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Background Great progress has been made in the diagnosis and treatment of membranous nephropathy (MN). However, a significant number of patients do not respond to immunosuppressive therapy and eventually progress to end-stage kidney disease. To investigate the mechanism of different outcome of MN, we performed single-cell sequencing to analyze the urine cells of patients with and without complete remission of MN. Methods Urine single-cell RNA sequencing was performed on 12 healthy controls (HC) and 15 patients with MN. The patients were divided into a complete remission group (CR, n  = 9) and a no remission group (NR, n  = 6). Results (i) Macrophages were the largest group in urine cells, comprising 48.02%, 68.96% and 20.95% in the HC, CR and NR groups, respectively. (ii) Urinary macrophages expressing FIColin-1 and S100 calcium-binding protein A8 were mainly found in the HC and CR groups, indicating that they were derived from bone marrow and peripheral blood, while the urinary macrophages expressing the regulator of G-protein signaling 1 (RGS1) and HLA-DPA1 , mainly found in the NR group, were derived from renal resident macrophages. (iii) In healthy adults, urine macrophages expressed the metallothionein family, indicating that they can regulate anti-inflammatory and proinflammatory functions bidirectionally. In the CR group, the urine macrophages showed strong proinflammatory properties. In the NR group, the urinary macrophages mainly associated with the level of proteinuria and the impaired renal function. Conclusions Our study firstly delineated the differences in urinary cell maps between healthy individuals and MN patients with CR or NR outcomes. Not only the origin but also the function of urine macrophages were different in the HC, CR and NR groups. [ABSTRACT FROM AUTHOR]

Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular [Ca.sup.2+] concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β53 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling., Introduction Podocyte injury and loss have emerged as a central pathological mechanism underlying glomerular injury and sclerosis (1). Although multiple signaling pathways have been implicated in podocyte injury (2, 3), [...]

Calcium/calcineurin signaling is critical for normal cellular physiology. Abnormalities in this pathway cause many diseases, including podocytopathy; therefore, understanding the mechanisms that underlie the regulation of calcium/calcineurin signaling is essential. Here, we showed that critical components of calcium/calcineurin signaling, including TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, are the targets of the microRNA-30 family (miR-30s). We found that these 5 genes are highly expressed as mRNA, but the level of the proteins is low in normal podocytes. Conversely, protein levels were markedly elevated in podocytes from rats treated with puromycin aminonucleoside (PAN) and from patients with focal segmental glomerulosclerosis (FSGS). In both FSGS patients and PAN-treated rats, miR-30s were downregulated in podocytes. In cultured podocytes, PAN or a miR-30 sponge increased TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3 expression; calcium influx; intracellular Ca2+ concentration; and calcineurin activity. Moreover, NFATC3 nuclear translocation, synaptopodin degradation, integrin β3 (ITGB3) activation, and actin fiber loss, which are downstream of calcium/calcineurin signaling, were induced by miR-30 reduction but blocked by the calcineurin inhibitor FK506. Podocyte-specific expression of the miR-30 sponge in mice increased calcium/calcineurin pathway component protein expression and calcineurin activity. The mice developed podocyte foot process effacement and proteinuria, which were prevented by FK506. miR-30s also regulated calcium/calcineurin signaling in cardiomyocytes. Together, our results identify miR-30s as essential regulators of calcium/calcineurin signaling. [ABSTRACT FROM AUTHOR]

Identification of genes essential for structure, function, and survival of a cell type is critical for understanding of the underlying mechanisms. Unfortunately, there is no efficient way to identify such genes. Studies by single-cell RNA sequencing have shown that gene expressions of single cells of the same type are highly heterogeneous. We therefore speculate that the genes expressed in all individual cells of the same type are essential for the cell type, including the housekeeping genes and cell type-specific essential genes. Based on this rationale, we design a high-throughput approach to identify podocyte essential genes. In this approach, mouse podocytes are subjected to ultra-deep single-cell RNA-seq, and the genes expressed in all single podocytes are sorted out and considered as the candidates of podocyte essential genes. The essentiality of these genes for podocytes is assessed by bioinformatics, cross-species conserved expression, association with injury/disease, inclusion of known essential genes, and experimental validation. By comparison with the essential genes of other cell types, podocyte-specific essential genes can be distinguished. This approach applies to any cell types. In this chapter, we describe the approach and detailed methods., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Triptolide is known to have a strong anti-proteinuric effect through direct protection of podocytes from injury and is used to treat glomerular diseases. However, the mechanism underlying its protective effect on podocytes remains elusive. MiR-30 family has recently been shown to be essential for structural and functional homeostasis of podocytes but is downregulated by injurious factors, leading to podocyte injury. In the present study, we explore whether Triptolide protects podocytes through preventing miR-30 downregulation. Since TGF-β signaling is a critical mediator in various podocyte injuries and we previously found that TGF-β induces podocyte injury through downregulating miR-30s, we thus used TGF-β-induced podocyte injury model to address the issue. We found that Triptolide is capable of protecting cultured podocytes from TGF-β-induced cytoskeletal injury and apoptosis, as expected. Consistently, Triptolide also prevented TGF-β-induced signaling activation of MAPK p38, NFkB (p65) and calcineurin/NFATC3, which are known to be downstream mediators of podocyte injury. Meanwhile, Triptolide was found to completely prevent TGF-β-induced miR-30 downregulation, indicating that Triptolide protects podocytes by sustaining miR-30 expression. Mechanistically, we found that Triptolide can prevent TGF-β-induced Smad2/3 phosphorylation/activation, which likely underlies miR-30 restoration by Triptolide. We also performed ex vivo study and found that Triptolide prevented TGF-β-induced miR-30 downregulation and Smad2/3 phosphorylation in the isolated glomeruli of mice or rats. Thus, our study has provided novel insights into the mechanism underlying the therapeutic effectiveness of Triptolide on podocytopathies., Competing Interests: None.