Urinary single-cell sequence analysis of the urinary macrophage in different outcomes of membranous nephropathy.

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.

Development and Validation of a Prognostic Nomogram to Predict 30-Day Mortality Risk in Patients with Sepsis-Induced Cardiorenal Syndrome.

Introduction: Sepsis-induced cardiorenal syndrome (sepsis-induced CRS) is a devastating medical condition that is frequently associated with a high fatality rate. In this study, we aimed to develop an individualized nomogram that may help clinicians assess 30-day mortality risk in patients diagnosed with sepsis-induced CRS. Methods: A total of 340 patients with sepsis-induced CRS admitted from January 2015 to May 2019 in Shanghai Tongji Hospital were used as a training cohort to develop a nomogram prognostic model. The model was constructed using multivariable logistic analyses and was then externally validated by an independent cohort of 103 patients diagnosed with sepsis-induced CRS from June 2019 to December 2020. The prognostic ability of the nomogram was assessed through discrimination, calibration, and accuracy. Results: Five prognostic factors were determined and included in the nomogram: age, Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, vasopressors, baseline serum creatinine, and the rate of change in myoglobin. Our prognostic nomogram showed well-fitted calibration curves and yielded strong discrimination power with the area under the curve of 0.879 and 0.912 in model development and validation, respectively. In addition, the nomogram prognostic model exhibited an evidently higher predictive accuracy than the SOFA score. Conclusions: We developed a prognostic nomogram model for patients with sepsis-induced CRS and externally validated the model in another independent cohort. The nomogram exhibited greater strength in predicting 30-day mortality risk than the SOFA score, which may help clinicians estimate short-term prognosis and modulate therapeutic strategies.

Clinical Features and Prognosis in ANCA Associated Vasculitis Patients with Acute Kidney Injury.

BACKGROUND: Renal involvement in patients with anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) often presents as acute kidney injury (AKI), which is closely related to the prognosis of critically ill patients. Nevertheless, there are few researches on the subgroup of AVV patients with AKI. The study aimed to explore clinical features and prognosis in AAV patients with AKI. METHODS: A retrospective analysis of AAV patients was conducted. The enrolled patients were grouped according to whether AKI on admission occurred or not. Demographic, clinical data and follow-up records were gathered from medical histories. Survival rates of AAV patients with AKI and risk factors of all AAV patients were studied. RESULTS: (1) In the AKI group, the levels of hemoglobin, evaluated glomerular filtration rate (eGFR), serum albumin and complement 3 were significantly lower (P<0.05); the proportions of microscopic polyangiitis (MPA) and levels of serum creatinine (SCr) on admission, red blood cell (RBC) counts in urine, 24-hour urine protein excretion (UPE) and Birmingham Vasculitis Activity Score (BVAS) were significantly higher (P<0.05). There was a significantly lower incidence of otorhinolaryngologic involvement in the AKI group (P<0.05). (2) There were significantly lower survival rates and renal survival rates in the AKI group (P<0.05). (3) Higher creatinine and AKI were risk factors for poor prognosis in AAV patients. CONCLUSION: AAV patients with AKI have more severe kidney damage, higher disease activity and worse prognosis. More attention should be paid to AAV patients with AKI for both remission induction and infection prevention.

A Single-Cell RNA-Seq-Based Approach for Genome-Wide Identification of Cell Essential Genes.

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.

Correction: Intellectual capital and the efficiency of SMEs in the transition economy China; Do financial resources strengthen the routes?

[This corrects the article DOI: 10.1371/journal.pone.0235462.].

Intellectual capital and the efficiency of SMEs in the transition economy China; Do financial resources strengthen the routes?

