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Tubular epithelial cells (TECs) exposed to hypoxia incite tubulointerstitial inflammation (TII), while the exact mechanism is unclear. In this study, we identified that hypoxia evoked tubule injury as evidenced by tubular hypoxia-inducible factor-1α and kidney injury molecule-1 (KIM-1) expression and that renal small extracellular vesicle (sEV) production was increased with the development of TII after ischemia-reperfusion injury (IRI). Intriguingly, KIM-1-positive tubules were surrounded by macrophages and co-localized with sEVs. In vitro, KIM-1 expression and sEV release were increased in hypoxic TECs and the hypoxia-induced inflammatory response was ameliorated when KIM-1 or Rab27a, a master regulator of sEV secretion, was silenced. Furthermore, KIM-1 was identified to mediate hypoxic TEC-derived sEV (Hypo-sEV) uptake by TECs. Phosphatidylserine (PS), a ligand of KIM-1, was present in Hypo-sEVs as detected by nanoflow cytometry. Correspondingly, the inflammatory response induced by exogenous Hypo-sEVs was attenuated when KIM-1 was knocked down. In vivo, exogenous-applied Hypo-sEVs localized to KIM-1-positive tubules and exacerbated TII in IRI mice. Our study demonstrated that KIM-1 expressed by injured tubules mediated sEV uptake via recognizing PS, which participated in the amplification of tubule inflammation induced by hypoxia, leading to the development of TII in ischemic acute kidney injury.

Background: Acute kidney injury (AKI) is a significant public health problem with high morbidity and mortality. Unfortunately, no definitive treatment is currently available for AKI. RNA interference (RNAi) provides a new and potent method for gene therapy to tackle this dilemma. Methods We engineered red blood cell-derived extracellular vesicles (REVs) with targeting peptides and therapeutic siRNAs to treat experimental AKI in a mouse model following renal ischemia/reperfusion (I/R) injury and unilateral ureteral obstruction (UUO). Phage display identified peptides that bind to the kidney injury molecule-1 (Kim-1). RNA-sequencing (RNA-seq) characterized the transcriptome of ischemic kidney to explore potential therapeutic targets. Results REVs targeted with Kim-1-binding LTH peptide (REVLTH) efficiently homed to and accumulated at the injured tubules in kidney following I/R injury. We identified transcription factors P65 and Snai1 that drive inflammation and fibrosis as potential therapeutic targets. Taking advantage of the established REVLTH, siRNAs targeting P65 and Snai1 were efficiently delivered to ischemic kidney and consequently blocked the expression of P-p65 and Snai1 in tubules. Moreover, dual suppression of P65 and Snai1 significantly improved I/R- and UUO-induced kidney injury by alleviating tubulointerstitial inflammation and fibrosis, and potently abrogated the transition to chronic kidney disease. Conclusions A red blood cell-derived extracellular vesicle platform targeted Kim-1 in acutely injured mouse kidney and delivered siRNAs for transcription factors P65 and Snai1, alleviating inflammation and fibrosis in the tubules.

Background Urinary sediment messenger RNAs (mRNAs) have been shown as novel biomarkers of kidney disease. We aimed to identify targeted urinary mRNAs in diabetic nephropathy (DN) based on bioinformatics analysis and clinical validation. Methods Microarray studies of DN were searched in the GEO database and Nephroseq platform. Gene modules negatively correlated with estimated glomerular filtration rate (eGFR) were identified by informatics methods. Hub genes were screened within the selected modules. In validation cohorts, a quantitative polymerase chain reaction assay was used to compare the expression levels of candidate mRNAs. Patients with renal biopsy–confirmed DN were then followed up for a median time of 21 months. End-stage renal disease (ESRD) was defined as the primary endpoint. Multivariate Cox proportional hazards regression was developed to evaluate the prognostic values of candidate mRNAs. Results Bioinformatics analysis revealed four chemokines (CCL5, CXCL1, CXLC6 and CXCL12) as candidate mRNAs negatively correlated with eGFR, of which CCL5 and CXCL1 mRNA levels were upregulated in the urinary sediment of patients with DN. In addition, urinary sediment mRNA of CXCL1 was negatively correlated with eGFR (r = −0.2275, P = 0.0301) and CCL5 level was negatively correlated with eGFR (r = −0.4388, P Conclusions Urinary sediment CCL5 and CXCL1 mRNAs were upregulated in DN patients and associated with a decline in renal function and degree of renal interstitial fibrosis. Urinary sediment CCL5 mRNA could be used as a potential prognostic biomarker of DN.

