High Renal Mast Cell Density Is Associated with Poor Prognosis in Patients with Immunoglobulin A Nephropathy.

INTRODUCTION: This observational cohort study evaluated the prognostic value of mast cells in the pathogenesis and progression of IgA nephropathy. METHODS: A total of 76 adult IgAN patients were enrolled into this study from Jan 2007 and June 2010. Immunohistochemistry and immunofluorescence were used to identify tryptase-positive mast cells in renal biopsy samples. Patients were classified into Tryptasehigh and Tryptaselow groups. Depending on an average of 96-month follow-up, the predictive value of tryptase-positive mast cells in IgAN progression was analyzed. RESULTS: Tryptase-positive mast cells were found frequently in IgAN kidneys while rarely observed in normal kidneys. We also found IgAN patients in Tryptasehigh group presented both severe clinical and pathological renal manifestations. Furthermore, Tryptasehigh group contained more interstitial macrophages and lymphocytes infiltration than Tryptaselow group. Higher tryptase-positive cells density is associated with poor prognosis in patients with IgAN. CONCLUSIONS: High renal mast cells density is associated with severe renal lesions and poor prognosis in patients with Immunoglobulin A nephropathy. High renal mast cells density might be used as a predictor of poor prognosis in patients with IgAN.

Depletion of macrophages with clodronate liposomes partially attenuates renal fibrosis on AKI-CKD transition.

Clodronate liposomes are bisphosphonates encapsulated by liposomes that are known to induce macrophage depletion in vivo. In a previous study, clodronate liposomes improved renal ischemia/reperfusion (I/R) injury in mice, which may be due to effects on macrophage phenotypes. However, how inflammatory cytokines secretion participates is unknown. In this study, we investigated the effect of macrophages in the I/R kidney by depleting macrophages with clodronate liposomes and changing inflammatory cytokines. C57BL/6 mice underwent I/R injury with or without clodronate liposomes administration on Days 5 and 15. Tubular injury, collagen deposition, and fibrosis were detected and analyzed by histological staining, immunocytochemistry (IHC), flow cytometry (FACS), and reverse transcription-polymerase chain reaction (RT-PCR). Inflammatory cytokines were detected and analyzed by Western blotting and RT-PCR. We found that clodronate liposomes alleviated renal fibrosis and tissue damage on both Days 5 and 15. KIM-1, IL-10, and TGF-ß were reduced significantly in the clodronate liposomes treatment group. However, TNF-α was not different between the clodronate liposomes treatment group and the phosphate-buffered saline treatment group on either Day 5 or Day 15. Thus, clodronate liposomes can alleviate renal fibrosis and tissue damage and reduce the inflammatory cytokines IL-10 and TGF-ß, suggesting that clodronate liposomes alleviate renal fibrosis may because of M1/M2 polarization.

Lopinavir/ritonavir is associated with pneumonia resolution in COVID-19 patients with influenza coinfection: A retrospective matched-pair cohort study.

During the early stages of the pandemic, some coronavirus disease (COVID-19) patients were misdiagnosed as having influenza, which aroused the concern that some deaths attributed to influenza were actually COVID-19-related. However, little is known about whether coinfection with influenza contributes to severity of COVID-19 pneumonia, and the optimal therapeutic strategy for these patients. We retrospectively studied 128 hospitalized patients with COVID-19 pneumonia. All patients were positive severe acute respiratory syndrome coronavirus 2 positive by nucleic acid detection. Sixty-four cases were coinfected with influenza A/B and the other 64 were influenza negative, matched by age, sex, and days from onset of symptoms. Among the 64 coinfected patients, 54 (84.4%) were coinfected with influenza A, and 10 (15.6%) with influenza B. The median duration of viral shedding time from admission was longer for patients with influenza coinfection (17.0 days) than for those without influenza coinfection (12.0 days) (P < .001). The multivariable Cox proportional hazards model showed that the hazards ratio of resolution in lung involvement was 1.878 (P = .020) for patients administered lopinavir/ritonavir, compared with those not administered lopinavir/ritonavir (95% confidence interval: 1.103-3.196). Among influenza coinfected patients, those treated with lopinavir/ritonavir exhibited faster pneumonia resolution within 2 weeks after symptom onset (37% vs 1%; P = .001). There was no difference in lung involvement between influenza coinfected and noninfected groups. Lopinavir/ritonavir eliminated the difference of lung involvement between influenza coinfected and noninfected groups, indicating that lopinavir/ritonavir is associated with pneumonia resolution in COVID-19.