Intellectual capital has been grabbed the attention of researchers due to its momentous role in sustainable competitive advantage and organizational success. There is a growing catalog of related assessments, publications and reviews that display the direct and indirect role of intellectual capital in business success and profitability. Despite the bourgeoning literature, studies have not yet unleashed the influence of each dimension of intellectual capital; human capital, structural capital and customer capital on SMEs' efficiency with financial resources as a moderator. The present study fills the gap and assesses if financial resources strengthen the paths between the dimensions of intellectual capital and SMEs' efficiency. A survey method was used and collected evidence from 264 Chinese SMEs. The findings exhibit that human capital directly enhances SMEs' efficiency but the presence of financial resources as a moderator weakens the influence. However, social capital and customer capital do not directly improve SMEs' efficiency but financial resources reinforce the paths social and customer capital and SMEs efficiency. This research recommends that owners and managers of SMEs need to use their financial resources complementary with structural and customer capital while human capital should be used exclusively.

Warming deferentially altered multidimensional soil legacy induced by past land use history.

The legacy effects of previous land use and climate history may affect current soil function. However, the manner in which these legacy effects of land use are modulated by the subsequent climate remains unclear. For this reason, we investigated how the legacies of soil multiple functions left by conversion of grassland to agricultural management were mediated by climate warming with a reciprocal transplant approach. The overall legacy was further separated into the contributions by changes in the abiotic properties of the soil (abiotic process) and microbial community (biotic process). We here hypothesized that warming may mediate the legacy effects of previous land use, mainly by changing biotic processes. Results indicated that warming significantly influenced the total legacies of soil respiration and three exoenzyme activities representing recalcitrant carbon, nitrogen, and phosphorus cycling, but did not affect the total legacy of ß-1,4-glucosidase activity, which is involved in labile carbon cycling. The relative contributions of abiotic and biotic processes to the warming effects on the total legacy depended on the type of soil function. The effects of warming on land use change legacies were derived from altered bacterial community structure. The results of the present study suggest that climate conditions could interact with land use legacy to determine the ecosystem functions in a process-specific way.

Triptolide protects podocytes from TGF-ß-induced injury by preventing miR-30 downregulation.

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-ß signaling is a critical mediator in various podocyte injuries and we previously found that TGF-ß induces podocyte injury through downregulating miR-30s, we thus used TGF-ß-induced podocyte injury model to address the issue. We found that Triptolide is capable of protecting cultured podocytes from TGF-ß-induced cytoskeletal injury and apoptosis, as expected. Consistently, Triptolide also prevented TGF-ß-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-ß-induced miR-30 downregulation, indicating that Triptolide protects podocytes by sustaining miR-30 expression. Mechanistically, we found that Triptolide can prevent TGF-ß-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-ß-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.

Genome-wide identification of genes essential for podocyte cytoskeletons based on single-cell RNA sequencing.

Gene expression differs substantially among individual cells of the same type. We speculate that genes that are expressed in all but a portion of cells of a given cell type would be likely essential and required for either the cell survival (housekeeping) or for the cell type's unique structure and function, enabling the organism to survive. Here, we performed RNA-seq of 20 mouse podocytes using the Fluidigm C1 system and identified 335 genes that were expressed in all of them. Among them, 239 genes were also expressed in mesangial and endothelial cells and were involved in energy metabolism, protein synthesis, etc., as housekeeping genes. In contrast, 92 genes were preferentially expressed in podocytes (over five-fold versus expression in mesangial and endothelial cells) and are, therefore, the essential candidate genes specific for podocytes. Assessments by bioinformatics, conserved expression in human podocytes, and association with injury/disease all support the essentiality of these genes for podocytes. Factually, 27 of the 92 genes are already known to be essential for podocyte structure and function. Thirty-seven novel genes were functionally analyzed by siRNA silencing, and we found that a deficiency of 30 genes led to either cytoskeletal injury (FGFR1, AOX1, AIF1L, HAUS8, RAB3B, LPIN2, GOLIM4, CERS6, ARHGEF18, ARPC1A, SRGAP1, ITGB5, ILDR2, MPP5, TSC22D1, DNAJC11, SEPT10, MOCS2, FNBP1L, and TMOD3) or significant downregulation of CD2AP and synaptopodin (IFT80, MYOM2, ANXA4, CYB5R4, GPC1, ZNF277, NSF, ITGAV, CRYAB, and MTSS1). Thus, the list of genes essential for podocyte cytoskeletons is expanded by single-cell RNA sequencing. It appears that podocyte-specific essential genes are mainly associated with podocyte cytoskeletons.

