Primary Renal Tubular Epithelial Cells Research Articles

Single-cell RNA-sequencing of BK polyomavirus replication in primary human renal proximal tubular epithelial cells identifies specific transcriptome signatures and a novel mitochondrial stress pattern.

BK polyomavirus (BKPyV) contributes to premature renal failure in 10%-20% of kidney transplant recipients. Current treatment relies on reducing immunosuppression to regain BKPyV-specific immune control. Subsequently, declining allograft function may result from persisting viral cytopathology, BKPyV-specific immune reconstitution, or alloimmunity/rejection, all being poorly distinguishable by current histological or molecular approaches. To reduce the complexity encountered in BKPyV-replicating kidneys, we analyzed differentially expressed genes (DEGs) in primary human renal proximal tubular epithelial cells at 24 and 48 h post-infection (hpi) using single-cell RNA-sequencing (10x-Genomics-3´ kit). At 24 hpi, viral transcript reads predominantly mapped to the early viral gene region (EVGR) and shifted to >100-fold higher late viral gene region (LVGR) levels at 48 hpi, matching the sequential bi-directional viral protein expression from the circular double-stranded BKPyV-DNA genome. Besides expected coverage "hills" at viral 3´-poly-A sites, unexpected "spike" and "pulse" reads resulted from off-target TSO priming. "Spike" and "pulse" patterns were rare for the mostly unidirectional reads mapping to the circular mitochondrial genome. Bioinformatic curation removed "spikes" and "pulses" and reclassified 10% of DEGs in renal proximal tubular epithelial cells (RPTECs). Up-regulated gene ontologies included S and G2/M phase, double-stranded DNA repair, proximal tubulopathy, and renal tubular dysfunction, whereas allograft rejection, antigen presentation, innate immunity, translation, and autophagy were down-regulated. BKPyV-LVGR expression induced a novel mitochondrial cell stress pattern consisting of discordant up-regulation and down-regulation of mitochondria-encoded and nucleus-encoded mitochondrial genes, respectively. We explored which top-scoring gene sets of late-phase BKPyV-replicating RPTECs can identify BKPyV-associated nephropathy in kidney transplant biopsies. The results should facilitate distinguishing BKPyV-associated pathology from other entities in kidney transplant biopsies.IMPORTANCEBK polyomavirus (BKPyV) infects more than 90% of the general population and then persists in the reno-urinary tract. Subsequently, low-level urinary shedding is seen in 10% of healthy BKPyV-seropositive persons, indicating that BKPyV replication occurs despite the presence of virus-specific cellular and humoral immunity. Notably, transplantation of donor kidneys with low-level BKPyV replication is a risk factor for progression to high-level BKPyV viruria, new-onset BKPyV-DNAemia and biopsy-proven BKPyV nephropathy. Here, we identify a short list of robust up- and down-regulated nucleus-encoded differentially expressed genes potentially allowing to discriminate viral from allograft immune damage. By carefully curating viral and mitochondrial transcriptomes, we identify a novel virus-associated mitochondrial stress pattern of up-regulated mitochondria-encoded and down-regulated nucleus-encoded mitochondrial transcripts which heralds the BKPyV-agnoprotein-mediated immune escape by breakdown of the mitochondrial membrane potential and network and mitophagy. The results may prove useful when assessing the role of BKPyV replication in kidney transplant patients with suspected acute rejection and/or BKPyV nephropathy.

Dapagliflozin Protects against Cisplatin-Induced AKI on Primary Human Renal Proximal Tubular Epithelial Cells (hRPTECs)

Dapagliflozin Protects against Cisplatin-Induced AKI on Primary Human Renal Proximal Tubular Epithelial Cells (hRPTECs)

Malate dehydrogenase-2 inhibition shields renal tubular epithelial cells from anoxia-reoxygenation injury by reducing reactive oxygen species.

Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.