Severe glomerular C3 deposition indicates severe renal lesions and a poor prognosis in patients with immunoglobulin A nephropathy.

AIMS: Glomerular complement 3 (C3) deposition is often observed in renal biopsies of patients with IgA nephropathy (IgAN); however, the relationship between the intensity of C3 deposition and the long-term prognosis of IgAN has rarely been reported. In this retrospective study, we aimed to evaluate the prognostic value of glomerular C3 deposition for IgAN progression. METHODS AND RESULTS: From June 2009 to June 2010, a total of 136 adult patients with IgAN were enrolled in the study. According to the intensity of glomerular C3 deposition, patients were divided into a glomerular C3high group (34 patients) and a glomerular C3low group (102 patients). The levels of clinical parameters, glomerular immune complexes, histopathological features, and serum cytokines of the two groups were compared. On the basis of an average of 105 months of follow-up, the predictive value of glomerular C3 deposition for IgAN progression was also investigated. Patients in the C3high group had more severe glomerular IgA, IgG, IgM, and complement factor H deposition, a higher percentage of mesangial hypercellularity (M1), and higher levels of segmental glomerulosclerosis (S1), tubular atrophy/interstitial fibrosis (T2), and crescents (C2) than those in the C3low group. Renal biopsies in the C3high group showed higher densities of interstitial inflammatory cells and higher levels of serum interferon-γ than those in the C3low group. Multivariate Cox regression analysis revealed that a higher intensity of glomerular C3 deposition remained as an independent predictor of serum creatinine doubling and end-stage renal disease. CONCLUSIONS: A high intensity of glomerular C3 deposition is associated with the severity of renal lesions, and predicts long-term poor renal survival for IgAN patients.

Renal Involvement and Early Prognosis in Patients with COVID-19 Pneumonia.

BACKGROUND: Some patients with COVID-19 pneumonia also present with kidney injury, and autopsy findings of patients who died from the illness sometimes show renal damage. However, little is known about the clinical characteristics of kidney-related complications, including hematuria, proteinuria, and AKI. METHODS: In this retrospective, single-center study in China, we analyzed data from electronic medical records of 333 hospitalized patients with COVID-19 pneumonia, including information about clinical, laboratory, radiologic, and other characteristics, as well as information about renal outcomes. RESULTS: We found that 251 of the 333 patients (75.4%) had abnormal urine dipstick tests or AKI. Of 198 patients with renal involvement for the median duration of 12 days, 118 (59.6%) experienced remission of pneumonia during this period, and 111 of 162 (68.5%) patients experienced remission of proteinuria. Among 35 patients who developed AKI (with AKI identified by criteria expanded somewhat beyond the 2012 Kidney Disease: Improving Global Outcomes definition), 16 (45.7%) experienced complete recovery of kidney function. We suspect that most AKI cases were intrinsic AKI. Patients with renal involvement had higher overall mortality compared with those without renal involvement (28 of 251 [11.2%] versus one of 82 [1.2%], respectively). Stepwise multivariate binary logistic regression analyses showed that severity of pneumonia was the risk factor most commonly associated with lower odds of proteinuric or hematuric remission and recovery from AKI. CONCLUSIONS: Renal abnormalities occurred in the majority of patients with COVID-19 pneumonia. Although proteinuria, hematuria, and AKI often resolved in such patients within 3 weeks after the onset of symptoms, renal complications in COVID-19 were associated with higher mortality.

High renal DC-SIGN+ cell density is associated with severe renal lesions and poor prognosis in patients with immunoglobulin A nephropathy.