Single-cell RNA-sequence analysis of mouse glomerular mesangial cells uncovers mesangial cell essential genes.

Mesangial cells are essential for the structure and function of glomeruli, but the mechanisms underlying these roles are not well understood. Here, we performed a single-cell RNA-sequence (RNA-seq) analysis of mouse mesangial cells using the Fluidigm C1 platform. We found that gene expression in individual mesangial cells was tremendously heterogeneous, with mean correlation coefficients of 0.20, and most mesangial genes were actually expressed in only a portion of mesangial cells and are therefore presumably dispensable. In contrast, 1,045 genes were expressed in every single mesangial cell and were considered mesangial cell essential genes. A gene ontology analysis revealed a significant enrichment of genes associated with the endothelium, supporting the inference that mesangial cells function as pericytes. Among 58 endothelium-associated genes, 18 encode proteins that are secreted and may be directly involved in endothelial homeostasis. Importantly, 11 (Angpt2, Anxa5, Axl, Ecm1, Eng, Fn1, Mfge8, Msn, Nrp1, Serpine2, and Sparc) were upregulated, while 2 (Apoe and Fgf1) were downregulated in various glomerulopathies. The enrichment of genes associated with other reported functions of mesangial cells was also found. Furthermore, we identified 173 genes specifically expressed in every mesangial cell in glomeruli from the mesangial cell essential gene list. Finally, based on single mesangial cell RNA-seq results, we found that commonly used glomerular cell type markers, including Fhl2, Igfbp5, Wt1, Tek/Tie2, Kdr/Flk1, Flt1/Vegfr1, and Cd34, are actually not specific. Thus, single mesangial cell RNA-seq analysis has provided insights into the functions and underlying mechanisms of mesangial cells.

Toll-like Receptor 9 Can be Activated by Endogenous Mitochondrial DNA to Induce Podocyte Apoptosis.

Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases.

MicroRNA-30 family members regulate calcium/calcineurin signaling in podocytes.

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 ß3 (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.

Circulating Mitochondrial DAMPs Are Not Effective Inducers of Proteinuria and Kidney Injury in Rodents.

Mitochondria in eukaryotic cells are derived from bacteria in evolution. Like bacteria, mitochondria contain DNA with unmethylated CpG motifs and formyl peptides, both of which have recently been shown to be damage associated molecular patterns (DAMPs) and induce immune response and cell injury. Based on the facts that circulating mitochondrial DAMPs (mtDAMPs) are increased in the patients of trauma or burn injury who also have proteinuria, that mtDAMPs can activate immune cells which in turn secrete glomerular permeability factors, that renal intrinsic cells express a variety of DAMP receptors, and that mtDAMPs can directly increase endothelial cell permeability in vitro, we hypothesized that mtDAMPs may be novel circulating factors inducing proteinuria and kidney injury. We tested this hypothesis by directly injecting mtDAMPs into rodents and examining urinary protein and kidney histology. We prepared mtDAMP samples, including mitochondrial DNA (mtDNA) and mitochondrial debris (MTD), from rodent liver. In mice, injection of mtDNA for 20 µg/ml initial concentration in circulation (much higher than the clinical range), did not cause any renal manifestations. However, an increased dose leading to 45 µg/ml initial concentration in circulation resulted in a transient, slight increase in urinary albumin. In rats, MTD injection resulting in 450 µg/ml initial concentration of MTD protein in circulation, which was much higher than the clinical range, caused mild, transient proteinuria and lung lesions. Multiple injections of such large amount of either mtDNA or MTD into rodents on 3 consecutive days also failed in inducing proteinuria and kidney injury. In summary, clinical levels of circulating mtDAMPs do not induce proteinuria and clinically irrelevant high levels of mtDAMPs cause only a transient and slight increase in urinary protein in rodents, suggesting that circulating mtDAMPs may not be responsible for the proteinuria and kidney injury in patients with trauma, burn injury, and other diseases.

