Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-β released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.

Poor graft function (PGF) is a severe complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). Murine studies have demonstrated that effective haematopoiesis depends on the specific bone marrow (BM) microenvironment. Increasing evidence shows that BM macrophages (MФs), which constitute an important component of BM immune microenvironment, are indispensable for the regulation of haematopoietic stem cells (HSCs) in the BM. However, little is known about the number and function of BM MФs or whether they directly interact with HSCs in PGF patients. In the current prospective case-control study, PGF patients showed a significant increase in classically activated inflammatory MФs (M1; 2·18 ± 0·11% vs. 0·82 ± 0·06%, P < 0·0001), a striking reduction in alternatively activated anti-inflammatory MФs (M2; 3·02 ± 0·31% vs. 21·89 ± 0·90%, P < 0·0001), resulting in a markedly increased M1/M2 ratio (0·82 ± 0·06 vs. 0·06 ± 0·002; P < 0·0001) in the BM compared with good graft function patients. Meanwhile, standard monocyte subsets were altered in PGF patients. Dysfunctional BM MФs, which were characterized by reduced proliferation, migration and phagocytosis, were evident in PGF patients. Furthermore, BM MФs from PGF patients with high tumour necrosis factor-α and interleukin 12 levels and low transforming growth factor-β levels, led to impaired BM CD34 cell function. In summary, our data indicate that an unbalanced BM M1/M2 ratio and dysfunctional MФs may contribute to the occurrence of PGF following allo-HSCT.

Graft-versus-host disease (GVHD) is a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT) that is frequently associated with bone marrow (BM) suppression, and clinical management is challenging. BM endothelial progenitor cells (EPCs) play crucial roles in the regulation of haematopoiesis and thrombopoiesis. However, little is known regarding the functional roles of BM EPCs in acute GVHD (aGVHD) patients. In the current prospective case-control study, reduced and dysfunctional BM EPCs, characterized by decreased migration and angiogenesis capacities and increased levels of reactive oxygen species (ROS) and apoptosis, were found in aGVHD patients compared with those without aGVHD. Moreover, lower frequency and increased levels of ROS, apoptosis and DNA damage, but reduced colony-forming unit-plating efficiency were found in BM CD34 + cells of aGVHD patients compared with those without aGVHD. The severity of aGVHD and GVHD-mediated cytopenia was associated with BM EPC impairment in aGVHD patients. In addition, the EPC impairment positively correlated with ROS level. Taken together, our results suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Although these findings require validation, our data indicate that improvement of BM EPCs may represent a promising therapeutic approach for aGVHD patients.

Background: Endothelial cells (ECs) function as an instructive platform to support haematopoietic stem cell (HSC) homeostasis. Our recent studies found that impaired bone marrow (BM) ECs are responsible for the defective haematopoiesis in patients with poor graft function (PGF), which is characterised by pancytopenia post-allotransplant. Although activated autophagy was reported to benefit ECs, whether EC autophagy plays a critical role in supporting HSCs and its effect on PGF patients post-allotransplant remain unclear. Methods: To evaluate whether the autophagy status of ECs modulates their ability to support haematopoiesis, human umbilical vein endothelial cells (HUVECs) and primary BM ECs derived from healthy donors were subjected to knockdown or overexpression of Beclin-1 (an autophagy-related protein). Moreover, BM ECs derived from PGF patients were studied. Findings: Beclin-1 knockdown significantly reduced the haematopoiesis-supporting ability of ECs by suppressing autophagy, which could be restored by activating autophagy via Beclin-1 upregulation. Moreover, autophagy positively regulated haematopoiesis-related genes in HUVECs. Subsequently, a prospective casecontrol study demonstrated that defective autophagy reduced Beclin-1 expression and the colony-forming unit (CFU) plating efficiency in BM ECs from PGF patients compared to matched patients with good graft function. Rapamycin, an autophagy activator, quantitatively and functionally improved BM ECs from PGF patients in vitro and enhanced their ability to support HSCs by activating the Beclin-1 pathway. Interpretation: Our results suggest that the autophagy status of ECs modulates their ability to support haematopoiesis by regulating the Beclin-1 pathway. Defective autophagy in BM ECs may be involved in the pathogenesis of PGF post-allotransplant. Rapamycin provides a promising therapeutic approach for PGF patients. Funding: Please see funding sources.

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MicroRNAs (miRNAs) possess an important regulating effect among numerous renal diseases, while their functions in the process of epithelial-to-mesenchymal transition (EMT) after podocyte injury remain unclear. The purpose of our study is to identify the potential functions of miR-30a in EMT of podocytes and explore the underlying mechanisms of miR-30a in the impaired podocytes. The results revealed that downregulation of miR-30a in podocyte injury animal models and patients, highly induced the mesenchymal markers of EMT including Collagen I, Fibronectin and Snail. Furthermore, overexpression of miR-30a enhances epithelial markers (E-cadherin) but diminished mesenchymal markers (Collagen I, Fibronectin and Snail) in podocytes. In addition, we established miR-30a target NFATc3, an important transcription factor of Non-canonical Wnt signaling pathway. More importantly, our findings demonstrated that the augmentation of miR-30a level in podocytes inhibits the nuclear translocation of NFATc3 to protect cytoskeleton disorder or rearrangement. In summary, we uncovered the protective function of miR30a targeting NFATc3 in the regulation of podocyte injury response to EMT.

BackgroundThe renal tubules, which have distant metabolic features and functions in different segments, reabsorb >99% of approximately 180 l of water and 25,000 mmol of Na + daily. Defective metabolism in renal tubules is involved in the pathobiology of kidney diseases. However, the mechanisms underlying the metabolic regulation in renal tubules remain to be defined.MethodsWe quantitatively compared the proteomes of the isolated proximal tubules (PT) and distal tubules (DT) from C57BL/6 mouse using tandem mass tag (TMT) labeling-based quantitative mass spectrometry. Bioinformatics analysis of the differentially expressed proteins revealed the significant differences between PT and DT in metabolism pathway. We also performed in vitro and in vivo assays to investigate the molecular mechanism underlying the distant metabolic features in PT and DT.FindingsWe demonstrate that the renal proximal tubule (PT) has high expression of lipid metabolism enzymes, which is transcriptionally upregulated by abundantly expressed PPARα/γ. In contrast, the renal distal tubule (DT) has elevated glycolytic enzyme expression, which is mediated by highly expressed c-Myc. Importantly, PPARγ transcriptionally enhances the protease iRhom2 expression in PT, which suppresses EGF expression and secretion and subsequent EGFR-dependent glycolytic gene expression and glycolysis. PPARγ inhibition reduces iRhom2 expression and increases EGF and GLUT1 expression in PT in mice, resulting in renal tubule hypertrophy, tubulointerstitial fibrosis and damaged kidney functions, which are rescued by 2-deoxy-d-glucose treatment.InterpretationThese findings delineate instrumental mechanisms underlying the active lipid metabolism and suppressed glycolysis in PT and active glycolysis in DT and reveal critical roles for PPARs and c-Myc in maintaining renal metabolic homeostasis. FUND: This work was supported by the National Natural Science Foundation of China (grants 81572076 and 81873932; to Q.Z.), the Applied Development Program of the Science and Technology Committee of Chongqing (cstc2014yykfB10003; Q.Z.), the Program of Populace Creativities Workshops of the Science and Technology Committee of Chongqing (Q.Z.), the special demonstration programs for innovation and application of techniques (cstc2018jscx-mszdX0022) from the Science and Technology Committee of Chongqing (Q.Z.).

Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.