BACKGROUND AND AIMS: In this observational cohort study, we assessed the prognostic value of DC-SIGN+ cells in the pathogenesis and progression of IgA nephropathy (IgAN). METHODS AND RESULTS: A total of 139 adult IgAN patients were enrolled into this study from June 2009 to June 2010. We characterised DC-SIGN+ cells by immunohistochemistry or immunofluorescence in renal biopsy tissue. Correlations between the DC-SIGN, intercellular adhesion molecule 3 (ICAM-3), CD4 and CD8 were evaluated. Patients were classified into the DC-SIGNhigh and DC-SIGNlow groups. Depending on an average of 100-month follow-up, the predictive value of DC-SIGN+ cells in IgAN progression was analysed. DC-SIGN+ cells were found frequently in IgAN kidneys while rarely observed in normal kidneys, and almost all DC-SIGN+ cells expressed MHC-II. We also found that DC-SIGN+ cells were adjacent to ICAM-3-positive CD4+ and CD8+ lymphocytes. The density of DC-SIGN+ cells was positively and linearly correlated with the density of ICAM-3+ cells, CD4+ cells and CD8+ cells in renal biopsy tissues. In the DC-SIGNhigh group, the degree of renal lesion and inflammatory cell infiltration was more severe compared to the DC-SIGNlow group. Patients in the DC-SIGNhigh group also had increased incidences of deteriorating renal function during the follow up compared to patients in the DC-SIGNlow group. CONCLUSIONS: DC-SIGN+ cells probably served as a potential contributor to exacerbate local inflammatory response. The density of DC-SIGN+ cells was associated with the severity of renal lesions of the patients. High renal DC-SIGN+ cell density might be used as a predictor of poor prognosis in patients with IgAN.

Association of plasma macrophage colony-stimulating factor with cardiovascular morbidity and all-cause mortality in chronic hemodialysis patients.

BACKGROUND: Cardiovascular disease (CVD) events are the main cause of death in long-term hemodialysis (HD) patients. Macrophage colony- stimulating factor (M-CSF) is actively involved in the formation of atherosclerosis and causes plaque instability, thrombosis and the development of acute coronary syndromes. However, little information is available on the role of M-CSF in HD patients. We aimed to investigate the association between plasma M-CSF levels and CVD events as well as all-cause mortality in patients undergoing long-term HD. METHODS: Fifty two HD patients and 8 healthy controls were recruited in this study. HD patients were followed up from September 2014 to May 2017. The primary end point was CVD event, the secondary outcome was death from any cause. Patients were divided into two groups with low and high M-CSF levels based on the optimal cut-off value determined by the ROC curve. Cox regression analyses were used to assess the predictive value of plasma M-CSF for CVD events and all-cause mortality in HD patients. We tested the levels of plasma M-CSF and other inflammatory cytokines in surviving HD patients using ELISA or CBA kit. RESULTS: The average plasma level of M-CSF in 52 patients was approximately twice that of healthy controls (992.4 vs. 427.2 pg/mL; p <  0.05). During 32 months of follow-up, 26 patients (50.0%) had at least one CVD event and 8 patients (15.4%) died. The mean plasma M-CSF concentration increased in survivors after follow-up compared to that detected at baseline (1277.8 ± 693.3 vs. 997.2 ± 417.4 pg/mL; p <  0.05). Multivariate Cox regression analysis showed that plasma M-CSF is an independent risk factor for CVD events in HD patients (p <  0.05). In the Cox regression model after adjusting for gender and age, high M-CSF levels were related to an increased risk of all-cause death (p <  0.05). We also found that M-CSF levels were positively correlated with IL-6 and IL-18 levels (both p < 0.05), which are the major pathogentic cytokines that contribute to HD-related CVD events. CONCLUSION: M-CSF is a prognostic factor for CVD events and all-cause mortality in HD patients.

Macrophages Regulate Unilateral Ureteral Obstruction-Induced Renal Lymphangiogenesis through C-C Motif Chemokine Receptor 2-Dependent Phosphatidylinositol 3-Kinase-AKT-Mechanistic Target of Rapamycin Signaling and Hypoxia-Inducible Factor-1α/Vascular Endothelial Growth Factor-C Expression.

