Identification of a Novel ECM Remodeling Macrophage Subset in AKI to CKD Transition by Integrative Spatial and Single-Cell Analysis.

The transition from acute kidney injury (AKI) to chronic kidney disease (CKD) is a critical clinical issue. Although previous studies have suggested macrophages as a key player in promoting inflammation and fibrosis during this transition, the heterogeneity and dynamic characterization of macrophages are still poorly understood. Here, we used integrated single-cell RNA sequencing and spatial transcriptomic to characterize the spatiotemporal heterogeneity of macrophages in murine AKI-to-CKD model of unilateral ischemia-reperfusion injury. A marked increase in macrophage infiltration at day 1 was followed by a second peak at day 14 post AKI. Spatiotemporal profiling revealed that injured tubules and macrophages co-localized early after AKI, whereas in late chronic stages had spatial proximity to fibroblasts. Further pseudotime analysis revealed two distinct lineages of macrophages in this transition: renal resident macrophages differentiated into the pro-repair subsets, whereas infiltrating monocyte-derived macrophages contributed to chronic inflammation and fibrosis. A novel macrophage subset, extracellular matrix remodeling-associated macrophages (EAMs) originating from monocytes, linked to renal fibrogenesis and communicated with fibroblasts via insulin-like growth factors (IGF) signalling. In sum, our study identified the spatiotemporal dynamics of macrophage heterogeneity with a unique subset of EAMs in AKI-to-CKD transition, which could be a potential therapeutic target for preventing CKD development.

Renal tubular epithelial cells response to injury in acute kidney injury.

Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid and significant decrease in renal function that can arise from various etiologies, and is associated with high morbidity and mortality. The renal tubular epithelial cells (TECs) represent the central cell type affected by AKI, and their notable regenerative capacity is critical for the recovery of renal function in afflicted patients. The adaptive repair process initiated by surviving TECs following mild AKI facilitates full renal recovery. Conversely, when injury is severe or persistent, it allows the TECs to undergo pathological responses, abnormal adaptive repair and phenotypic transformation, which will lead to the development of renal fibrosis. Given the implications of TECs fate after injury in renal outcomes, a deeper understanding of these mechanisms is necessary to identify promising therapeutic targets and biomarkers of the repair process in the human kidney.

Role of sphingolipid metabolism signaling in a novel mouse model of renal osteodystrophy based on transcriptomic approach.

BACKGROUND: Renal osteodystrophy (ROD) is a skeletal pathology associated with chronic kidney disease-mineral and bone disorder (CKD-MBD) that is characterized by aberrant bone mineralization and remodeling. ROD increases the risk of fracture and mortality in CKD patients. The underlying mechanisms of ROD remain elusive, partially due to the absence of an appropriate animal model. To address this gap, we established a stable mouse model of ROD using an optimized adenine-enriched diet and conducted exploratory analyses through ribonucleic acid sequencing (RNA-seq). METHODS: Male 8-week-old C57BL/6J mice were randomly allocated into three groups: control group (n = 5), adenine and high-phosphate (HP) diet group (n = 20), and the optimized adenine-containing diet group (n = 20) for 12 weeks. We assessed the skeletal characteristics of model mice through blood biochemistry, microcomputed tomography (micro-CT), and bone histomorphometry. RNA-seq was utilized to profile gene expression changes of ROD. We elucidated the functions of differentially expressed genes (DEGs) using gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). DEGs were validated via quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: By the fifth week, adenine followed by an HP diet induced rapid weight loss and high mortality rates in the mouse group, precluding further model development. Mice with optimized adenine diet-induced ROD displayed significant abnormalities in serum creatinine and blood urea nitrogen levels, accompanied by pronounced hyperparathyroidism and hyperphosphatemia. The femur bone mineral density (BMD) of the model mice was lower than that of control mice, with substantial bone loss and cortical porosity. ROD mice exhibited substantial bone turnover with an increase in osteoblast and osteoclast markers. Transcriptomic profiling revealed 1907 genes with upregulated expression and 723 genes with downregulated expression in the femurs of ROD mice relative to those of control mice. Pathway analyses indicated significant enrichment of upregulated genes in the sphingolipid metabolism pathway. The significant upregulation of alkaline ceramidase 1 (Acer1), alkaline ceramidase 2 (Acer2), prosaposin-like 1 (Psapl1), adenosine A1 receptor (Adora1), and sphingosine-1-phosphate receptor 5 (S1pr5) were successfully validated in mouse femurs by qRT-PCR. CONCLUSIONS: Optimized adenine diet mouse model may be a valuable proxy for studying ROD. RNA-seq analysis revealed that the sphingolipid metabolism pathway is likely a key player in ROD pathogenesis, thereby providing new avenues for therapeutic intervention.

Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155.

BACKGROUND: Chronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence. METHODS: Among the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155-/- mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization. RESULTS: Compared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated ß-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs. CONCLUSIONS: Our work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

Identification of stable reference genes for relative quantification of long RNA expression in urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA-seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA-seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1-2, RPL4, GAPDH, RPS27A as candidate reference genes. RT-qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.

Mincle receptor in macrophage and neutrophil contributes to the unresolved inflammation during the transition from acute kidney injury to chronic kidney disease.

Background: Recent studies have demonstrated a strong association between acute kidney injury (AKI) and chronic kidney disease (CKD), while the unresolved inflammation is believed to be a driving force for this chronic transition process. As a transmembrane pattern recognition receptor, Mincle (macrophage-inducible C-type lectin, Clec4e) was identified to participate in the early immune response after AKI. However, the impact of Mincle on the chronic transition of AKI remains largely unclear. Methods: We performed single-cell RNA sequencing (scRNA-seq) with the unilateral ischemia-reperfusion (UIR) murine model of AKI at days 1, 3, 14 and 28 after injury. Potential effects and mechanism of Mincle on renal inflammation and fibrosis were further validated in vivo utilizing Mincle knockout mice. Results: The dynamic expression of Mincle in macrophages and neutrophils throughout the transition from AKI to CKD was observed. For both cell types, Mincle expression was significantly up-regulated on day 1 following AKI, with a second rise observed on day 14. Notably, we identified distinct subclusters of Minclehigh neutrophils and Minclehigh macrophages that exhibited time-dependent influx with dual peaks characterized with remarkable pro-inflammatory and pro-fibrotic functions. Moreover, we identified that Minclehigh neutrophils represented an "aged" mature neutrophil subset derived from the "fresh" mature neutrophil cluster in kidney. Additionally, we observed a synergistic mechanism whereby Mincle-expressing macrophages and neutrophils sustained renal inflammation by tumor necrosis factor (TNF) production. Mincle-deficient mice exhibited reduced renal injury and fibrosis following AKI. Conclusion: The present findings have unveiled combined persistence of Minclehigh neutrophils and macrophages during AKI-to-CKD transition, contributing to unresolved inflammation followed by fibrosis via TNF-α as a central pro-inflammatory cytokine. Targeting Mincle may offer a novel therapeutic strategy for preventing the transition from AKI to CKD.

Podocyte-derived soluble RARRES1 drives kidney disease progression through direct podocyte and proximal tubular injury.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single-cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

HIPK2 C-terminal domain inhibits NF-κB signaling and renal inflammation in kidney injury.

HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-ß/Smad3-mediated extracellular matrix accumulation, NF-κB-mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that caspase-6-mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) lead to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment could restrain the NF-κB transcriptional activity by cytoplasmic sequestration of p65 and the attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidneys of mice with unilateral ureteral obstruction and LPS-induced acute kidney injury. Collectively, our findings indicate that the HIPK2-CT is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential antiinflammatory agents to treat kidney disease.