Oxygen homeostasis disturbances play a critical role in the pathogenesis of acute kidney injury (AKI). The transcription factor hypoxia-inducible factor-1 (HIF-1) is a master regulator of adaptive responses to hypoxia. Aside from posttranslational hydroxylation, the mechanism of HIF-1 regulation in AKI remains largely unclear. In this study, the mechanism of HIF-α regulation in AKI was investigated. We found that tubular HIF-1α expression significantly increased at the transcriptional level in ischemia-reperfusion-, unilateral ureteral obstruction-, and sepsis-induced AKI models, which was closely associated with macrophage-dependent inflammation. Meanwhile, NF-κB, which plays a central role in the inflammation response, was involved in the increasing expression of HIF-1α in AKI, as evidenced by pharmacological modulation (NF-κB inhibitor BAY11-7082). Mechanistically, NF-κB directly bound to the HIF-1α promoter and enhanced its transcription, which occurred not only under hypoxic conditions but also under normoxic conditions. Moreover, the induced HIF-1α by inflammation protected against tubular injury in AKI. Thus, our findings not only provide novel insights into HIF-1 regulation in AKI but also offer to understand the pathophysiology of kidney diseases.NEW & NOTEWORTHY Here, the mechanism of hypoxia-inducible factor-α (HIF-α) regulation in acute kidney injury (AKI) was investigated. We found that tubular HIF-1α expression significantly increased at the transcriptional level, which was closely associated with macrophage-dependent inflammation. Meanwhile, NF-κB was involved in the increasing expression of HIF-1α in AKI. Mechanistically, NF-κB directly bound to the HIF-1α promoter and enhanced its transcription. Our findings not only provide novel insights into HIF-1 regulation in AKI but also offer to understand the pathophysiology of kidney diseases.

Background/Aims Diabetic nephropathy (DN) is one of the main causes of end-stage kidney disease worldwide. Emerging studies have suggested that its pathogenesis is distinct from nondiabetic renal diseases in many aspects. However, it still lacks a comprehensive understanding of the unique molecular mechanism of DN. Methods A total of 255 Affymetrix U133 microarray datasets (Affymetrix, Santa Calra, CA, USA) of human glomerular and tubulointerstitial tissues were collected. The 22 215 Affymetrix identifiers shared by the Human Genome U133 Plus 2.0 and U133A Array were extracted to facilitate dataset pooling. Next, a linear model was constructed and the empirical Bayes method was used to select the differentially expressed genes (DEGs) of each kidney disease. Based on these DEG sets, the unique DEGs of DN were identified and further analyzed using gene ontology and pathway enrichment analysis. Finally, the protein–protein interaction networks (PINs) were constructed and hub genes were selected to further refine the results. Results A total of 129 and 1251 unique DEGs were identified in the diabetic glomerulus (upregulated n = 83 and downregulated n = 203) and the diabetic tubulointerstitium (upregulated n = 399 and downregulated n = 874), respectively. Enrichment analysis revealed that the DEGs in the diabetic glomerulus were significantly associated with the extracellular matrix, cell growth, regulation of blood coagulation, cholesterol homeostasis, intrinsic apoptotic signaling pathway and renal filtration cell differentiation. In the diabetic tubulointerstitium, the significantly enriched biological processes and pathways included metabolism, the advanced glycation end products–receptor for advanced glycation end products signaling pathway in diabetic complications, the epidermal growth factor receptor (EGFR) signaling pathway, the FoxO signaling pathway, autophagy and ferroptosis. By constructing PINs, several nodes, such as AGR2, CSNK2A1, EGFR and HSPD1, were identified as hub genes, which might play key roles in regulating the development of DN. Conclusions Our study not only reveals the unique molecular mechanism of DN but also provides a valuable resource for biomarker and therapeutic target discovery. Some of our findings are promising and should be explored in future work.

Mesenchymal stem cells-derived exosomes (MSC-exos) have attracted great interest as a cell-free therapy for acute kidney injury (AKI). However, the in vivo biodistribution of MSC-exos in ischemic AKI has not been established. The potential of MSC-exos in promoting tubular repair and the underlying mechanisms remain largely unknown. Methods: Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of human umbilical cord mesenchymal stem cells (hucMSCs) derived exosomes. The biodistribution of MSC-exos in murine ischemia/reperfusion (I/R) induced AKI was imaged by the IVIS spectrum imaging system. The therapeutic efficacy of MSC-exos was investigated in renal I/R injury. The cell cycle arrest, proliferation and apoptosis of tubular epithelial cells (TECs) were evaluated in vivo and in HK-2 cells. The exosomal miRNAs of MSC-exos were profiled by high-throughput miRNA sequencing. One of the most enriched miRNA in MSC-exos was knockdown by transfecting miRNA inhibitor to hucMSCs. Then we investigated whether this candidate miRNA was involved in MSC-exos-mediated tubular repair. Results: Ex vivo imaging showed that MSC-exos was efficiently homing to the ischemic kidney and predominantly accumulated in proximal tubules by virtue of the VLA-4 and LFA-1 on MSC-exos surface. MSC-exos alleviated murine ischemic AKI and decreased the renal tubules injury in a dose-dependent manner. Furthermore, MSC-exos significantly attenuated the cell cycle arrest and apoptosis of TECs both in vivo and in vitro. Mechanistically, miR-125b-5p, which was highly enriched in MSC-exos, repressed the protein expression of p53 in TECs, leading to not only the up-regulation of CDK1 and Cyclin B1 to rescue G2/M arrest, but also the modulation of Bcl-2 and Bax to inhibit TEC apoptosis. Finally, inhibiting miR-125b-5p could mitigate the protective effects of MSC-exos in I/R mice. Conclusion: MSC-exos exhibit preferential tropism to injured kidney and localize to proximal tubules in ischemic AKI. We demonstrate that MSC-exos ameliorate ischemic AKI and promote tubular repair by targeting the cell cycle arrest and apoptosis of TECs through miR-125b-5p/p53 pathway. This study provides a novel insight into the role of MSC-exos in renal tubule repair and highlights the potential of MSC-exos as a promising therapeutic strategy for AKI.