Lymphangiogenesis occurs during renal fibrosis in patients with chronic kidney diseases and vascular endothelial growth factor (VEGF)-C is required for the formation of lymphatic vessels; however, the underlying mechanisms remain unclear. We demonstrate that macrophages can regulate unilateral ureteral obstruction (UUO)-induced renal lymphangiogenesis by expressing high levels of VEGF-C by C-C motif chemokine receptor 2 (CCR2)-mediated signaling. Mice deficient in Ccr2 manifested repressed lymphangiogenesis along with attenuated renal injury and fibrosis after UUO induction. The infiltrated macrophages after UUO induction generated a microenvironment in favor of lymphangiogenesis, which likely depended on Ccr2 expression. Mechanistic studies revealed that CCR2 is required for macrophages to activate phosphatidylinositol 3-kinase (PI3K)-AKT-mechanistic target of rapamycin (mTOR) signaling in response to its ligand monocyte chemoattractant protein 1 stimulation, whereas hypoxia-inducible factor (HIF)-1α is downstream of PI3K-AKT-mTOR signaling. HIF-1α directly bound to the VEGF-C promoter to drive its expression to enhance lymphangiogenesis. Collectively, we characterized a novel regulatory network in macrophages, in which CCR2 activates PI3K-AKT-mTOR signaling to mediate HIF-1α expression, which then drives VEGF-C expression to promote lymphangiogenesis.

A more accurate method acquirement by a comparison of the prediction equations for estimating glomerular filtration rate in Chinese patients with obstructive nephropathy.

BACKGROUND: Researchers have developed several equations to predict glomerular filtration rate (GFR) in patients with chronic kidney diseases (CKD). However, there are scarcely any studies performed to discern the best equation to estimate GFR in patients with pure obstructive nephropathy. In present study, we assessed the suitability of six prediction equations and compared their performance in eGFR evaluation for Chinese patients with obstructive nephropathy. METHODS: A total of 245 adult patients with obstructive nephropathy were enrolled. We evaluated the performance of the 3 Modification of Diet in Renal Disease equations (MDRD) (the original MDRD7, 7MDRD; the abbreviated MDRD, aMDRD; and re-expressed abbreviated MDRD, re-aMDRD) and 3 Chronic Kidney Disease Epidemiology Collaboration equations (CKD-EPI) (CKD-EPI equation based on creatinine alone, CKD-EPIcr; CKD-EPI equation based on cystatin C alone, CKD-EPIcys; CKD-EPI equation based on combined creatinine-cystatin, CKD-EPIcr-cys). The measured GFR (mGFR) by 99mTc-DTPA renal dynamic imaging method was used as the reference GFR. RESULTS: The mean age of the study population was 51.61 ± 14.17 and 131 were male (53.47 %). The mean measured GFR was 66.54 ± 23.99 ml/min/1.73 m2. Overall, the CKD-EPIcr-cys equation gave the best performance with the best correlation (R = 0.72) and agreement (-34.87, 40.83). CKD-EPIcr-cys equation also exhibited the highest accuracy (69.39 %, P < 0.01) and diagnostic efficacy (ROCAUC = 0.874) with the smallest bias (2.98, P < 0.01). In the subgroup of the lowest GFR, CKD-EPIcys equation exhibited the highest accuracy (52.69 %) and the smallest bias (0.27). In the youngest age subgroup, CKD-EPIcys equation had the highest accuracy (71.64 %) and the smallest bias (-1.24). In other subgroups stratified by GFR, age and gender, CKD-EPIcr-cys equation remained the best performance. CONCLUSION: The 3 CKD-EPI equations performed better than the 3 MDRD equations in estimating GFR in Chinese obstructive nephropathy patients; while the CKD-EPI equation based on combined creatinine-cystatin C provided the best estimation of GFR.

Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting ß-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells.

Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. ß-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with ß-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating ß-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in ß-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-ß1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of ß-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-ß1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of ß-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and ß-catenin was detected. We also show that ß-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting ß-catenin translocation to the nucleus.

Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney.