Tubule-specific cyclin-dependent kinase 12 knockdown potentiates kidney injury through transcriptional elongation defects.

Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.

Therapeutic role of miR-26a on cardiaorenal injury in mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway.

BACKGROUND: Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD. METHODS: We generated a microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett's multiple comparison and t-test were used to analyze the data. RESULTS: Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes. CONCLUSIONS: Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.

Incidence, risk factors and outcome of postoperative acute kidney injury in China.

BACKGROUND: Postoperative acute kidney injury (AKI) is a common condition after surgery, however, the available data about nationwide epidemiology of postoperative AKI in China from large and high-quality studies are limited. This study aimed to determine the incidence, risk factors and outcomes of postoperative AKI among patients undergoing surgery in China. METHODS: This was a large, multicentre, retrospective study performed in 16 tertiary medical centres in China. Adult patients (≥18 years of age) who underwent surgical procedures from 1 January 2013 to 31 December 2019 were included. Postoperative AKI was defined by the Kidney Disease: Improving Global Outcomes creatinine criteria. The associations of AKI and in-hospital outcomes were investigated using logistic regression models adjusted for potential confounders. RESULTS: Among 520 707 patients included in our study, 25 830 (5.0%) patients developed postoperative AKI. The incidence of postoperative AKI varied by surgery type, which was highest in cardiac (34.6%), urologic (8.7%) and general (4.2%) surgeries. A total of 89.2% of postoperative AKI cases were detected in the first 2 postoperative days. However, only 584 (2.3%) patients with postoperative AKI were diagnosed with AKI on discharge. Risk factors for postoperative AKI included older age, male sex, lower baseline kidney function, pre-surgery hospital stay ≤3 days or >7 days, hypertension, diabetes mellitus and use of proton pump inhibitors or diuretics. The risk of in-hospital death increased with the stage of AKI. In addition, patients with postoperative AKI had longer lengths of hospital stay (12 versus 19 days) and were more likely to require intensive care unit care (13.1% versus 45.0%) and renal replacement therapy (0.4% versus 7.7%). CONCLUSIONS: Postoperative AKI was common across surgery type in China, particularly for patients undergoing cardiac surgery. Implementation and evaluation of an alarm system is important for the battle against postoperative AKI.

Measurement of urinary exosomal phospholipase A2 receptor is a sensitive method for diagnosis of PLA2R-associated membranous nephropathy.

Background: The discovery of phospholipase A2 receptor (PLA2R) and its antibody (aPLA2Rab) has paved the way for diagnosing PLA2R-associated membranous nephropathy (PLA2R-MN) with a high specificity of 98%. However, the sensitivity was only 40% to 83.9%, and there is ongoing discussion around determining the optimal threshold for diagnosis. Recent advancements in the use of exosomes, a novel form of "liquid biopsy," have shown great promise in identifying markers for various medical conditions. Methods: Protein mass spectrometry and western blot were applied to verify the existence of PLA2R antigen in the urine exosome. We then evaluated the efficacy of urinary exosomal PLA2R antigen alone or combined with serum aPLA2Rab level to diagnose PLA2R-MN. Results: The urinary exosomes contained a high abundance of PLA2R antigen as evidenced by protein mass spectrometry and western blot in 85 PLA2R-MN patients vs the disease controls (14 secondary MN patients, 22 non-MN patients and 4 PLA2R-negative MN patients) and 20 healthy controls. Of note, urinary exosomal PLA2R antigen abundance also had a good consistency with the PLA2R antigen level in the renal specimens of PLA2R-MN patients. The sensitivity of urinary exosomal PLA2R for diagnosing PLA2R-MN reached 95.4%, whereas the specificity was 63.3%. Combining detection of the urinary exosomal PLA2R and serum aPLA2Rab could develop a more sensitive diagnostic method for PLA2R-MN, especially for patients with serum aPLA2Rab ranging from 2 to 20 RU/mL. Conclusions: Measurement of urinary exosomal PLA2R could be a sensitive method for the diagnosis of PLA2R-MN.