Aldosterone exerts an enormous function on proximal tubular cells (PTC) senescence, which is a common pathomechanism contributing to renal dysfunction. Numerous studies have shown that oxidative stress is deeply involved in the pathophysiologic processes of chronic kidney diseases. The study aims to investigate whether autophagy could regulate the process of senescence through oxidative stress in PTC both in vivo and ex vivo. Our results suggested that aldosterone treatment increased the senescence and oxidative stress as evidenced by increased percent of SA-β-Gal positive cells, reactive oxygen species level, expression of NADPH oxidase 4 (NOX4) rather than NOX2, and the up-regulation of p21 in cultured PTC. Furthermore, the alternation of the expression of p62 and LC3-II/LC3-I demonstrated that aldosterone treatment remarkably influenced autophagic flux. NOX4 siRNA treatment or autophagy induction with rapamycin reduced the oxidative stress and senescence in aldosterone-induced PTC. On the contrary, inhibition of autophagy with chloroquine worsened these changes. Similar results were further confirmed in vivo. Our results suggested that autophagy may become a realistic therapeutic strategy against aldosterone-induced PTC injury via improving oxidative stress.

Background Peritoneal dialysis (PD) patients experience accelerated arterial aging, which is characterized by elastin degradation. Elastin-derived peptides (EDPs) are direct products of elastin fragmentation. This study tried to explore the association between serum EDPs and abdominal aortic calcification (AAC) in PD patients. Methods Serum levels of EDPs were analyzed in 126 eligible PD patients and 30 controls. PD patients were grouped according to the annularity of AAC evaluated by an abdominal computed tomography (CT) scan. Serum EDPs were analyzed in relation to the presence of AAC or severe AAC in PD patients by logistic regression analysis. Results Serum EDPs in PD patients were significantly higher than age-matched controls. In 126 PD patients, higher EDPs was associated with greater risk of present AAC (OR = 1.056, 95%CI 1.010–1.103) and severe AAC (OR = 1.062, 95%CI 1.004–1.123). A combination of EDPs substantially improved the accuracy of diagnostic performance for AAC and severe AAC. Conclusions EDPs can predict the presence and extent of AAC in PD patients, indicating its possible role to recognize PD patients at risk for AAC and severe AAC.

Purpose Glioblastoma (GBM) is the most lethal primary cancer in adult central nervous system, and new strategies are desperately needed. The secretory pathway kinase or kinase-like proteins (SPKKPs) have been shown to mediate multiple physiological functions by phosphorylating extracellular proteins and proteoglycans. However, their roles in cancers, especially GBM, remain poorly defined. Methods The least absolute shrinkage and selection operator (LASSO) regression was employed for establishing the SPKKPs signature for IDH wild type (wt) GBM prognosis. Integrative analyses with multiple datasets were employed to identify the core member of this gene family in glioma. The receiver operator characteristic (ROC) curves and immunohistochemistry were further used for evaluating its association with progressive malignancy in glioma and GBM patients' survival, respectively. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to interpret its functions in GBM, which were further verified in vitro. Results A SPKKPs classifier was constructed with 3 genes of this family. This signature could effectively distinguish IDH wt GBM survival. Family with sequence similarity 20 C (FAM20C) was further identified as the core member of this family in glioma. Elevated FAM20C expression was not only closely correlated with glioma malignancy progression and the mesenchymal subtype of GBM but also indicated unfavorable survival of GBM patients. FAM20C was also found to be associated with the disrupted immune response in GBM microenvironment and was required for the migration of glioma and immune cells. Conclusion These data indicate that the potential of FAM20C serving as a predictive molecule and a therapeutic target for GBM.