Laparoscopic management or laparoscopy combined with transvaginal management of type II cesarean scar pregnancy.

OBJECTIVE: To evaluate the clinical effectiveness of laparoscopic management of cesarean scar pregnancy (CSP) by deep implantation. BACKGROUND: A pregnancy implanting within the scar from a previous cesarean delivery is a rare condition of ectopic pregnancy. There are two different types of CSPs. Type I is caused by implantation of the amniotic sac on the scar with progression toward either the cervicoisthmic space or the uterine cavity. Type II (CSP-II) is caused by deep implantation into a previous CS defect with infiltrating growth into the uterine myometrium and bulging from the uterine serosal surface, which may result in uterine rupture and severe bleeding during the first trimester of pregnancy. Thus, timely management with an early and accurate diagnosis of CSP-II is important. However, laparoscopic management in CSP-II has not yet been evaluated. METHODS: Eleven patients with CSP-II underwent conservative laparoscopic surgery or laparoscopy combined with transvaginal bilateral uterine artery ligation and resection of the scar with gestational tissue and wound repair to preserve the uterus from March 2008 to November 2011. Patients with CSP-II were diagnosed using color Doppler sonography, and the diagnosis was confirmed by laparoscopy. The operation time, the blood loss during surgery, the levels of ß-human chorionic gonadotropin (ß-hCG) before surgery, the time taken for serum ß-hCG levels to return to <100 mIU/mL postoperatively, and the time for the uterine body to revert to its original state were retrospectively analyzed. RESULTS: All 11 operations were successfully performed using laparoscopy with preservation of the uterus. One patient underwent a dilation and curettage after laparoscopic bilateral uterine artery ligation. Eight patients were treated solely by laparoscopic bilateral uterine artery ligation and resection of the scar with gestational tissue and wound repair. The remaining two patients underwent laparoscopic bilateral uterine artery ligation and transvaginal resection of the CS with gestational tissue and wound repair because of dense adhesions and heavy bleeding. The average operation time was 85.5 (±17.5) minutes, and the blood loss was 250.0 (±221.4) mL. The blood serum level of ß-hCG returned to <100 mIU/mL in 16.4 (±5.3) days postoperatively. Among the 10 patients who underwent resection of CS and wound repair, the time for the uterus to revert to its original state (judged by ultra-sonography) was 10.8 (±3.0) days postoperatively. CONCLUSIONS: Laparoscopy can remove ectopic gestational tissue and allow subsequent wound repair, as well as provide diagnostic confirmation. Being a minimally invasive procedure, laparoscopic or laparoscopy combined with transvaginal bilateral uterine artery ligation and resection of the scar with gestational tissue and wound repair can become an effective alternative for the treatment of CSP-II.

Monobutyl phthalate induces the expression change of G-Protein-Coupled Receptor 30 in rat testicular Sertoli cells.

The aim of the study was to explore whether G-Protein-Coupled Receptor 30 (GPR30) was expressed in rat testicular Sertoli cells and to assess the impact of monobutyl phthalate (MBP) on the expression of GPR30 in Sertoli cells. By using RT-PCR, Western-Blot and immunofluorescent microscopy, the expression of GPR30 in rat Sertoli cells was found at both gene and protein level. Cultures of Sertoli cells were exposed to MBP (10- -1000 mM) or a vehicle. The results indicated that the expression of GPR30 increased at gene and protein levels in Sertoli cells following administration of MBP even at a relatively low concentration. We suggest that changes of GPR30 expression may play an important role in the effects of the xenoestrogen MBP on Sertoli cell function. (Folia Histochemica et Cytobiologica 2013, Vol. 51, No. 1, 18-24).