Renal histology of Fanconi syndrome associated with adefovir dipivoxil: A case report.

A sporadic occurrence of Fanconi syndrome associated with adefovir dipivoxil (ADV) has been reported, particularly when confirmed by renal biopsy. This study presents the case of a 53-year-old man who had been taking ADV 10 mg daily for 10 years to treat chronic hepatitis B (CHB) and subsequently developed Fanconi syndrome. The clinical manifestations included hypophosphatemic osteomalacia, glucosuria, renal tubular acidosis, low-molecular-weight proteinuria, and renal insufficiency. Renal biopsy revealed significant injury to proximal tubular epithelial cells, including vacuolar degeneration and regeneration of tubular epithelial cells. The ultrastructural pathology indicated severe morphological abnormalities of mitochondria, such as densely packed and enlarged mitochondria, with loss, blunting, and disordered arrangement of cristae. Following discontinuation of ADV and supplementation with oral phosphate, hypophosphatemia, glucosuria, and proteinuria were resolved. These findings support the previous hypothesis that ADV-induced nephrotoxicity may involve mitochondrial injury.

Computed tomography-determined skeletal muscle density predicts 3-year mortality in initial-dialysis patients in China.

BACKGROUND: Skeletal muscle mass and quality assessed by computed tomography (CT) images of the third lumbar vertebra (L3) level have been established as risk factors for poor clinical outcomes in several illnesses, but the relevance for dialysis patients is unclear. A few studies have suggested a correlation between CT-determined skeletal muscle mass and quality at the first lumbar vertebra (L1) level and adverse outcomes. Generally, chest CT does not reach beyond L1. We aimed to determine whether opportunistic CT scan (chest CT)-determined skeletal muscle mass and quality at L1 are associated with mortality in initial-dialysis patients. METHODS: This 3-year multicentric retrospective study included initial-dialysis patients from four centres between 2014 and 2017 in China. Unenhanced CT images of the L1 and L3 levels were obtained to assess skeletal muscle mass [by skeletal muscle index, (SMI), cm2 /m2 ] and quality [by skeletal muscle density (SMD), HU]. Skeletal muscle measures at L1 were compared with those at L3. The sex-specific optimal cutoff values of L1 SMI and L1 SMD were determined in relation to all-cause mortality. The outcomes were all-cause death and cardiac death. Cox regression models were applied to investigate the risk factors for death. RESULTS: A total of 485 patients were enrolled, of whom 257 had both L1 and L3 images. Pearson's correlation coefficient between L1 and L3 SMI was 0.84 (P < 0.001), and that between L1 and L3 SMD was 0.90 (P < 0.001). No significant association between L1 SMI and mortality was observed (P > 0.05). Low L1 SMD (n = 280, 57.73%) was diagnosed based on the optimal cutoff value (<39.56 HU for males and <33.06 HU for females). Multivariate regression analysis revealed that the low L1 SMD group had higher risks of all-cause death (hazard ratio 1.80; 95% confidence interval 1.05-3.11, P = 0.034) and cardiac death (hazard ratio 3.74; 95% confidence interval 1.43-9.79, P = 0.007). CONCLUSIONS: In initial-dialysis patients, there is high agreement between the L1 and L3 measures for SMI and SMD. Low SMD measured at L1, but not low SMI, is an independent predictor of both all-cause death and cardiac death.

Progression of Vascular Calcification and Clinical Outcomes in Patients Receiving Maintenance Dialysis.