Background: Protein-energy wasting (PEW) and inflammation have been recognized as two major factors associated with the poor prognosis of patients with maintenance haemodialysis (MHD). The aim of this study was to evaluate the effects of oral fat based high-energy supplements (Fresubin) on malnutrition and inflammation in MHD patients. Method: This study was open-label, prospective, nonrandomized and comparative. A total of 47 MHD patients with PEW were randomly assigned into 2 groups: a control group, and a Fresubin treatment group, in which patients received daily diet intake and Fresubin nutrient support 120 mL/d for 80 days. Laboratory data and anthropometric parameters were measured. Result: The results showed that compared with baseline, the level of serum albumin at 80 d in the Fresubin treatment group was significantly increased (35.4 ± 3.7 vs 38.9 ± 2.9, p = 0.003), while there was no significant difference in the control group. In addition, the magnitudes of changes in albumin (p = 0.009), serum prealbumin (p = 0.017), and abdominal circumference (p = 0.037) in the Fresubin treatment group were markedly increased compared with those in the control group. There was a downward trend in the inflammatory marker hypersensitive C-reactive protein in the Fresubin group (p =0.056), which decreased more dramatically than that in the control group (p = 0.026). Conclusion: Fresubin treatment is well tolerated, and improves malnutrition and inflammatory status in MHD patients with PEW.

Cyclin-dependent kinase 12 (CDK12) plays a critical role in regulating gene transcription. CDK12 inhibition is a potential anticancer therapeutic strategy. However, several clinical trials have shown that CDK inhibitors might cause renal dysfunction and electrolyte disorders. CDK12 is abundant in renal tubular epithelial cells (RTECs), but the exact role of CDK12 in renal physiology remains unclear. Genetic knockout of CDK12 in mouse RTECs causes polydipsia, polyuria, and hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced Na-K-2Cl cotransporter 2 (NKCC2) levels in the kidney. In addition, CKD12 knockout causes an increase in Slc12a1 (which encodes NKCC2) intronic polyadenylation events, which results in Slc12a1 truncated transcript production and NKCC2 downregulation. These findings provide novel insight into CDK12 being necessary for maintaining renal homeostasis by regulating NKCC2 transcription, which explains the critical water and electrolyte disturbance that occurs during the application of CDK12 inhibitors for cancer treatment. Therefore, there are safety concerns about the clinical use of these new anticancer drugs.

Immune cell infiltration mediates therapeutic response to immune therapies. The investigation on the genes regulating leukocyte migration may help us to understand the mechanisms regulating immune cell infiltration in tumor microenvironment. Here, we collected the data from Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) to analyze the expression of leukocyte migration related genes in glioblastoma (GBM). Lymphocyte specific protein 1 (LSP1) was identified as the only gene in this family which not only has an elevated expression, but also serve as an independent predictive factor for progressive malignancy in glioma. We further confirmed these results in clinical glioma samples by quantitative PCR, immunofluorescence, immunohistochemistry, and western blot. Moreover, LSP1 expression was closely related to the response to radio- and chemotherapy in GBM, and positively correlated with immunosuppressive cell populations, including M2 macrophages, neutrophil, and regulatory T cell. Additionally, elevated LSP-1 expression enhanced the expression of immunosuppression related genes like programmed cell death 1 (PD1) and leukocyte associated immunoglobulin like receptor 1 (LAIR1) in macrophages. LSP1 also promoted the migration of macrophages. Together, our study suggests a novel role of LSP1 contributing to immunosuppressive microenvironment in GBM and serving as a potential therapeutic target for it.

Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) are orally active first-in-class new generation drugs for renal anemia. This extensive meta-analysis of randomized controlled trials (RCTs) was designed to provide clear information on the efficacy and safety of HIF-PHIs on anemia in chronic kidney disease (CKD) patients. Searches included PubMed, Web of Science, Ovid MEDLINE, and Cochrane Library database up to October 2019. RCTs of patients with CKD comparing HIF-PHIs with erythropoiesis-stimulating agents (ESAs) or placebo in the treatment of anemia. The primary outcome was hemoglobin change from baseline (Hb CFB); the secondary outcomes included iron-related parameters and the occurrence of each adverse event. 26 trials in 17 articles were included, with a total of 2804 dialysis or patients with CKD. HIF-PHIs treatment produced a significant beneficial effect on Hb CFB compared with the placebo group (MD, 0.69; 95% CI, 0.36 to 1.02). However, this favored effect of HIF-PHIs treatment was not observed in subgroup analysis among trials compared with ESAs (MD, 0.06; 95% CI, −0.20 to 0.31). The significant reduction in hepcidin by HIF-PHIs was observed in all subgroups when compared with the placebo group, whereas this effect was observed only in NDD-CKD patients when compared with ESAs. HIF-PHIs increased the risk of nausea (RR, 2.20; 95% CI, 1.06 to 4.53) and diarrhea (RR, 1.75; 95% CI, 1.06 to 2.92). We conclude that orally given HIF-PHIs are at least as efficacious as ESAs treatment to correct anemia short term in patients with CKD. In addition, HIF-PHIs improved iron metabolism and utilization in patients with CKD.

