| The parietal epithelial cells(PECs)are a single layer of epithelial cells located on the basal membrane of the renal corpuscle.They form the second barrier of the glomerular filtration structure through the tight connections between PECs.PECs connect the glomerulus and renal tubules,allowing the primary urine produced by the glomerulus to be transported smoothly into the renal tubules.PECs have been found to have other characteristics besides their anatomical location.Studies have found that there may be a subpopulation of PECs that have features similar to podocytes and are located at the vascular pole of the glomerulus.These cells may have the ability to differentiate into podocytes.Another subpopulation of PECs may be able to differentiate into renal tubular epithelial cells and may be located at the urinary pole of the glomerulus.In addition,PECs can proliferate pathologically in focal segmental glomerulosclerosis and crescentic glomerulonephritis,which can promote deterioration of glomerular function.However,there is currently no clear understanding of PEC subpopulations,their therapeutic value in podocytes repair,or their pathogenic mechanisms in crescentic glomerulonephritis.Although heterogeneity has been found in cell morphology and marker protein expression among PECs,these classification methods based on morphology and markers cannot represent functional differences among different subpopulations of PECs.Our study based on single-cell analysis of mouse kidney identified PEC subpopulation composition and biological functions and further explored the role of different PEC subpopulations in podocytes injury repair and crescent formation.We found that Mif and Csflr were intervention targets for crescent formation in glomeruli and screened and verified that Caffeic acid and Sotuletinib could inhibit crescent formation by intervening in Mif and Csflr respectively in mice.Purpose:1.To explore the subpopulation composition and biological functions of PECs,identify subpopulations of progenitor cells involved in podocyte differentiation and renal tubular epithelial cell differentiation.2.To analyze the role of PECs in crescent formation,as well as the mechanism of PEC activation and proliferation,screen out targets with intervention value,and understand drugs with clinical application prospects.Methods:Single-cell transcriptome data of mouse glomeruli were retrieved,downloaded and analyzed,and single-cell analysis tools were used to determine the subpopulation composition of PECs,followed by identification of their biological functions using differential genes.Next,we analyzed the lineage transition relationship of PECs with podocytes and renal tubular epithelial cells using single-cell multi-algorithm,the molecular features of PECs involved in podocyte regeneration,and crucially,we analyzed the mechanism of PEC activation and proliferation in glomerular crescent formation using single-cell event flow strategy.Finally,using renal-tissue samples from patients and mouse models of crescentic glomerulonephritis,we identified pathogenic signals identified by single-cell analysis that were highly expressed in the glomerulus and screened for small-molecule inhibitors targeting pathogenic signals.The efficacy and safety of inhibitors in delaying the progression of crescentic glomerulonephritis were verified in a mouse model.Results:1.PECs single-cell grouping and functional identification.Mouse PECs are divided into 5 single-cell subgroups,among which PEC-A1 and PEC-A2 are subgroups of progenitor cells involved in podocyte regeneration.The characteristic of PEC-A1 is that it expresses a large number of podocyte marker genes such as Wt1 and Nphs2 while expressing common PECs marker genes,while PEC-A2 mainly expresses PEC marker genes such as Cldnl and Pax2.PEC-A3 only exists in crescentic glomerulonephritis and has extremely strong proliferation ability,which is a potential subgroup of PECs that form crescents.PEC-A4 is a subgroup of progenitor cells involved in renal tubular epithelial cell regeneration.The characteristic of PEC-B is that it has extremely strong extracellular matrix synthesis and secretion ability,and its proportion increases in various types of renal glomerular injury,suggesting that it may participate in various types of renal glomerular injury.2.Characteristics of podocyte progenitor cells in PECs.Podocyte regeneration in PECs is hierarchical,that is,PEC-A2 differentiates into PEC-A1 and then into podocytes.Wtl plays a promoting role in the differentiation of PEC-A2 to PEC-A1.In immature renal corpuscles(3 weeks old),PEC-A1 and PEC-A2 have stronger migration ability and epithelial cell differentiation potential,while they have been lost in mature renal corpuscles(12 weeks old).Moreover,mature renal corpuscles’ podocytes release Cxc112,1134,Mif.Pros1,and these 4 signal molecules may inhibit the differentiation ability of PECs into podocytes,while immature renal corpuscles do not.3.Participation of PECs in crescent formation.PEC-A3 has extremely strong proliferation ability in crescents and is a subgroup formed by the transformation of PEC-A4 and PECA2,among which Sox9 regulates the expression of Cd44 in PEC-A4 and PEC-A3 and inhibits the expression of Sox9 will reverse the direction of transformation from PEC-A4 to PEC-A3.Crescentic glomerulonephritis staining also verified that Sox9+PECs expressed Ki67 in large quantities at the same time,which increased with the progression of crescents.Upstream and downstream signal analysis suggests that podocytes,monocytes,T cells,endothelial cells and mesangial cells all release Mif,1134,Cxc112,Csfl and Tgfb2 signals targeting PEC subgroups in crescentic glomerulonephritis renal corpuscles,and these signals are expressed at higher levels in podocytes.4.Pharmacological inhibition of pathogenic signals delays the progression of crescentic glomerulonephritis in renal corpuscles.Mif,1134,Cxc112,Csfl and Tgfb2 are highly expressed in the renal corpuscles of patients with crescentic glomerulonephritis and mouse models.qPCR detection of various node-separated mouse renal corpuscles found that Mif 1134,Cxcll2,Csfl and Tgfb2 all appeared transiently with high expression early on.Using small molecule inhibitors caffeic acid for Mif ligand and Sotuletinib for Csf1r receptor to intervene crescentic glomerulonephritis mouse models from day 1 after modeling until day 7 for sampling.The results showed that intervention with Mif and Csfl signal transduction effectively reduced the BUN and sCr levels of mice’s kidney function while increasing blood Alb levels but had no statistically significant effect on proteinuria.At the same time,there was no significant effect on the expression of liver function ALT and AST.Renal tissue PAS staining found that after treatment mice’s crescentic glomerulonephritis renal corpuscles’ proportion of crescents and glomerulosclerosis was significantly reduced,and Cd44 staining also showed that the degree of activation of PECs was significantly reduced.Conclusion:1.PECs exhibit cellular heterogeneity in morphology,molecular characteristics,and function;and progenitor cells that differentiate into both podocytes and tubular epithelial cells are present in PECs.2.The progenitor cells of podocytes in PECs gradually lose their migration and differentiation abilities as the glomerulus develops and are inhibited from differentiating into podocytes by signals released by podocytes.3.Among the subgroups of PECs,PEC-A3 is a key subgroup involved in cellular crescent formation,and Sox9 expression promotes the appearance of PEC-A3 in crescentic glomerulonephritis.4.After anti-GBM antibody-induced glomerular injury,various cells dominated by podocytes release pathogenic signaling molecules such as Mif and Csf1 to stimulate normal subgroups of PECs,namely PEC-A4 and PEC-A2,to differentiate into PEC-A3,leading to crescentic glomerulonephritis.Targeted intervention with Mif and Csflr using caffeic acid and Sotuletinib can effectively inhibit the progression of crescentic glomerulonephritis. |