Human pulmonary microvascular endothelial cells respond to DAMPs from injured renal tubular cells.

Acute kidney injury (AKI) causes distant organ dysfunction through yet unknown mechanisms, leading to multiorgan failure and death. The lungs are one of the most common extrarenal organs affected by AKI, and combined lung and kidney injury has a mortality as high as 60%-80%. One mechanism that has been implicated in lung injury after AKI involves molecules released from injured kidney cells (DAMPs, or damage-associated molecular patterns) that promote a noninfectious inflammatory response by binding to pattern recognition receptors (PRRs) constitutively expressed on the pulmonary endothelium. To date there are limited data investigating the role of PRRs and DAMPs in the pulmonary endothelial response to AKI. Understanding these mechanisms holds great promise for therapeutics aimed at ameliorating the devastating effects of AKI. In this study, we stimulate primary human microvascular endothelial cells with DAMPs derived from injured primary renal tubular epithelial cells (RTECs) as an ex-vivo model of lung injury following AKI. We show that DAMPs derived from injured RTECs cause activation of Toll-Like Receptor and NOD-Like Receptor signaling pathways as well as increase human primary pulmonary microvascular endothelial cell (HMVEC) cytokine production, cell signaling activation, and permeability. We further show that cytokine production in HMVECs in response to DAMPs derived from RTECs is reduced by the inhibition of NOD1 and NOD2, which may have implications for future therapeutics. This paper adds to our understanding of PRR expression and function in pulmonary HMVECs and provides a foundation for future work aimed at developing therapeutic strategies to prevent lung injury following AKI.

Stevioside protects against acute kidney injury by inhibiting gasdermin D pathway

AbstractRecent studies indicate a significant upregulation of gasdermin D (GSDMD) in acute kidney injury (AKI), a severe medical condition characterized by high morbidity and mortality globally. In this study, we identified and validated the therapeutic effects of small molecule inhibitors targeting the GSDMD pathway for AKI treatment. Using a drug screening assay, we evaluated thousands of small molecules from DrugBank against Lipopolysaccharide (LPS) and Nigericin‐stimulated immortalized bone marrow‐derived macrophages (iBMDMs) to discern GSDMD pathway activators. We simulated AKI in primary renal tubular epithelial cells using hydrogen peroxide (H2O2) exposure. Furthermore, AKI in mouse models was induced via cisplatin and ischemia/reperfusion. Our findings highlight stevioside as a potent GSDMD activator exhibiting minimal toxicity. Experimental results, both in vitro and in vivo, demonstrate stevioside's significant potential in alleviating renal tubular epithelial cell injury and AKI histological damage. After stevioside treatment, a notable decrease in cleaved GSDMD‐N terminal levels was observed coupled with diminished inflammatory factor release. This observation was consistent in both cisplatin‐ and ischemia/reperfusion‐induced AKI mouse models. Collectively, our research suggests that stevioside could be a promising candidate for modulating GSDMD signaling in AKI treatment.

#2023 The role of neutrophil extracellular traps in cisplatin-induced acute kidney injury