The discovery of hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitor (PHI) has revolutionized the treatment strategy for renal anemia. However, the presence of multiple transcription targets of HIF raises safety concerns regarding HIF-PHI. Here, we explored the dose-dependent effect of MK-8617 (MK), a kind of HIF-PHI, on renal fibrosis. MK was administered by oral gavage to mice for 12 wk at doses of 1.5, 5, and 12.5 mg/kg. In vitro, the human proximal tubule epithelial cell line HK-2 was treated with increasing doses of MK administration. Transcriptome profiling was performed, and fibrogenesis was evaluated. The dose-dependent biphasic effects of MK on tubulointerstitial fibrosis (TIF) were observed in chronic kidney disease mice. Accordingly, high-dose MK treatment could significantly enhance TIF. Using RNA-sequencing, combined with in vivo and in vitro experiments, we found that Krüppel-like factor 5 (KLF5) expression level was significantly increased in the proximal tubular cells, which could be transcriptionally regulated by HIF-1α with high-dose MK treatment but not low-dose MK. Furthermore, our study clarified that HIF-1α–KLF5–TGF-β1 signaling activation is the potential mechanism of high-dose MK-induced TIF, as knockdown of KLF5 reduced TIF in vivo. Collectively, our study demonstrates that high-dose MK treatment initiates TIF by activating HIF-1α–KLF5–TGF-β1 signaling. These findings provide novel insights into TIF induction by high-dose MK (HIF-PHI), suggesting that the safety dosage window needs to be emphasized in future clinical applications.—Li, Z.-L., Lv, L.-L., Wang, B., Tang, T.-T., Feng, Y., Cao, J.-Y., Jiang, L.-Q., Sun, Y.-B., Liu, H., Zhang, X.-L., Ma, K.-L., Tang, R.-N., Liu, B.-C. The profibrotic effects of MK-8617 on tubulointerstitial fibrosis mediated by the KLF5 regulating pathway.

Foot process effacement is an important feature of early diabetic nephropathy (DN) which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62 mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase 3 and caspase 8. Interestingly, the expression of VDR in podocytes was decreased under diabetic condition which impaired autophagic flux through down-regulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking p62-mediated autophagy pathway in DN.

Background and Aims In recent years, it has been found that targeted mRNA detection of sediment cells in urine can be used as novel biomarkers for the diagnosis of diabetic nephropathy (DN). However, its value in predicting the progression of DN is not clear. The purpose of this study is to seek for urinary mRNA markers that evaluate the prognosis of DN through systems biological screening, clinical verification and prospective studies. Method GEO database and “Nephroseq” platform were searched, and the transcriptome data of DN glomeruli and tubules and their clinical information were obtained. Weighted gene co-expression network analysis (WGCNA) combined with gene ontology (GO) annotation and KEGG pathway enrichment analysis were used to screen the hub genes negatively related to eGFR, and the hub genes were used as candidate markers for mRNA detection in urine sediment in DN patients. A total of 91 patients with DN diagnosed by renal biopsy were included, and 60 patients with type 2 diabetes and 61 healthy people were selected as the control groups. The mRNA expression of candidate molecules was detected by Taqman probe quantitative PCR, and the correlation between mRNA expression and eGFR and urinary protein levels were analyzed. Patients with DN were followed up for a median time of 21 months, and the primary end point was defied as end stage renal disease or eGFR decreasing by more than 50%. Multivariate Cox regression was used to evaluate the value of mRNA in predicting DN progression. Results GSE30528 and GSE30529 datasets were selected for analysis, including mRNA expression data of 9 cases of DN and 13 cases of normal glomeruli; and 10 cases of DN and 12 cases of normal tubules respectively. The clinical data of the patients in this study, including gender, race, age and eGFR, were searched on the Nephroseq platform. The gene modules negatively related to eGFR were screened by WGCNA. GO and KEGG analysis showed that the main function of the gene modules in both datasets were related to the activation of inflammatory cells and chemokines pathway. Through the screening of hub genes and the comparison of expression levels, CCL5, CXCL1, CXCL6 and CXCL12 were finally obtained as candidate genes. Quantitative PCR showed that the levels of CCL5 and CXCL1 was significantly increased in DN group, CCL5 was negatively correlated with eGFR and positively correlated with urinary protein level, while CXCL1 was negatively correlated with eGFR, but had no significant correlation with urinary protein level. Multivariate Cox regression showed that eGFR, urinary protein level, degree of renal fibrosis and urinary CCL5 were independent risk factors of primary end point. Conclusion The activation of chemokine signal pathway in renal tissue is involved in the progression of DN. Urinary CCL5 mRNA can independently predict the prognosis of DN and may be served as a novel biomarker for the progression of DN.

Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg(−1)·d(−1), i.p.) was administered starting on day 5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.

Using extracellular vesicles for targeted delivery of IL-10 is an efficient therapeutic strategy against ischemic AKI., Recently, extracellular vesicles (EVs) have been attracting strong research interest for use as natural drug delivery systems. We report an approach to manufacturing interleukin-10 (IL-10)–loaded EVs (IL-10+ EVs) by engineering macrophages for treating ischemic acute kidney injury (AKI). Delivery of IL-10 via EVs enhanced not only the stability of IL-10, but also its targeting to the kidney due to the adhesive components on the EV surface. Treatment with IL-10+ EVs significantly ameliorated renal tubular injury and inflammation caused by ischemia/reperfusion injury, and potently prevented the transition to chronic kidney disease. Mechanistically, IL-10+ EVs targeted tubular epithelial cells, and suppressed mammalian target of rapamycin signaling, thereby promoting mitophagy to maintain mitochondrial fitness. Moreover, IL-10+ EVs efficiently drove M2 macrophage polarization by targeting macrophages in the tubulointerstitium. Our study demonstrates that EVs can serve as a promising delivery platform to manipulate IL-10 for the effective treatment of ischemic AKI.

Hypoxia promotes tubulointerstitial inflammation in the kidney. Although hypoxia inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia, the exact mechanisms through which HIF-1α modulates the induction of tubulointerstitial inflammation are still largely unclear. We demonstrated tubulointerstitial inflammation and increased tubular HIF-1α expression in murine models of ischemia/reperfusion injury and unilateral ureteral obstruction. Increased expression of HIF-1α in tubular epithelial cells was associated with selective shedding of microRNA-23a (miRNA-23a)-enriched exosomes in vivo and systemic inhibition of miRNA-23a prior to ischemia/reperfusion injury attenuated tubulointerstitial inflammation. In vitro, uptake of miRNA-23a-enriched exosomes by macrophages triggered their reprogramming into a pro-inflammatory state via suppression of the ubiquitin editor A20. To confirm the effect of miRNA-23a-containing exosomes on tubulointerstitial inflammation, we exposed tubular epithelial cells to hypoxic conditions to promote the release of miRNA-23a-containing exosomes. Injection of these miRNA-23a-enriched exosomes into uninjured renal parenchyma resulted in increased inflammatory infiltration in vivo. Taken together, our studies demonstrate that the HIF-1α-dependent release of miRNA-23a-enriched exosomes from hypoxic tubular epithelial cells activates macrophages to promote tubulointerstitial inflammation. Blockade of exosome-mediated miRNA-23a transfer between tubular epithelial cells and macrophages may serve as a novel therapeutic approach to ameliorate tubulointerstitial inflammation.

Background Exosomes derived from mesenchymal stem cells (MSC-exos) have been demonstrated with great potential in the treatment of multiple human diseases including acute kidney injury (AKI) by virtue of their intrinsic cargoes. However, there are major challenges of low yield and the lack of an established biomanufacturing platform to efficiently produce MSC-exos, thereby limiting their therapeutic application. Here, we aimed to establish a novel strategy to produce MSC-exos with a hollow fiber bioreactor-based three-dimensional (3D) culture system and evaluate the therapeutic efficacy of 3D-exosomes (3D-exos) on AKI. Methods Mesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in two-dimensional (2D) flasks. 2 × 108 MSCs were inoculated into the hollow fiber bioreactor for 3D culture. The culture supernatants were collected every 1 or 2 days for isolating exosomes. Exosomes from 2D (2D-exos) and 3D cultures were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis of exosome markers. The yield of exosomes from 2 × 108 MSCs seeded in 2D and 3D culture system was compared, based on protein quantification. The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro. Results 3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers of 3D-exos when compared to those of 2D-exos. Compared with conventional 2D culture, the 3D culture system increased total exosome production up to 19.4-fold. 3D-exos were more concentrated in the harvested supernatants (15.5-fold) than 2D-exos, which led to a higher exosome collection efficiency of 3D culture system. In vivo, both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function, attenuated pathological changes of renal tubules, reduced inflammatory factors, and repressed T cell and macrophage infiltration. Impressively, 3D-exos were more effective than 2D-exos. Moreover, 3D-exos were taken up by tubular epithelial cells (TECs) with improved efficiency, thereby exhibiting superior anti-inflammatory effect and improved viability of TECs in vitro. Conclusions In summary, our findings demonstrate that the hollow fiber 3D culture system provides an efficient strategy for the continuous production of MSC-exos which has enhanced therapeutic potential for cisplatin-induced AKI.