Importance: Baseline findings from the China Dialysis Calcification Study (CDCS) revealed a high prevalence of vascular calcification (VC) among patients with end-stage kidney disease; however, data on VC progression were limited. Objectives: To understand the progression of VC at different anatomical sites, identify risk factors for VC progression, and assess the association of VC progression with the risk of cardiovascular events and death among patients receiving maintenance dialysis. Design, Setting, and Participants: This cohort study was a 4-year follow-up assessment of participants in the CDCS, a nationwide multicenter prospective cohort study involving patients aged 18 to 74 years who were undergoing hemodialysis or peritoneal dialysis. Participants were recruited from 24 centers across China between May 1, 2014, and April 30, 2015, and followed up for 4 years. A total of 1489 patients receiving maintenance dialysis were included in the current analysis. Data were analyzed from September 1 to December 31, 2021. Exposures: Patient demographic characteristics and medical history; high-sensitivity C-reactive protein laboratory values; serum calcium, phosphorus, and intact parathyroid hormone (iPTH) values; and previous or concomitant use of medications. Main Outcomes and Measures: The primary outcome was progression of VC at 3 different anatomical sites (coronary artery, abdominal aorta, and cardiac valves) and identification of risk factors for VC progression. Participants received assessments of coronary artery calcification (CAC), abdominal aortic calcification (AAC), and cardiac valve calcification (CVC) at baseline, 24 months, 36 months, and 48 months. Secondary outcomes included (1) the association between VC progression and the risk of all-cause death, cardiovascular (CV)-related death, and a composite of all-cause death and nonfatal CV events and (2) the association between achievement of serum calcium, phosphorus, and iPTH target levels and the risk of VC progression. Results: Among 1489 patients, the median (IQR) age was 51.0 (41.0-60.0) years; 59.5% of patients were male. By the end of 4-year follow-up, progression of total VC was observed in 86.5% of patients; 69.6% of patients had CAC progression, 72.4% had AAC progression, and 33.4% had CVC progression. Common risk factors for VC progression at the 3 different anatomical sites were older age and higher fibroblast growth factor 23 levels. Progression of CAC was associated with a higher risk of all-cause death (model 1 [adjusted for age, sex, and body mass index]: hazard ratio [HR], 1.97 [95% CI, 1.16-3.33]; model 2 [adjusted for all factors in model 1 plus smoking status, history of diabetes, and mean arterial pressure]: HR, 1.89 [95% CI, 1.11-3.21]; model 3 [adjusted for all factors in model 2 plus calcium, phosphorus, intact parathyroid hormone, and fibroblast growth factor 23 levels and calcium-based phosphate binder use]: HR, 1.92 [95% CI, 1.11-3.31]) and the composite of all-cause death and nonfatal CV events (model 1: HR, 1.98 [95% CI, 1.19-3.31]; model 2: HR, 1.91 [95% CI, 1.14-3.21]; model 3: HR, 1.95 [95% CI, 1.14-3.33]) after adjusting for all confounding factors except the presence of baseline calcification. Among the 3 targets of calcium, phosphorus, and iPTH, patients who achieved no target levels (model 1: odds ratio [OR], 4.75 [95% CI, 2.65-8.52]; model 2: OR, 4.81 [95% CI, 2.67-8.66]; model 3 [for this analysis, adjusted for all factors in model 2 plus fibroblast growth factor 23 level and calcium-based phosphate binder use]: OR, 2.76 [95% CI, 1.48-5.16]), 1 target level (model 1: OR, 3.71 [95% CI, 2.35-5.88]; model 2: OR, 3.62 [95% CI, 2.26-5.78]; model 3: OR, 2.19 [95% CI, 1.33-3.61]), or 2 target levels (model 1: OR, 2.73 [95% CI, 1.74-4.26]; model 2: OR, 2.69 [95% CI, 1.71-4.25]; model 3: OR, 1.72 [95% CI, 1.06-2.79]) had higher odds of CAC progression compared with patients who achieved all 3 target levels. Conclusions and Relevance: In this study, VC progressed rapidly in patients undergoing dialysis, with different VC types associated with different rates of prevalence and progression. Consistent achievement of serum calcium, phosphorus, and iPTH target levels was associated with a lower risk of CAC progression. These results may be useful for increasing patient awareness and developing appropriate strategies to improve the management of chronic kidney disease-mineral and bone disorder among patients undergoing dialysis.

