bluuebunny/crossref_metadata_2025_split huggingface dataset:
#1548 The involvement of soluble fms-like tyrosine kinase-1 in the transition from acute kidney injury to chronic kidney disease
Authors: Hikari Tasaki , Tsushima Hideo , Eriguchi Masahiro , Uemura Takayuki , Tamaki Hiroyuki , Nishimoto Masatoshi , Kosugi Takaaki , Samejima Ken-Ichi , Tsuruya Kazuhiko 
Published: May 2024

Abstract

Background and Aims
Fms-like tyrosine kinase-1 (Flt-1) is a receptor for vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and, playing a crucial role in angiogenesis and inflammation pathways. Soluble Flt-1 (sFlt-1) acts as a decoy for both VEGF and PlGF, regulating Flt-1-mediated angiogenesis and inflammation. We investigated the involvement of sFlt-1 in the transition from acute kidney injury to chronic kidney disease (CKD).

Method
Wild-type (WT) and sFlt-1 knock-out (KO) mice underwent ischemia-reperfusion injury (IRI) treatment. A unilateral 23-minute IRI and contralateral nephrectomy were performed to examine kidney function (AKI to CKD) for 2 weeks. To explore the downstream signaling pathway for sFlt-1 in the kidney, a unilateral 28-minute IRI was performed, and angiogenesis and cytokine expression arrays (Proteome Profiler Array Kits) were used to compare the relative expressions between 28-minute IRI and the contralateral untreated kidney cortices in both WT and sFlt-1 KO mice.

Results
A plasma sFlt-1 concentration was elevated just after IRI and reached its peak after 12 hours. Blood urea nitrogen (BUN) and plasma creatinine (pCre) levels peaked at 24 hours after IRI. The peak levels of BUN and pCre were significantly lower in sFlt-1 KO compared to those in WT mice (BUN 43.23 ± 5.9 mg/dL and 82.7 ± 13.0 mg/dL, p = 0.008, pCre 0.18 ± 0.03 mg/dL and 0.31 ± 0.04 mg/dL, p = 0.008 in KO and WT mice, respectively). Urinary KIM-1 (uKIM-1) levels 12 hours after IRI in WT mice were higher than those in sFlt-1 KO mice (WT vs sFlt-1 KO; uKIM-1 4.82 ± 0.83 pg/day vs 2.39 ± 0.98 pg/day, p = 0.09). Histopathological evaluation of the kidneys showed reduced monocyte infiltration in sFlt-1 KO mice compared to WT mice 14 days after IRI. In cytokine expression assay of kidney cortices 3 days after IRI, IRI-induced increased expressions of tissue repairs and immune response proteins such as TIMP-1, C5/C5a, and TREM-1 were alleviated in sFlt-1 KO mice compared to those in WT mice. In the Angiogenesis expression array of kidney cortices 1 day after IRI, there were no significant differences in the levels of IRI-induced increased expression of anti-/pro-angiogenesis factors, including TIMP-1, Cyr61, SerpinE1, angiogenin, platelet factor 4, collagen factor 3, MMP-9, and SDF-1, between WT and sFlt-1 KO mice.

Conclusion
The acute and chronic kidney damages after IRI was reduced in sFlt-1 KO mice compared to WT mice. The protective effect on the kidneys by eliminating sFlt-1 would be associated with immune response signaling such as TIMP-1, C5/C5a, and TREM-1 but not with anti-/pro-angiogenesis factors.
#1457 Human renal proximal tubule-on-a-chip model to study prevention of ischemia and reperfusion injury
Authors: Tool Laura , Vormann Marianne , Ohbuchi Masato , Gijzen Linda , Van Vught Remko , Hankemeier Thomas , Kiyonaga Fumiko , Kawabe Tetsuhiro , Goto Takayuki , Fujimori Akira , Lanz Henriëtte , Tetsuka Kazuhiro 
Published: May 2024

Abstract

Background and Aims
Renal ischemia/reperfusion injury (rIRI) is characterized by restriction of blood supply followed by restoration of blood flow and re-oxygenation and is a leading cause of acute kidney injury (AKI). While animal models are useful for studying systemic manifestations of AKI, their translational relevance is limited. Human in vitro models provide valuable mechanistic insights into rIRI, but often fail to incorporate crucial aspects such as reperfusion injury. To address these limitations, this study aims to develop an advanced in vitro model to study rIRI comprising a perfused three-dimensional (3D) human renal proximal tubule-on-a-chip.