Objective. Lupus nephritis (LN) is a major and severe complication of systemic lupus erythematosus (SLE). Neutrophil gelatinase-associated lipocalin (NGAL), as a promising next-generation biomarker in clinical nephrology, has received extensive attention. However, its diagnostic performance in LN has high variability. Therefore, we performed an updated meta-analysis to further evaluate the diagnostic accuracy of urinary NGAL (uNGAL).Materials and Methods. PubMed, Embase, and Cochrane Library were searched from inception to October 27, 2019. Meta-analysis was performed with a bivariate random effects model. Additionally, the summary receiver operating characteristic (SROC) curves were established. The sources of heterogeneity were explored by meta-regression, subgroup analysis, and sensitivity analysis. Publication bias was assessed using the Deeks test.Results. 19 articles consisting of 21 eligible studies were included. In diagnosing LN, the estimates (95% confidence interval (CI)) were as follows: sensitivity, 0.84 (0.71-0.91); specificity, 0.91 (0.70-0.98); and the SROC-AUC value, 0.92 (0.90-0.94). In identifying active LN, the estimates were as follows: sensitivity, 0.72 (0.56-0.84); specificity, 0.71 (0.51-0.84); and the AUC value, 0.77 (0.74-0.81). With respect to predicting renal flare, the estimates were as follows: sensitivity, 0.80 (0.57-0.92); specificity, 0.67 (0.58-0.75); and the AUC value, 0.74 (0.70-0.78). For the studies to distinguish proliferative LN, the estimates were as follows: sensitivity, 0.87 (0.66-0.97), and specificity, 0.69 (0.39-0.91). Deeks’ funnel plot suggested that there was no significant publication bias.Conclusions. Our meta-analysis indicates that uNGAL was a useful biomarker for diagnosis, estimation of activity, and prediction of renal flare of LN. In addition, the usefulness of uNGAL to distinguish pathological types of LN needs to be further investigated.

Background: Recently, cinacalcet (CINA) has been shown to be effective for attenuating bone loss in the treatment of secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease (CKD), which might be associated with the reduction in serum parathyroid hormone (PTH) levels. However, the exact mechanism is largely unclear. Emerging studies have revealed that an increased number of bone marrow adipocytes (BMAs) are involved in bone loss and the endothelial-to-adipocyte transition via the endothelial-to-mesenchymal transition (EndMT) might play a key role in this pathological process. Here, we assessed whether CINA could attenuate bone loss via inhibiting endothelial-to-adipocyte transition in CKD rats. Methods: A rat model of CKD was induced by adenine and a high phosphorus diet. CINA was orally administrated to CKD animals (10 mg/kg once a day). Dual energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and bone mechanical tests were used to determine the skeletal changes. The bone marrow expression of EndMT markers was also examined. The effect of elevated PTH levels on the endothelial-to-adipocyte transition was studied in endothelial cells (ECs). Results: Elevation of serum PTH levels, remarkable bone loss and increased numbers of BMAs were observed in rats with CKD compared with the controls, and these changes were attenuated after treatment with CINA. Furthermore, the CINA treatment abolished the upregulation of mesenchymal markers (FSP1 and α-SMA) and the downregulation of an endothelial marker (CD31) in bone tissues from rats with CKD. The serum PTH concentrations were correlated with the bone marrow protein levels of these EndMT-related proteins. An in vitro treatment in ECs demonstrated that PTH induced the EndMT in a concentration- and time-dependent manner. Accordingly, ECs treated with PTH exhibited adipogenic potential following growth in adipogenic culture medium. Conclusions: Our study indicated CINA treatment attenuated bone loss in CKD rats, which might be associated with inhibiting PTH-induced endothelial-to-adipocyte transition in CKD rats.

