research Center for Child Health and Disorders, Chongqing, Chongqing, China
Background
Podocytes exhibit a unique cytoskeleton architecture, which is closely related to their function in maintaining the kidney filtration barrier. As the core structure of the skeleton network, microtubules (MTs) play a key role in supporting and maintaining the skeleton network, but there is a lack of understanding of podocyte MTs.
Methods
Single cell transcriptome sequencing and spatial transcriptome sequencing were performed on children 's kidneys. Immunofluorescence (IFA), immunohistochemical (IHC) and western blot (WB) were used to determine the expression of TPPP3, α-tubulin, acetylated tubulin (Ace-tubulin), actin microfilaments. TPPP3-siRNA was used to knock down podocytes, and WB was used to verify the knock-out results.
Results
Single-cell combined spatial transcriptome sequencing of children’s kidneys revealed that human podocytes significantly expressed tubulin-related molecule TPPP3. The results of IHC, IFA and WB confirmed that TPPP3 was specifically expressed in the nucleus and cytoplasm of human glomerular podocytes. Surprisingly, TPPP3 was not expressed in kidneys and podocytes of mice and rats. The expression of TPPP3 was significantly increased in HPC differentiation stage compared with HPC in proliferative stage without podocyte molecular characteristics. When TPPP3 was knocked down by siRNA, the results of cell fluorescence and WB showed that the Ace-tubulin was significantly decreased, and the filamentous structure of Ace-tubulin and α-tubulin was broken, the continuity was destroyed. It is worth noting that knockdown of TPPP3 in HPCs directly leads to decreased actin (F-actin) and structural disorder. Furthermore, using drug intervention, increasing the level of microtubule acetylation can partially restore the microtubule structure damage caused by knockdown of TPPP3. In addition, the results of IFA and IHC confirmed that the expression of TPPP3 in glomeruli of MCD and FSGS patients was decreased. The in vitro podocyte injury model suggested that the expression of slit membrane protein Podocin and TPPP3 was decreased.
Conclusion
Human podocytes specifically expressed microtubule-associated protein TPPP3, which maid be involved in regulating the level of acetylated tubulin and affecting the cytoskeleton such as actin microfilaments in podocytes