In a study published in Advanced Science, the teams led by Prof. CHEN Luonan from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, and Prof. MOU Shan from the Department of Nephrology, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, collaborated to report the activation of the bone morphogenetic protein-2 (BMP2) pathway in the podocytes from patients with primary membranous nephropathy (PMN), which is mediated by complement components and results in the production of type IV collagen and thickening of glomerular basement membrane (GBM). Additionally, with the DNB (dynamic network biomarker) method, the research predicted a key cell state related to the critical transition of PMN.
Chronic kidney disease (CKD) is a severe burden on human health, and proteinuria represents one of its key clinical features. Massive proteinuria (>3.5 g/24 hours) causes nephrotic syndrome (NS), resulting in severe systemic infection, fatal thromboembolism, acute kidney injury, and metabolic disorders, which seriously affects the long-term prognosis of patients and can lead to the end-stage renal disease.
PMN is an autoimmune glomerular disease, whose major clinical manifestation is proteinuria, and represents the most frequent primary cause of NS in adults. Elucidating the pathogenesis and signaling pathways of PMN is of great significance for its diagnosis and treatment.
The histological structure of the kidney is complex as it has multiple cell types, including the inherent cell types of glomeruli, renal tubules, renal interstitium, and infiltrated immune cells. PMN is caused by autoantibodies targeting autoantigens on podocytes, which combine to form immune complexes and activate the complement system, resulting in the impairment of the renal filtration barrier and the abnormal leakage of proteins into the urine to form proteinuria.
Podocyte is a rare cell type in the kidney, thus, their abnormal signals in diseases are hard to detect. The study included kidney core needle biopsy samples from 11 PMN patients and 7 healthy kidney transplant donors for single-cell RNA sequencing. After refined clustering, the podocyte cluster with sufficient abudance was determined.
Bioinformatics analysis revealed upregulated expression of BMP2 in podocytes of PMN patients together with type IV collagen subunits COL4A3 and COL4A4, the main components of GBM matrix, which may be associated with GBM thickening, the core pathological change of PMN.
BMP2 belongs to the transforming growth factor-β superfamily, by phosphorylating SMADs, BMP2 regulates downstream gene to express extracellular matrix (ECM) production. Therefore, the researchers cultured immortalized mouse podocyte cell lines in vitro and stimulated them with BMP2 recombinant protein, and found that the BMP2/pSMAD1/COL4 pathway was activated in podocytes with increased expression of Col4a3 and Col4a4, which matched the results of single-cell analysis. Meanwhile, immunostaining was performed on kidney sections to verify the changes in PMN podocytes at the protein level. Further exploration into the upstream of this pathway unraveled that complement components C3a and C5b-9 can promote the expression of BMP2 and activate the downstream pathway, thereby associating the activation of the complement system with the thickening of GBM in the pathogenesis of PMN.
Considering that podocytes are main target cells of PMN, and there may be critical state transitions during the occurrence and development of PMN, the researchers delineated 3 continuous states of PMN podocytes based on pseudo-time analysis, and used the DNB algorithm originally developed by Chen’s lab to predict a state of podocytes near/at the tipping point of PMN pathological transition. Within the tipping state of podocytes, strong fluctuations of the gene regulatory network related to BMP2 were observed, which may affect the expression of downstream ECM components.
Moreover, as a renal-limited autoimmune disease, immune cells in the PMN kidneys present specific alterations. The study found that there is increased macrophage infiltration in the PMN kidney, which may be differentiated from classical monocytes and eventually exhibit an M2-polarized anti-inflammatory phenotype, and perform complex functions through the expression of apolipoproteins, antigen processing and presentation molecules, and chemokines.
In addition, B cells were activated in PMN kidneys with augmented expression of HLA-II and immunoglobulin IgG molecules. Cell talk analysis showed a delicate and complex regulatory network between infiltrated immune cells and renal inherent cells, and between the immune cells themselves in PMN kidneys.
Overall, the current study provides new insights into the role of podocytes and immune cells in the pathomechanisms of PMN, contributing to the complete understanding of PMN pathophysiology.
Contact：lnchen@sibcb.ac.cn
Reference：http://doi.org/10.1002/advs.202404151
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