Relationship between the triglyceride-glucose index and coronary artery calcification in asymptomatic, non-diabetic patients undergoing maintenance hemodialysis.

OBJECTIVE: Coronary artery calcification (CAC) is positively and independently associated with cardiovascular disease (CVD) in patients undergoing maintenance hemodialysis (MHD). Insulin resistance is independently associated with CAC and is an important risk factor for CVD. The triglyceride-glucose (TyG) index is a reliable biomarker of insulin resistance. This cross-sectional, observational study aimed to investigate the relationship between the TyG index and CAC in asymptomatic non-diabetic patients undergoing MHD. METHODS: The quantitative coronary artery calcification score (CACS) was calculated and expressed using the Agatston score. The TyG index was calculated as ln [fasting triglyceride (mg/dL) × fasting glucose (mg/dL)/2]. Multiple Poisson regression analysis, Spearman correlation analysis, and receiver operating characteristic (ROC) curves were used to investigate the relationship between the TyG index and CAC. RESULTS: The 151 patients were divided into three groups according to the tertiles of the TyG index. With an increase in the TyG index, the CACS significantly increased (Spearman's rho = 0.414, p < 0.001). Poisson regression analysis indicated that the TyG index was independently related to the presence of CAC (prevalence ratio, 1.281 [95% confidence interval, 1.121-1.465], p < 0.001). Furthermore, ROC curve analysis showed that the TyG index was of value in predicting the CAC in asymptomatic non-diabetic patients undergoing MHD, with an area under the curve of 0.667 (p = 0.010). CONCLUSION: The TyG index is independently related to the presence of CAC in asymptomatic, non-diabetic patients undergoing MHD.

Quercetin alleviates tubulointerstitial inflammation by inhibiting exosomes-mediated crosstalk between tubular epithelial cells and macrophages.

BACKGROUND: Tubulointerstitial inflammation (TII) is a critical pathological feature of kidney disease leading to renal fibrosis, and its treatment remains a major clinical challenge. We sought to explore the role of quercetin, a potential exosomes inhibitor, in exosomes release and TII. METHODS: The effects of quercetin on exosomes release and TII were examined by two TII mouse models: the unilateral ureteral obstruction (UUO) models and the LPS-induced mouse models. In vitro, exosomes-mediated crosstalk between tubular epithelial cells (TECs) and macrophages was performed to investigate the mechanisms by which quercetin inhibited exosomes and TII. RESULTS: In this study, we found that exosomes-mediated crosstalk between TECs and macrophages contributed to the development of TII. In vitro, exosomes released from LPS-stimulated TECs induced increased expression of inflammatory cytokines and fibrotic markers in Raw264·7 cells and vice versa. Interestingly, heat shock protein 70 (Hsp70) or Hsp90 proteins could control exosomes release from TECs and macrophages both in vivo and in vitro. Importantly, quercetin, a previously recognized heat shock protein inhibitor, could significantly reduce exosomes release in TII models by down-regulating Hsp70 or Hsp90. Quercetin abrogated exosomes-mediated intercellular communication, which attenuated TII and renal fibrosis accordingly. CONCLUSION: Quercetin could serve as a novel strategy for treatment of tubulointerstitial inflammation by inhibiting the exosomes-mediated crosstalk between tubules and macrophages.

Comprehensive analysis of the roles of fatty acid transport related proteins in clear cell renal cell carcinoma.