Biliverdin reductase A is an enzyme, with serine/threonine/tyrosine kinase activation, converting biliverdin (BV) to bilirubin (BR) in heme degradation pathway. It has been reported to have anti-inflammatory and antioxidant effect in monocytes and human glioblastoma. However, the function of BVRA in polarized macrophage was unknown. This study aimed to investigate the effect of BVRA on macrophage activation and polarization in injured renal microenvironment. Classically activated macrophages (M1macrophages) and alternative activation of macrophages (M2 macrophages) polarization of murine bone marrow derived macrophage was induced by GM-CSF and M-CSF. M1 polarization was associated with a significant down-regulation of BVRA and Interleukin-10 (IL-10), and increased secretion of TNF-α. We also found IL-10 expression was increased in BVRA over-expressed macrophages, while it decreased in BVRA knockdown macrophages. In contrast, BVRA over-expressed or knockdown macrophages had no effect on TNF-α expression level, indicating BVRA mediated IL-10 expression in macrophages. Furthermore, we observed in macrophages infected with recombinant adenoviruses BVRA gene, which BVRA over-expressed enhanced both INOS and ARG-1 mRNA expression, resulting in a specific macrophage phenotype. Through in vivo study, we found BVRA positive macrophages largely existed in mice renal ischemia perfusion injury. With the treatment of the regular cytokines GM-CSF, M-CSF or LPS, excreted in the injured renal microenvironment, IL-10 secretion was significantly increased in BVRA over-expressed macrophages. In conclusion, the BVRA positive macrophage is a source of anti-inflammatory cytokine IL-10 in injured kidney, which may provide a potential target for treatment of kidney disease.

Lymphatic vessels in patients with crescentic glomerulonephritis: association with renal pathology and prognosis.

BACKGROUND: Various immune cells, including T cells, B cells, macrophages, and neutrophils contribute to the development of crescentic glomerulonephritis. Previous animal studies have suggested that lymphangiogenesis is involved in the migration of inflammatory cells and the activation of adaptive immunity. However, the extent of the association between lymphatic vessels and crescentic glomerulonephritis severity and prognosis remains unknown. METHODS AND RESULTS: In this study, we assessed lymphatic vessel density in 71 patients with crescentic glomerulonephritis who underwent renal biopsies between June 2017 and June 2022. By immunohistochemistry and immunofluorescence, we identified increased lymphatic vessel density in the kidneys of patients with crescentic glomerulonephritis compared to controls. Lymphatic vessels were categorized as total, periglomerular, and interstitial. Spearman's rank correlation analysis showed a positive correlation between total and periglomerular lymphatic vessel density and glomerular crescent proportion. High lymphatic vessel density (total and periglomerular) correlated with declining kidney function, increased proteinuria, and severe glomerular and interstitial pathology. Interstitial lymphatic vessel density had minimal relationship with renal lesions. After a median duration of 13 months of follow-up, higher total and periglomerular lymphatic vessel density was associated with poorer prognosis. Transcriptomic analysis revealed increased immune cell activation and migration in crescentic glomerulonephritis patients compared to healthy controls. Periglomerular lymphatic vessels might play a significant role in immune cell infiltration and renal injury. CONCLUSION: Elevated lymphatic vessel density in patients with crescentic glomerulonephritis is associated with poor prognosis and may serve as a predictive factor for adverse outcomes in these patients.

Erbin interacts with Sema4C and inhibits Sema4C-induced epithelial-mesenchymal transition in HK2 cells.

Erbin, a member of Leucine-rich repeat and PDZ-containing protein family, was found to inhibit TGF-ß-induced epithelial-mesenchymal transition (EMT) in our previous study. However, the mechanism of Erbin in regulating EMT is unclear. Semaphorin protein Sema4C, with PDZ binding site at C-terminal has been recognized as a positive regulator of EMT. Here, we aimed to examine the interaction between Erbin and Sema4C. HK2 cells were treated with TGF-ß1, or transfected with Erbin and (or) Sema4C. Interaction of Erbin and Sema4C was identified by immunoprecipitation. RT-PCR was used to detect the expression of Erbin and Sema4C at mRNA level after transfection. The expression levels of Erbin, Sema4C, and markers of EMT were measured by using Western blotting or ELISA. After HK2 cells were stimulated with 10 ng/mL TGF-ß1 for 72 h, the protein expression levels of Erbin and Sema4C were both up-regulated, and immunoprecipitation results showed Erbin interacted with Sema4C in HK2 cells both at endogenous and exogenous levels. Furthermore, overexpression of Sema4C suppressed E-cadherin, induced vimentin and promoted fibronectin secretion, indicating Sema4C promotes the process of EMT. However, HK2 cells overexpressing Erbin were resistant to Sema4C-induced EMT. In contrast, Erbin specific siRNA promoted EMT induced by Sema4C. Taken together, these results suggest that Erbin can interact with Sema4C, and co-expression of Erbin blocks the process of Sema4C-induced EMT.