Abstract Background and Aims Acute kidney injury (AKI) is a public health problem that seriously endangers human health. The exact pathogenesis of AKI has not been fully elucidated. Previous studies have shown that the immune system plays a key role in the inflammatory response of AKI in the acute phase, especially innate immunity. Recent discoveries have highlighted that neutrophils, in particular the neutrophil extracellular traps (NETs), have central roles in the occurrence and development of AKI. However, the specific role and mechanism of NETs in AKI of different etiologies are remain unclear. This study aimed to investigate the role of NETs in the development of cisplatin-induced AKI and provide potential therapeutic targets for AKI treatment. Method Wild-type (WT) and NETs-deficient (PAD4-/-) mouse models of AKI were constructed by intraperitoneal injection of cisplatin. Mice were weighed at 24, 48, and 72 hours after modeling, and sacrificed at 72 hours. Serum and kidney specimens were collected for renal function analysis, pathological, immunohistochemical staining, and molecular biology experiments. Mouse bone marrow neutrophils were isolated from healthy mouse. NETs were induced in vitro by adding phorbol 12 myristate 13-acetate (PMA). Primary renal tubular epithelial cells (TECs) of mouse kidney were extracted, and the cells were collected after 24 h of cisplatin and cisplatin combined with NETs intervention, respectively. The inflammatory indicators such as TNF-α, IL-1β, MCP-1, and renal injury indicators such as KIM-1 and Ngal were detected by RT-PCR. Results Compared with the WT-cisplatin group, the PAD4-/- -cisplatin group showed a reduced degree of weight loss after modeling, lower serum creatinine and urea nitrogen levels, and significantly improved renal function. The PAD4-/- -cisplatin group showed a significant decrease in the inflammatory indicators TNF-α, IL-1β, IL-6, and renal injury indicators KIM-1 and Ngal. Cisplatin intervention for 6 hours could induce the generation of NETs. Compared with the cisplatin intervention group, the expression of inflammatory indicators TNF-α, IL-1β, and MCP-1 were significantly higher in the cisplatin combined with NETs intervention TECs group. Conclusion Cisplatin directly induces neutrophils to generate NETs, which can exacerbate cisplatin-induced injury to TECs in vitro. The inhibition of NETs by PAD4-/- improves cisplatin-induced AKI. The inhibition of NETs may be a novel target for the treatment of cisplatin AKI.

#753 Targeted anti-IL-1β bacteroides fragilis outer membrane vesicles alleviate inflammatory injury in diabetic nephropathy

Abstract Background and Aims It is accepted that diabetic nephropathy (DN) is not only a disease caused by impaired glucose metabolism, but also a chronic inflammatory disease. The interleukin-1β (IL-1β) plays an important role in inflammation-mediated renal injury in DN. IL-1β blocking therapy can prevent inflammatory cytokine-mediated kidney injury. However, the high cost and systemic side effects of IL-1β antibodies limit its application. Therefore, firstly this study aimed to construct kidney-targeting outer membrane vesicles (OMVs) of Bacteroides fragilis loaded with IL-1β single-chain variable fragment (scFv). These OMVs derived from human commensal non-toxigenic Bacteroides fragilis have been proven to play an immunosuppressive role in the intestine and distant organs. This targeted drug delivery platform built with this as a carrier is expected to relieve kidney inflammation in the development of DN. Methods We recombinantly expressed the scFv based on the Gevokizumab sequence in Escherichia coli, and loaded it into the OMVs derived from Bacteroides fragilis by multiple ultrasounds. The targeting peptide with the sequence (KKEEE)3{Lys(N3)} was connected to the membrane surface through a copper-free catalyzed click chemistry reaction. The package, which consisted of OMVs and the scFv targeting IL-1β in the kidney (OMV-KTP-scFv) was obtained. OMV-KTP-scFv treated in HK-2 cells and healthy mice respectively, and evaluated its in vitro and in vivo drug safety. The concentration of IL-1β scFv in blood and kidney tissues at different time points were measured using streptozotocin (STZ)-induced diabetic mice. DID-labeled OMVs was used for ex vivo infrared imaging of various organs to evaluate the delivery efficiency of the targeted carrier. In vitro, OMV-KTP-scFv was used to prevent inflammation in mouse primary renal tubular epithelial cells induced by high glucose, and in vivo to alleviate kidney injury in STZ-induced diabetic mice. Results We successfully prepared OMV-KTP-scFv and confirmed the co-localization of OMVs, KTP, and scFv using super-resolution microscopy. The prepared OMV-KTP-scFv had high stability, effectively extended the plasma half-life of scFv, and could deliver scFv accurately to the proximal renal tubules. Furthermore, OMV-KTP-scFv also had good safety in vitro and in vivo, and no obvious biotoxicity was found during long-term in vivo administration. In addition, our results show that OMV-KTP-scFv significantly inhibited the expression of inflammatory factors in renal tubular cells of DN, reduced the infiltration of renal interstitial inflammatory cells, and alleviated renal tubular injury. Conclusion Our findings demonstrated that OMVs of Bacteroides fragilis targeting the kidney can deliver the IL-1β scFv to the renal tubulointerstitium and reduce the renal interstitial injury in DN by antagonizing local inflammation.