OBJECTIVE: This study aimed to explore the clinical significance of fatty acid transport-related protein (FATRP) in patients with clear cell renal cell carcinoma(ccRCC). METHODS: RNA-seq data and corresponding clinical data of ccRCC were obtained from TCGA data portal. Seventeen key FATRP genes were comprehensively investigated using bioinformatics approaches to systematically investigate their expression patterns in ccRCC. In addition, the correlation between the expression levels of these genes and clinicopathological features in ccRCC was further explored. RESULTS: Among the 17 key FATRP genes, only FABP5, FABP6, and FABP7 could be regarded as ideal biomarkers for ccRCC, as they were highly expressed in ccRCC tumor tissues, and positively correlates with tumor progression and poor prognosis. FABP6 had the highest copy number variations (CNV) events (63.07 %), and ccRCC patients with FABP6 amplification had a better prognosis than the unaltered group. DNA methylation levels of FABP6 and FABP7 were downregulated in ccRCC tumor tissues compared to those in normal tissues. FABP5 showed the opposite results. Moreover, a novel four FATRP gene (FABP1, FABP5, FABP7, FATP2) and three clinical parameter (age, stage, and grade) prediction model was constructed and that comprised a significant independent prognostic signature. CONCLUSIONS: Only a few FATRP genes are upregulated in ccRCC tumor tissue, and positively correlate with tumor progression and poor prognosis. The accuracy of a single gene of these FATRP genes as predictors of progression and prognosis of ccRCC is limited. The performance of the novel prediction model proposed by this study was much better than that of any single gene.

Detecting Neonatal AKI by Serum Cystatin C.

SIGNIFICANCE STATEMENT: Serum creatinine is not a sensitive biomarker for neonatal AKI because it is confounded by maternal creatinine level, gestational age, and neonatal muscle mass. In this multicenter cohort study of 52,333 hospitalized Chinese neonates, the authors proposed serum cystatin C-related criteria (CyNA) for neonatal AKI. They found that cystatin C (Cys-C) is a robust and sensitive biomarker for identifying AKI in neonates who are at an elevated risk of in-hospital mortality and that CyNA detects 6.5 times as many cases as the modified Kidney Disease Improving Global Outcomes creatinine criteria. They also show that AKI can be detected using a single test of Cys-C. These findings suggest that CyNA shows promise as a powerful and easily applicable tool for detecting AKI in neonates. BACKGROUND: Serum creatinine is not a sensitive biomarker for AKI in neonates. A better biomarker-based criterion for neonatal AKI is needed. METHODS: In this large multicenter cohort study, we estimated the upper normal limit (UNL) and reference change value (RCV) of serum cystatin C (Cys-C) in neonates and proposed cystatin C-based criteria (CyNA) for detecting neonatal AKI using these values as the cutoffs. We assessed the association of CyNA-detected AKI with the risk of in-hospital death and compared CyNA performance versus performance of modified Kidney Disease Improving Global Outcomes (KDIGO) creatinine criteria. RESULTS: In this study of 52,333 hospitalized neonates in China, Cys-C level did not vary with gestational age and birth weight and remained relatively stable during the neonatal period. CyNA criteria define AKI by a serum Cys-C of ≥2.2 mg/L (UNL) or an increase in Cys-C of ≥25% (RCV) during the neonatal period. Among 45,839 neonates with measurements of both Cys-C and creatinine, 4513 (9.8%) had AKI detected by CyNA only, 373 (0.8%) by KDIGO only, and 381 (0.8%) by both criteria. Compared with neonates without AKI by both criteria, neonates with AKI detected by CyNA alone had an increased risk of in-hospital mortality (hazard ratio [HR], 2.86; 95% confidence interval [95% CI], 2.02 to 4.04). Neonates with AKI detected by both criteria had an even higher risk of in-hospital mortality (HR, 4.86; 95% CI, 2.84 to 8.29). CONCLUSIONS: Serum Cys-C is a robust and sensitive biomarker for detecting neonatal AKI. Compared with modified KDIGO creatinine criteria, CyNA is 6.5 times more sensitive in identifying neonates at elevated risk of in-hospital mortality.

KIM-1 augments hypoxia-induced tubulointerstitial inflammation through uptake of small extracellular vesicles by tubular epithelial cells.

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.