Method
Our proximal tubule-on-a-chip model comprises a renal proximal tubule alongside a endothelial vessel, separated by extracellular matrix. A microfluidic platform, the OrganoPlate 3-Lane 40, was used to establish the model. The resulting coculture was characterized in terms of its three-dimensional structure, protein expression, and response to nephrotoxins. Subsequently, we simulated rIRI by manipulating oxygen levels, nutrient availability, and perfusion flow settings. The potential protective effect of adenosine was evaluated in the coculture model during ischemia and reperfusion. The extent of injury was assessed through morphological evaluation, caspase 3/7 activation, and cell viability measurements.

Results
The combination of low oxygen, reduced glucose, and interrupted flow exerted a potent disruptive effect on the proximal tubules, which was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia–reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase-3/7 activation.

Conclusion
We successfully developed a human in vitro model to study rIRI utilizing a coculture of a proximal tubule and a blood vessel on-a-chip. This model allowed us to characterize the renoprotective effect of adenosine. The robustness of the model and assays, coupled with the platform's high throughput capabilities, position it as a promising tool for advancing pathophysiological research and facilitating the development of innovative therapeutic modalities aimed at treating rIRI and improving AKI outcomes.
#2529 The role of the circadian clock system in the transition from acute kidney injury to chronic kidney disease
Authors: Park Sang Hoon , Jo Sang Kyung , Kim Myung Gyu 
Published: May 2024

Abstract

Background and Aims
Recent studies suggest that circadian rhythms are an important factor in maintaining healthy kidney functions; however, its role in acute kidney injury (AKI) remains unclear.

Method
To investigate the role of tubule-specific clock gene disturbance in hypoxic tubular injury, we created human proximal tubule-specific BMAL1 KO cells using CRISPR-Cas9 gene editing. In mice, we also determined whether time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, can have beneficial effect on recovery of ischemia reperfusion injury (IRI) or its progression to chronic kidney disease (CKD).

Results
BMAL1 loss in tubule cells resulted in the upregulation of cell cycle regulatory gene (p21) and inflammatory genes (TNF-α and CCL4) mRNA expression, suggesting that BMAL1 is a critical regulator of these genes. Exposure to hypoxia for 48 h resulted in more increased expressions of p21 in BMAL 1 KO cells than in WT HK-2 cells, leading to higher expression of TGF-b. In addition, loss of BMAl-1 led to significantly increased vimentin and decreased E-cadherin expression upon TGF-b stimulation, suggesting an aggravated epithelial-mesenchymal transition upon BMAL1 deletion. To clarify the role of clock system in AKI, we also tested whether TRF can have a beneficial effect during recovery phase after IRI. Despite limited access to food, TRF for 4 weeks after IRI had no effect on the total intake or weight of mice. However, we observed that TRF significantly improved renal function on day 21 and reduced renal fibrosis on day 28, which suggests that enhancing clock gene oscillations through restoring fasting/feeding cycle has a protective effect in the AKI to CKD transition.

Conclusion
We identified an important role of clock system in hypoxic tubular injury and the effects of treatment targeting circadian rhythm in AKI. Our results can provide a new perspective for developing novel therapeutic strategies for AKI.
#435 Endothelial activation and inflammation in chronic kidney disease (CKD): role of neutrophil extracellular traps (NETs)
Authors: Junho Carolina Victoria Cruz , Vondenhoff Sonja , Martin Philipp , Noels Heidi 
Published: May 2024

Abstract

Background and Aims
Advanced chronic kidney disease (CKD), especially in stages 4-5, amplifies cardiovascular disease (CVD) risk by around 50%. This heightened susceptibility is linked to a 1.5 to 3-fold increase in venous thromboembolism incidence, driven by coagulation dysregulation and heightened inflammation. During inflammation, neutrophils can release extracellular traps (NETs), that was described contribute to endothelial damage, through adhesion molecule elevation, and tissue factor (TF) secretion, exacerbating thrombosis. Additionally, NETs interact with von Willebrand Factor (vWF) and factor XII, further amplifying thrombus formation and the activation of the intrinsic coagulation pathway. The aim of this study was to observe whether NETs may contribute to increased CVD risk in CKD patients by inducing endothelial activation and thromboinflammatory responses to a larger extent compared to NETs from healthy donors.

Method
For that, neutrophils were isolated from healthy donors’ and CKD patients’ (stages 3-5 HD) peripheral blood, and NETs were collected by stimulating neutrophils with PMA for 3.5 hours. Human aortic endothelial cells (HAoECs) were subsequently exposed to NETs for 3 to 24 hours, followed by gene expression analysis with a focus on inflammation, endothelial activation, and prothrombotic markers. Additionally, protein analysis of the supernatant was conducted using ELISA. All experiments were performed under ethical protocol EK 225-21 from the University Hospital of Aachen.