Identification of driver genes in lupus nephritis based on comprehensive bioinformatics and machine learning.

Background: Lupus nephritis (LN) is a common and severe glomerulonephritis that often occurs as an organ manifestation of systemic lupus erythematosus (SLE). However, the complex pathological mechanisms associated with LN have hindered the progress of targeted therapies. Methods: We analyzed glomerular tissues from 133 patients with LN and 51 normal controls using data obtained from the GEO database. Differentially expressed genes (DEGs) were identified and subjected to enrichment analysis. Weighted gene co-expression network analysis (WGCNA) was utilized to identify key gene modules. The least absolute shrinkage and selection operator (LASSO) and random forest were used to identify hub genes. We also analyzed immune cell infiltration using CIBERSORT. Additionally, we investigated the relationships between hub genes and clinicopathological features, as well as examined the distribution and expression of hub genes in the kidney. Results: A total of 270 DEGs were identified in LN. Using weighted gene co-expression network analysis (WGCNA), we clustered these DEGs into 14 modules. Among them, the turquoise module displayed a significant correlation with LN (cor=0.88, p<0.0001). Machine learning techniques identified four hub genes, namely CD53 (AUC=0.995), TGFBI (AUC=0.997), MS4A6A (AUC=0.994), and HERC6 (AUC=0.999), which are involved in inflammation response and immune activation. CIBERSORT analysis suggested that these hub genes may contribute to immune cell infiltration. Furthermore, these hub genes exhibited strong correlations with the classification, renal function, and proteinuria of LN. Interestingly, the highest hub gene expression score was observed in macrophages. Conclusion: CD53, TGFBI, MS4A6A, and HERC6 have emerged as promising candidate driver genes for LN. These hub genes hold the potential to offer valuable insights into the molecular diagnosis and treatment of LN.

CD4+ T Lymphocytes, especially Th2 cells, contribute to the progress of renal fibrosis.

BACKGROUND: Renal tubulointerstitial fibrosis is the final common stage of renal failure. CD4+ T lymphocyte recruitment and activation after injury could be the very important early event that mediates the onset of renal fibrogenesis. But the role of CD4+ T lymphocytes in renal fibrosis is controversial and its cellular mechanism needs to be further investigated. METHODS: Biopsy specimens were from patients with minimal-change or IgA nephropathy. Mouse renal fibrosis was induced by unilateral ureteral obstruction (UUO). CD4+ T lymphocytes of wild BALB/c mice were depleted with anti-CD4 antibody. BALB/c Nu/Nu mice were reconstituted with polarized Th1 or Th2 cells by tail vein injection. RESULTS: Our study demonstrated that massive CD4+ T lymphocytes infiltrated fibrotic kidneys of patients. The depletion of CD4+ T lymphocytes inhibited UUO-induced mouse renal fibrosis. In the process of UUO-induced renal fibrosis, the ratios of Th2/Th1 increased with time. Results have also shown that Th2-reconstituted mice developed renal fibrosis more easily than Th1-reconstituted mice, which manifested by interstitial expansion and collagen deposition, higher expression of α-SMA and vimentin and increased expression of fibronectin, TGF-ß and collagen I. We also found that CD4+ T cells from Th1-reconstitued mice tended to secrete IL-4 and IL-13 Th2-like cytokines. CONCLUSION: In conclusion, our study demonstrated the importance of CD4+ T lymphocytes in renal fibrosis and gave the first direct evidence that Th2 cells play a pivotal role in UUO-induced renal fibrosis. Inhibition of CD4+ T lymphocyte differentiation to Th2 would be a potential therapeutic intervention to prevent renal fibrosis.

Blocking connexin 43 and its promotion of ATP release from renal tubular epithelial cells ameliorates renal fibrosis.