Although glucocorticoids are the mainstays in the treatment of renal diseases for decades, the dose dependent side effects have largely restricted their clinical use. Microvesicles (MVs) are small lipid-based membrane-bound particles generated by virtually all cells. Here we show that RAW 264.7 macrophage cell-derived MVs can be used as vectors to deliver dexamethasone (named as MV-DEX) targeting the inflamed kidney efficiently. Methods: RAW macrophages were incubated with dexamethasone and then MV-DEX was isolated from the supernatants by centrifugation method. Nanoparticle tracking analysis, transmission electron microscopy, western blot and high-performance liquid chromatography were used to analyze the properties of MV-DEX. The LC-MS/MS was applied to investigate the protein compositions of MV-DEX. Based on the murine models of LPS- or Adriamycin (ADR)-induced nephropathy or in-vitro culture of glomerular endothelial cells, the inflammation-targeting characteristics and the therapeutic efficacy of MV-DEX was examined. Finally, we assessed the side effects of chronic glucocorticoid therapy in MV-DEX-treated mice. Results: Proteomic analysis revealed distinct integrin expression patterns on the MV-DEX surface, in which the integrin αLβ2 (LFA-1) and α4β1 (VAL-4) enabled them to adhere to the inflamed kidney. Compared to free DEX treatment, equimolar doses of MV-DEX significantly attenuated renal injury with an enhanced therapeutic efficacy against renal inflammation and fibrosis in murine models of LPS- or ADR-induced nephropathy. In vitro, MV-DEX with about one-fifth of the doses of free DEX achieved significant anti-inflammatory efficacy by inhibiting NF-κB activity. Mechanistically, MV-DEX could package and deliver glucocorticoid receptors to renal cells, thereby, increasing cellular levels of the receptor and improving cell sensitivity to glucocorticoids. Notably, delivering DEX in MVs significantly reduced the side effects of chronic glucocorticoid therapy (e.g., hyperglycemia, suppression of HPA axis). Conclusion: In summary, macrophage-derived MVs efficiently deliver DEX into the inflamed kidney and exhibit a superior capacity to suppress renal inflammation and fibrosis without apparent glucocorticoid adverse effects. Our findings demonstrate the effectiveness and security of a novel drug delivery strategy with promising clinical applications.

Renal anemia, mainly caused by the deficiencies of erythropoietin (EPO) and iron metabolism disorder, is one of the most common complications of chronic kidney disease. Hypoxia-inducible factor (HIF) is a class of transcription factors responsible for maintaining homeostasis during oxygen deprivation. In normoxia, HIF is degraded by prolyl hydroxylase (PHD). While under hypoxic conditions, the hydroxylation activity of PHD is inhibited, and the cellular concentration of HIF is elevated, resulting in an increase in endogenous EPO production and iron absorption. Therefore, this regulating pathway, also termed as the HIF-PHD axis, has become a promising therapeutic target of treating renal anemia. Several innovative drugs acting as selective HIF-PHD inhibitors have been successfully developed in the past years, and some of them are undergoing clinical trials. In this review, we will introduce the definition and regulatory mechanism of HIF-PHD axis, as well as current insights into its physiologic and therapeutic role in renal anemia.

Glioblastoma (GBM) is the most lethal cancer in central nervous system. It is urgently needed to look for novel therapeutics for GBM. Oncostatin M receptor (OSMR) is a cytokine receptor gene of IL-6 family and has been reported to be involved in regulating GBM tumorigenesis. However, the role of OSMR regulating the disrupted immune response in GBM need to be further investigated. Three gene expression profiles, Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) data set (GSE16011), were enrolled in our study and used for OSMR expression and survival analysis. The expression of OSMR was further verified with immunohistochemistry and western blot analysis in glioma tissues. Microenvironment cell populations-counter (MCP-counter) was applied for analyzing the relationship between OSMR expression and nontumor cells. The functions of OSMR in GBM was investigated by Gene Ontology, Gene set enrichment analysis (GSEA), gene set variation analysis and so on. The analysis of cytokine receptor activity-related genes in glioma identifies OSMR as a gene with an independent predictive factor for progressive malignancy in GBM. Furthermore, OSMR expression is a prognostic marker in the response prediction to radiotherapy and chemotherapy. OSMR contributes to the regulation of local immune response and extracellular matrix process in GBM. Our findings define an important role of OSMR in the regulation of local immune response in GBM, which may suggest OSMR as a possible biomarker in developing new therapeutic immune strategies in GBM.

The mechanisms that underlie the profibrotic effect of interleukin (IL)-1β are complicated and not fully understood. Recent evidence has suggested the involvement of the calcium-sensing receptor (CaSR) in tubular injury. Therefore, the current study aimed to investigate whether CaSR mediates IL-1β-induced collagen expression in cultured mouse inner medullary collecting duct cells (mIMCD3) and to determine the possible downstream signaling effector. The results showed that IL-1β significantly upregulated the expression of type I and III collagens in a concentration- and time-dependent manner. Moreover, CaSR was expressed in mIMCD3 cells, and its expression was increased by increasing the concentrations and times of IL-1β treatment. Selective inhibitors (Calhex231 or NPS2143) or the siRNA of CaSR attenuated the enhanced expression of type I and III collagens. Furthermore, IL-1β increased nuclear β-catenin protein levels and decreased cytoplasmic β-catenin expression in cells. In contrast, blockage of CaSR by the pharmacological antagonists or siRNA could partially attenuate such changes in the IL-1β-induced nuclear translocation of β-catenin. DKK1, an inhibitor of β-catenin nuclear translocation, further inhibited the expression of type I and III collagens in cells treated with IL-1β plus CaSR antagonist. In summary, these data demonstrated that IL-1β-induced collagen I and III expressions in collecting duct cells might be partially mediated by CaSR and the downstream nuclear translocation of β-catenin.

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein—adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)—in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.