Abstract
Reticulon 3 (RTN3) is an endoplasmic reticulum protein that has previously been shown to play roles in neurodegenerative diseases, but little is known about its function in the kidneys. The aim of the present study was to clarify the roles of RTN3 in chronic kidney disease (CKD) and kidney fibrosis. In this study, RTN3 levels were measured in kidney tissues from healthy controls and CKD or kidney fibrosis patients. An RTN3-null mouse model was generated to explore the pathophysiological roles of RTN3 in the kidneys. The underlying mechanisms were studied in primary proximal tubular epithelial cells and HEK293 cells in vitro. The results showed that (1) a reduction in RTN3 in mice induces CKD and kidney fibrosis; (2) decreased RTN3 expression is found in patients with CKD; (3) RTN3 plays critical roles in regulating collagen biosynthesis and mitochondrial function; and (4) mechanistically, RTN3 regulates these phenotypes by interacting with GC-Rich Promoter Binding Protein 1 (GPBP1), which activates the IGF2-JAK2-STAT3 pathway. Our study indicates that RTN3 might play crucial roles in CKD and kidney fibrosis and that a reduction in RTN3 in the kidneys might be a risk factor for CKD and kidney fibrosis.
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Introduction
Chronic kidney disease (CKD) is a type of kidney disease in which there is gradual loss of kidney function over a period of months or years1. It affects 10–15% of the population worldwide and is now recognized as the most rapidly increasing contributor to the global burden of disease. The costs related to CKD and end-stage renal disease (the terminal manifestation of CKD) are an enormous burden for all healthcare systems around the world2. Usually, CKD does not cause symptoms until it reaches an advanced stage. At the middle and late stages of CKD, edema, fatigue, vomiting, loss of appetite, or confusion may develop3. In the clinic, the recommended diagnosis and testing procedures for CKD include blood pressure measurement, urine testing, and serum creatinine measurement2. Although many early-onset CKD cases were not previously considered to be of genetic origin, studies in recent years have discovered that approximately 20% of CKD cases may be associated with genetic factors4. Thus far, more than 200 candidate genes, including collagen-related genes, mitochondria-related genes, and ion channel-related genes, have been identified for 70% of CKD cases associated with genetic factors4.
The reticulon (RTN) protein family consists of RTN1 through RTN4 in mammals, and its members have a signature C-terminal RTN homolog domain (RHD)5. Biochemically, RTNs can shape the structure of the tubular endoplasmic reticulum (ER) due to the ω- (wedge-shaped) membrane topology in the N- and C-terminal domains6. Functionally, RTNs have been found to regulate neurite outgrowth and negatively modulate the activity of Alzheimer’s β-secretase, and they are pathologically linked to axonopathy in hereditary spastic paraplegias7,8,9. Interestingly, the functions of RTNs in human peripheral organs are still not clear. Previously, we found that increased RTN3 can lead to obesity and hypertriglyceridemia via interaction of RTN3 with heat shock protein family A (Hsp70) member 531, an important risk factor linked to diabetic kidney disease (DKD). In addition, upregulation of IGF2 has been proven to lead to Perlman syndrome, a disease related to kidney injury, in mouse nephron progenitor cells15. In proximal tubule cells, increased IGF levels promote fibronectin expression through a pathway involving Nox-dependent ROS generation and Akt signaling32. In our study, the expression of IGF2 was dramatically increased in RTN3-null mice with CKD, which indicated that kidney injury might have occurred via the IGF2 pathway.
JAK2-STAT3 activation has been detected in several types of kidney disease, such as acute kidney injury, CKD and DKD33,34,35. Podocyte-specific JAK2 overexpression has been found to accelerate disease progression in a DKD mouse model36, and a STAT3 inhibitor (S3I-201) can attenuate fibrosis and inflammation in UUO kidneys37. In a CKD mouse model, activation of the JAK2-STAT3 pathway has been found to induce oxidative stress and aggravate CKD38. In acute kidney injury and DKD, activation of the JAK2-STAT3 pathway also promotes the immune inflammatory response and induces apoptosis33. Furthermore, administration of JAK2 inhibitors, including baricitinib and Huang Gan formula, effectively improves kidney function in CKD and DKD, which indicates that JAK2 inhibitors might be useful as new therapies for CKD and DKD38,39.
Oxidative stress is harmful to cells due to excessive generation of ROS, which has been proven to lead to CKD40. Disruption of mitochondrial structure and function may promote the oxidative stress response41,42. In our study, we detected disruption of mitochondria in RTN3-null mouse kidneys. Previous studies have revealed that activation of the JAK2-STAT3 pathway may induce mitochondrial dysfunction and oxidative stress by disrupting the balance between BCL2 and BAX43,44. A specific JAK2 inhibitor can attenuate TNF-α-induced oxidative stress in renal tubular epithelial cells45. Hence, the mitochondrial dysfunction in RTN3-null mouse kidneys described in our current study may also have occurred via the JAK2-STAT3 pathway.
In summary, our study suggests that the RTN3-null mouse model might be an ideal model for research on CKD and kidney fibrosis because the model recapitulates features of CKD. Reduced RTN3 expression is a potential risk factor for glomerulosclerosis and tubulointerstitium in the kidney because it promotes collagen synthesis and aggregation and impairs mitochondrial structure and function, partly through alteration of the localization of GPBP1 and activation of the IGF2-JAK2-STAT3 pathway. Hence, our findings shed light on the importance of the relationship between ER proteins and CKD/kidney fibrosis in humans and animals. Collectively, our data suggest that RTN3 is a key molecule in the kidneys.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
The authors thank the patients and their families for participating in this study. We also thank BerryGenomics (Bei **g, China) for providing mass spectrometric and RNA-seq-related technical assistance. This study was supported by the National Natural Science Foundation of China (82170598, 82070738, 81970403 and 82000427), Natural Science Foundation of Hunan Province (2020JJ5785 and 2021JJ31015) and Research Project of the Hunan Provincial Health Commission (202103012102, 202103050563, and 202104022248).
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L.-L.F., R.D., and J.-S.L. wrote the draft of the manuscript and performed the cell and molecular experiments; J.-S.L. enrolled the patients’ samples; J.-Y.J. and C.-Y.W. performed HE, PAS staining, and IHC; Y.D. performed animal feeding; W-X.H. performed cell culture; and R.-Q.Y. and R.X. designed and supported the project. All authors approved the final manuscript.
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Fan, LL., Du, R., Liu, JS. et al. Loss of RTN3 phenocopies chronic kidney disease and results in activation of the IGF2-JAK2 pathway in proximal tubular epithelial cells. Exp Mol Med 54, 653–661 (2022). https://doi.org/10.1038/s12276-022-00763-7
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DOI: https://doi.org/10.1038/s12276-022-00763-7
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