Whether metabolites derived from injured renal tubular epithelial cells (TECs) participate in renal fibrosis is poorly explored. After TEC injury, various metabolites are released and among the most potent is adenosine triphosphate (ATP), which is released via ATP-permeable channels. In these hemichannels, connexin 43 (Cx43) is the most common member. However, its role in renal interstitial fibrosis (RIF) has not been fully examined. We analyzed renal samples from patients with obstructive nephropathy and mice with unilateral ureteral obstruction (UUO). Cx43-KSP mice were generated to deplete Cx43 in TECs. Through transcriptomics, metabolomics, and single-cell sequencing multi-omics analysis, the relationship among tubular Cx43, ATP, and macrophages in renal fibrosis was explored. The expression of Cx43 in TECs was upregulated in both patients and mice with obstructive nephropathy. Knockdown of Cx43 in TECs or using Cx43-specific inhibitors reduced UUO-induced inflammation and fibrosis in mice. Single-cell RNA sequencing showed that ATP specific receptors, including P2rx4 and P2rx7, were distributed mainly on macrophages. We found that P2rx4- or P2rx7-positive macrophages underwent pyroptosis after UUO, and in vitro ATP directly induced pyroptosis by macrophages. The administration of P2 receptor or P2X7 receptor blockers to UUO mice inhibited macrophage pyroptosis and demonstrated a similar degree of renoprotection as Cx43 genetic depletion. Further, we found that GAP 26 (a Cx43 hemichannel inhibitor) and A-839977 (an inhibitor of the pyroptosis receptor) alleviated UUO-induced fibrosis, while BzATP (the agonist of pyroptosis receptor) exacerbated fibrosis. Single-cell sequencing demonstrated that the pyroptotic macrophages upregulated the release of CXCL10, which activated intrarenal fibroblasts. Cx43 mediates the release of ATP from TECs during renal injury, inducing peritubular macrophage pyroptosis, which subsequently leads to the release of CXCL10 and activation of intrarenal fibroblasts and acceleration of renal fibrosis.

Role of Sema4C in TGF-ß1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells.

BACKGROUND: The p38 mitogen-activated protein kinase (p38 MAPK) is an important intracellular signal transduction pathway involved in TGF-ß1-induced epithelial-mesenchymal transition (EMT). Sema4C, a member of the semaphorin family, was found to be essential for the activation of p38 MAPK. However, the role of Sema4C in promoting TGF-ß1-induced EMT is unclear. METHODS: Renal fibrosis was induced by 5/6 subtotal nephrectomy rat model. In vitro, Sema4C was induced in human proximal tubular epithelial cells (HKC) by treatment with TGF-ß1, or was inhibited by siRNA or was over-expressed by Sema4C transfection. The selective p38 MAPK inhibitor, SB203580, was administered to inhibit the p38 pathway. The expression of Sema4C, the markers of EMT, p38 phosphorylation and fibronectin secretion were measured by western blotting, immunohistochemistry, immunocytochemistry or enzyme-linked immunosorbent assay. RESULTS: The expression of Sema4C increased in HKC cells that were treated with TGF-ß1. Knockdown of Sema4C potently inhibited phosphorylation of p38 MAPK and reversed TGF-ß1-induced EMT. Over-expression of Sema4C via Sema4C transfection elicited p38 MAPK phosphorylation and promoted EMT. The effects of Sema4C during EMT were blocked by a p38-specific inhibitor. In vivo, the expression of Sema4C increased in the tubular epithelia of 5/6-nephrectomized rats and human fibrotic renal tissue, and similar localization of phosphorylated p38 and Sema4C was demonstrated by immunohistochemistry on serial sections. CONCLUSIONS: Our findings suggest that Sema4C plays an important role in TGF-ß1-induced EMT through activation of p38 MAPK in proximal tubular epithelial cells.

Interleukin-7 Is Associated with Clinical and Pathological Activities in Immunoglobulin A Nephropathy and Protects the Renal Proximal Tubule Epithelium from Cellular Fibrosis.