Results
Exposure to both healthy NETs as well as the ones isolated from CKD patients resulted in a notable upregulation of endothelial activation (VCAM-1), prothrombotic (TF), and inflammatory (IL-6) gene expression within 3 hours, without difference between healthy and CKD conditions. On the other hand, when examining the cellular supernatant, a significantly higher increase in vWF secretion was observed upon treatment with CKD-NETs compared to healthy NETs. Furthermore, while secretion of the inflammatory cytokine IL-6 presented a comparable increase upon treatment with NETs derived from healthy and CKD neutrophils, IL-1β secretion was markedly enhanced after CKD-NET treatment in comparison to healthy NETs.

Conclusion
In summary, our findings suggest that NETs exert a significant influence on endothelial cells, contributing to increased thromboinflammatory activation. Notably, CKD-NETs evoked a more rapid impact on the endothelium, accompanied by increased secretion of IL-1β and vWF, and underlying mechanisms will be further investigated.
#1326 Impairment of ACSS2 aggravates renal fibrosis and cell senescence during aging
Authors: Fu Ping , Xiang Ting , LI Lingzhi , LI Jinxi , MA Liang 
Published: May 2024

Abstract

Background and Aims
Age-related changes in kidney function, structure, and their relationships have been extensively recognized recently. Here, we report that acyl-CoA synthetase short chain family member 2 (Acss2) as a regulator of lipid metabolism, play an important role in age-associated renal fibrosis.

Method
21-month-old Acss2 knockout (Acss2−/−) mice were used as the renal aging models, and H2O2 also was used to induced renal tubular epithelial cells (TCMK-1) senescence. Serum creatinine (Cr) and urinary protein creatinine ratio (UACR) were measured to evaluate kidney function, and renal tissue lesions were observed by HE staining and Masson staining. The expression of gene/protein in relation to inflammation, fibrosis and cell senescence were measured by qPCR, western blot. SA-β-gal staining also used to assess cell senescence.

Results
The results indicate that Acss2 was decreased in aging mice, Acss2 knockout impaired renal function as detected by increased UACR. Additionally, HE and Masson staining showed that Acss2 deficiency was associated with more glomerular sclerosis and fibrosis (Fig. 1). RT-qPCR and Western blotting also revealed that Acss2 enhanced kidney fibrosis and cell senescence, accompanied by an increase in SA-β-gal-positive senescent cells (Fig. 2). These phenomena were most apparent in the TCMK cells treated with Acss2 siRNA and H2O2 (Fig. 3). What's more, KEGG analysis showed that fatty acid metabolism changes significantly during aging in kidney, and Acss2 knockout could impact the expression of lipid metabolism genes (Fig. 4)

Conclusion
Acss2 is associated with renal aging, and it may played a role in renal fibrosis and cell senescence by regulating fatty acid metabolism.
#2000 Analysis between site-specific N-glycosylation of monoclonal immunoglobulin and complement system activation
Authors: Lu Wanhong , LI Huixian , Xie Xinfang 
Published: May 2024

Abstract

Background and Aims
Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma (MM) and monoclonal gammopathy of renal significance (MGRS). Hypocomplementemia and deposition of complement components in the kidney is observed in some patients with MM or MGRS. Previous studies have suggested that glycosylation of the immunoglobulin was associated with complement-mediated-effector activities. Whereas, site-specific N-glycosylation characterization for monoclonal immunoglobulin in MM or MGRS and its effects on complement system activation still remains unclear.

Method
We separated plasma monoclonal IgG1 and IgA immunoglobulin from patients with MM and human plasma immunoglobulin from healthy controls (HC). Purified IgG and IgA molecules were digested by trypsin. Tryptic peptides without enrichment of intact N-glycopeptides were analyzed using a combination of electron-transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (EThcD-sceHCD-MS/MS). N-glycosylation characterization was compared between monoclonal immunoglobulin from MM patients and healthy controls and its association with complement level was evaluated.

