Endoplasmic Reticulum Stress and Proteasome Pathway Involvement in Human Podocyte Injury with a Truncated COL4A3 Mutation
Collagen type IV (COL4)-related nephropathy encompasses a spectrum of kidney diseases, including Alport syndrome (AS) and thin basement membrane nephropathy, which are among the most common hereditary glomerular diseases. These conditions are primarily caused by mutations in the genes encoding the type IV collagen α3, α4, and α5 chains (COL4A3, COL4A4, and COL4A5, respectively). The glomerular basement membrane (GBM) is a critical structure in the kidney, and its integrity is essential for proper renal function. The COL4 α3/α4/α5 chains are synthesized and secreted by podocytes, which are specialized epithelial cells that play a crucial role in maintaining the filtration barrier of the kidney. Mutations in these genes can lead to a reduction or complete loss of normal COL4A3-5, resulting in an unstable GBM, abnormal substitution of GBM components, and eventual podocyte injury.
In this study, we aimed to explore the mechanisms by which specific COL4A3 mutations lead to podocyte injury, focusing on the role of endoplasmic reticulum stress (ERS) and the proteasome pathway. We investigated four COL4A3 mutations, including three missense mutations (G619R, G801R, and C1616Y) and a frameshift mutation (COL4A3 c.4317delA) that results in a truncated NC1 domain. Our findings revealed that the truncated COL4A3 mutation induces excessive ERS and apoptosis in podocytes, and we identified the proteasome pathway as a potential therapeutic target for COL4A3-related nephropathy.
Background and Rationale
COL4-related nephropathy is characterized by mutations in the COL4A3, COL4A4, and COL4A5 genes, which encode the α3, α4, and α5 chains of type IV collagen, respectively. These chains form a heterotrimeric structure that is essential for the stability and function of the GBM. Mutations in these genes can lead to a spectrum of renal diseases, ranging from mild hematuria to severe glomerulosclerosis and end-stage renal disease. The severity of the disease often correlates with the type and location of the mutation, with truncating mutations typically resulting in more severe phenotypes than missense mutations.
Podocytes are highly specialized cells that play a critical role in maintaining the integrity of the GBM. They are particularly susceptible to injury from genetic mutations, immune disorders, and metabolic disturbances. Persistent podocyte injury can lead to the activation of apoptosis-related genes, resulting in glomerular dysfunction and progressive kidney disease. Understanding the molecular mechanisms underlying podocyte injury in COL4-related nephropathy is essential for developing targeted therapies.
Methods
To investigate the mechanisms of podocyte injury caused by COL4A3 mutations, we constructed lentiviral plasmids containing wild-type (WT) and four mutant COL4A3 sequences (G619R, G801R, C1616Y, and COL4A3 c.4317delA). These plasmids were stably transfected into human podocytes, and the expression levels of COL4A3 mRNA and protein were assessed using real-time polymerase chain reaction (PCR) and Western blotting, respectively. We also treated the podocytes with MG132, a proteasome inhibitor, and brefeldin A, a transport protein inhibitor, to explore the role of the proteasome and endoplasmic reticulum transport pathways in podocyte injury.
To validate our findings in human podocytes, we established a COL4A3 knockout mouse monoclonal podocyte line using CRISPR/Cas9 technology. This model allowed us to study the effects of a truncating mutation in the NC1 domain of COL4A3 on podocyte function and survival.
Results
Our data showed that COL4A3 mRNA was significantly overexpressed in podocytes stably transfected with the lentiviral plasmids. However, the protein level of COL4A3 was significantly increased in all groups except the COL4A3 c.4317delA group, which exhibited a truncated protein. This suggests that the truncated COL4A3 mutation is subject to specific mechanisms that inhibit its expression at the protein level.
We observed that the COL4A3 c.4317delA group exhibited excessive ERS and apoptosis compared to the other test groups. Treatment with MG132 significantly increased the intracellular expression of the truncated COL4A3 protein and reduced the levels of ERS and apoptosis in the COL4A3 c.4317delA group. These findings indicate that the proteasome pathway plays a critical role in the degradation of the truncated COL4A3 protein and that proteasome inhibition could be a potential therapeutic strategy for COL4A3-related nephropathy.
In the COL4A3 knockout mouse podocyte model, we successfully generated a monoclonal cell line with a truncating mutation in the NC1 domain (COL4A3 c.4852insA). This model exhibited higher levels of ERS and apoptosis compared to the WT group, further supporting the role of ERS and apoptosis in podocyte injury caused by truncating COL4A3 mutations.
Discussion
Our study provides new insights into the mechanisms of podocyte injury in COL4-related nephropathy. We demonstrated that truncating mutations in the NC1 domain of COL4A3 induce excessive ERS and apoptosis in podocytes, whereas missense mutations do not have the same effect. This difference in the severity of podocyte injury may explain the genotype-phenotype correlation observed in COL4-related nephropathy, with truncating mutations typically resulting in more severe disease phenotypes.
The proteasome pathway was identified as a key player in the degradation of the truncated COL4A3 protein. Treatment with MG132, a proteasome inhibitor, reversed the lower expression of the truncated protein and reduced the levels of ERS and apoptosis in podocytes. These findings suggest that proteasome inhibition could be a potential therapeutic strategy for patients with severe truncating COL4A3 mutations.
The use of CRISPR/Cas9 technology to generate a COL4A3 knockout mouse podocyte model provided additional validation of our findings. This model allowed us to study the effects of a truncating mutation in the NC1 domain of COL4A3 in a more physiologically relevant context, further supporting the role of ERS and apoptosis in podocyte injury.
Conclusion
In conclusion, our study demonstrates that truncating mutations in the NC1 domain of COL4A3 induce excessive ERS and apoptosis in podocytes, leading to podocyte injury and progressive kidney disease. The proteasome pathway plays a critical role in the degradation of the truncated COL4A3 protein, and proteasome inhibition could be a potential therapeutic strategy for patients with severe truncating COL4A3 mutations. These findings provide new insights into the molecular mechanisms underlying COL4-related nephropathy and highlight potential avenues for targeted therapy.
doi.org/10.1097/CM9.0000000000000294
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