OBJECTIVE: Diagnosis of immunoglobulin A nephropathy (IgAN) requires the evaluation of renal biopsy specimens. However, renal biopsy is an invasive procedure and is not frequently performed for various reasons. Thus, recognized noninvasive biomarkers for predicting IgAN progression are urgently needed. METHODS: In the present study, we included 86 IgAN patients with renal biopsy from June 2015 to May 2016 and had their plasma interleukin-7 (IL-7) level measured with ELISA. The association between the plasma IL-7 level and clinico-pathological characteristics was analyzed. Immunohistochemical staining was used to assay the in situ expression of IL-7 in vivo. Western blotting was performed to examine the production of extracellular matrix, p-mTOR and the markers of autophagy under the treatment of IL-7 after TGF-ß1 stimulation in renal tubular epithelial cells. RESULTS: IL-7 was significantly decreased in patients with IgAN compared to healthy subjects (2.3077 vs. 8.6294 pg/mL, P<0.0001). There was a significant difference in the plasma IL-7 level between tubular atrophy/interstitial fibrosis T0 and T2 classes (P=0.0064). A lower plasma IL-7 value in patients at the time of biopsy indicated a poor renal outcome. In addition, IL-7 was over-expressed in renal tubular epithelial cells and significantly attenuated transforming growth factor ßl-induced extracellular matrix production by suppression of cellular autophagy via activation of mTOR1 signaling. CONCLUSION: These results suggested that IL-7 might be a noninvasive biomarker for predicating IgAN. It protected renal proximal tubular epithelial cells from cellular fibrosis by inhibiting autophagy via mTORl signaling.

Separated parabiont reveals the fate and lifespan of peripheral-derived immune cells in normal and ischaemia-induced injured kidneys.

Immune cell infiltration plays a key role in acute kidney injury (AKI) to chronic kidney disease (CKD) progression. T lymphocytes, neutrophils, monocytes/macrophages and other immune cells regulate inflammation, tissue remodelling and repair. To determine the kinetics of accumulation of various immune cell populations, we established an animal model combining parabiosis and separation surgery to explore the fate and lifespan of peripheral leucocytes that migrate to the kidney. We found that peripheral T lymphocytes could survive for a long time (more than 14 days), whereas peripheral neutrophils survived for a short time in both healthy and ischaemia-induced damaged kidneys. Nearly half of the peripheral-derived macrophages disappeared after 14 days in normal kidneys, while their existing time in the inflammatory kidneys was prolonged. A fraction of F4/80high macrophages were renewed from the circulating monocyte pool. In addition, we found that after renal ischaemia reperfusion, neutrophils increased significantly in the early phase, and T lymphocytes mainly accumulated in the late stage, whereas macrophages infiltrated throughout AKI-CKD progression and were sustained longer in injured as opposed to normal kidneys. In conclusion, peripheral-derived macrophages, T lymphocytes and neutrophils exhibit different lifespans in the kidney, which may play different roles during AKI-CKD progression.

Tertiary lymphoid organs are associated with the progression of kidney damage and regulated by interleukin-17A.

Background: Tertiary lymphoid organs (TLOs) occur after multiple chronic kidney injuries. interleukin-17A (IL-17A) has been reported to associate with the development of TLOs in inflammatory diseases. However, regulation of the renal TLOs and its clinical significance to the pathogenesis of chronic kidney injury are unknown. Methods: To evaluate the clinical significance and regulation of renal TLOs, we analyzed the progression of patients with kidney damage based on the existence and absence of TLOs in a larger multicenter cohort. We also blocked the recruitment of lymphocyte cells into the kidney by FTY720 (fingolimod) in vivo. Besides, we used aged IL-17A genetic knocked out mice and IL-17A-neutralizing antibody to explore the role of IL-17A in renal TLOs formation. Results: We demonstrated that renal TLOs of IgA nephropathy patients were associated with disease severity and were independent risk factors for renal progression after adjustment for age, sex, mean arterial pressure, proteinuria and, baseline eGFR and MEST-C score, especially in the early stage. Plasma levels of TLO-related chemokines CXCL13, CCL19, and CCL21 were higher in patients with renal TLOs. Inhibiting the formation of renal TLOs by FTY720 could reduce the intrarenal inflammation and fibrosis, and early intervention was found to be more effective. IL-17A was increased in renal TLOs models, and genetic depletion of IL-17A or treatment with anti-IL-17A antibody resulted in a marked reduction of the TLOs formation as well as alleviation of renal inflammation and fibrosis. Conclusion: These results indicate that TLOs are associated with the progression of kidney damage and regulated by IL-17A and may be effective targets for the treatment of kidney damage.