Results
A total of 46 IgG1 digestion samples (HC, n = 16; MM, n = 30) and 38 IgA1 digestion samples (HC, n = 16; MM, n = 22) were analyzed using the EThcDsceHCD-MS/MS. On average, 34 and 37 unique N-glycopeptides of IgG1, 19 unique N-glycans per IgG were identified from HC and MM. The relative abundance of fucosylated N-glycans of IgG1 in MM was significantly higher than HC (94.7% vs 88.8%, p = 0.002). Other types of N-glycans including sialylated, hybrid and complex were similar between the two groups. The types of N-glycans including fucosylated, sialylated, hybrid and complex were similar between patients with hypocomplementemia and normal complement levels. Regarding IgA1, on average, 63 and 52 unique N-glycopeptides of IgA1, 40 and 35 unique N-glycans per IgA were identified from HC and MM. The relative abundance of fucosylated (41.0% vs 26.4%, p &lt; 0.001), sialylated (61.0% vs 45.6%, p = 0.003), mannose (2.44% vs 0.28%, p &lt; 0.001) N-glycans of IgA1 in MM was significantly higher than HC. In MM, the relative abundance of mannose N-glycans of IgA1 in patients with hypocomplementemia was significantly lower than patients with normal complement levels (1.69% vs 7.52%, p &lt; 0.001). The relative abundance of fucosylated and sialylated N-glycans were similar between patients with hypocomplementemia and normal complement levels.

Conclusion
Site-specific N-glycosylation characterization of monoclonal IgG1 and IgA1 in MM patients were differentially expressed from healthy controls. Certain specific N-glycan types were associated with complement levels. These findings laid a foundation for future investigations of their biological effects.
#2037 Mitochondrial fumarate promotes ischemia/reperfusion-induced tubular injury
Authors: Liu Bicheng , LI Zuolin 
Published: May 2024

Abstract

Background and Aims
Mitochondrial dysfunction, a characteristic pathological feature of renal Ischemic/reperfusion injury (I/RI), predisposes tubular epithelial cells to maintain an inflammatory microenvironment, however, the exact mechanisms through which mitochondrial dysfunction modulates the induction of tubular injury remains incompletely understood.

Method
ESI-QTRAP-MS/MS approach was used to characterize the targeted metabolic profiling of kidney with I/RI. Tubule injury, mitochondrial dysfunction and fumarate level were evaluated using qPCR, transmission electron microscopy, ELISA, and immunohistochemistry.

Results
We demonstrated that tubule injury occurs at the phase of reperfusion in murine models of I/RI. Meanwhile, the enhanced glycolysis and mitochondrial dysfunction are found to be associated with tubule injury. Further, we found that tubular fumarate, which resulted from fumarate hydratase deficiency and released from dysfunctional mitochondria, promotes tubular injury. Mechanistically, fumarate induces tubular injury through causing disturbance of glutathione (GSH) hemostasis. Suppression of GSH with buthionine sulphoximine administration could deteriorate the fumarate inhibition-mediated tubule injury recovery. Reactive oxygen species/NF-κB signaling activation plays a vital role in fumarate-mediated tubule injury.

Conclusion
Our studies demonstrated that the mitochondrial-derived fumarate promotes tubular epithelial cells injury in renal I/RI. This finding represents a previously unrecognized mechanism for hypoxia-induced TECs injury. Blockade of fumarate-mediated ROS/NF-κB signaling activation may serve as a novel therapeutic approach to ameliorate hypoxic tubule injury.
#2394 Inhibition of ACSS2-mediated H3K9 crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence
Authors: LI Lingzhi , Xiang Ting , Fu Ping , MA Liang 
Published: May 2024

Abstract

Background and Aims
Histone lysine crotonylation (Kcr), a novel posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis.

Method
Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University.ACSS2 knockout mice and tubular specific knock out of ACSS2 mice in C57BL/6N background and wild-type littermates (WT) were used to induce the FAN and UUO model or as control groups (n = 6 each). In the parallel study, C57BL/6N mice were randomly divided into three groups: control, FAN/UUO and FAN/UUO+ACSS2 inhibitor (n = 6 each). At day 7, mice were sacrificed and the serum creatinine, BUN and urinary albumin/creatinine ratio (ACR) were tested. Renal histological injury was measured by HE and Masson's trichrome staining. SA-β-gal staining was used to check cellular senescence. Renal tissues of the mice were analyzed by western blot and qPCR assay for the expression of ACSS2, H3K9cr, fibrosis and cellular senescence related proteins and genes. RNA-sequencing and ChIP-sequencing combined with in vitro cell experiments were used to figure out the underlying mechanism.

Results
In this study, we reported that histone Kcr of tubular epithelial cells was abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) was found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys revealed that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis.

Conclusion
Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.
#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells
Authors: Garmaa Gantsetseg , Manzéger Anna , Kökény Gábor 
Published: May 2024

Abstract

Background and Aims
Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown.

Method
Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p &lt; 0.05.

Results
The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p &lt; 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p &lt; 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p &lt; 0.05).

Conclusion
Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.