Primary goose kidney tubular epithelial cells for goose astrovirus genotype 2 infection: establishment and RNA sequencing analysis

Goose astrovirus genotype 2 (GAstV-2) mainly causes gout in goslings; therefore, it is a major pathogen threatening to goose flocks. However, the mechanisms underlying host-GAstV-2 interactions remain unclear because host cells suitable for GAstV-2 replication have been unavailable. We previously noted that GAstV-2 is primarily located in goose renal epithelial cells, where it causes kidney damage. Therefore, here, we derived goose primary renal tubular epithelial (RTE) cells (GRTE cells) from the kidneys of goose embryos after collagenase I digestion. After culture in Dulbecco's modified Eagle medium/Nutrient mixture F-12 with 10% fetal bovine serum (FBS), the isolated cells had polygonal with roadstone-like morphology; they were identified to be epithelial cells based on the presence of cytokeratin 18 expression detected through immunofluorescence assay (IFA). GAstV-2 infection in GRTE cells led to no obvious cytopathic effects; the maximum amounts of infectious virions were observed 48 h post infection through IFA and quantitative PCR. Next, RNA-seq was performed to identify and map post-GAstV-2 infection differentially expressed genes. The downregulated pathways were mainly related to metabolism, including tryptophan metabolism, drug metabolism by cytochrome P450, xenobiotic metabolism by cytochrome P450, retinol metabolism, butanoate metabolism, starch and sucrose metabolism, ascorbate and aldarate metabolism, and drug metabolism by other enzymes and peroxisome. In contrast, the upregulated pathways were mostly related to the host cell defense and proliferation, including extracellular matrix-receptor interaction, complement and coagulation cascades, phagosome, PI3K-Akt signaling pathway, human T-lymphotropic virus 1 infection, lysosome, and tumor necrosis factor signaling pathway. In conclusion, we developed a GRTE cell line for GAstV-2 replication and analyzed the potential host-GAstV-2 interactions through RNA-seq; our results may aid in further investigating the pathogenic mechanisms underlying GAstV-2 infection and provide strategies for its prevention and control.

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2mg/ml trypsin + 2.5mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15min, 30min, 45min, and 60min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Effect of 7-ketocholesterol incorporation on substrate binding affinity and turnover rate of the organic cation transporter 2 (OCT2)

The organic cation transporter 2 (OCT2) is pivotal in the renal elimination of several positively charged molecules. OCT2 mode of transport is profoundly influenced by the level of membrane cholesterol. The aim of this study was to investigate the effect of oxidized cholesterol on OCT2 transport activity in human embryonic kidney 293 cells stably transfected with OCT2 (OCT2-HEK293) and in primary renal proximal tubular epithelial cells (RPTEC). Cholesterol was exchanged with 7-ketocholesterol, the main product of cholesterol auto-oxidation, by exposing cells to sterol-saturated methyl-β-cyclodextrin (mβcd). After a 30 min-exposure, approximately 50% of the endogenous cholesterol was replaced by 7-ketocholesterol without significant changes in total sterol level. In the presence of 7-ketocholesterol, [3H]1-methyl-4-phenylpyridinium (MPP+) uptake was significantly reduced in both cell lines. 7-ketocholesterol incorporation did not affect lipid raft integrity, nor OCT2 surface expression and spatial organization. The inhibitory effect of 7-ketocholesterol on MPP+ uptake was abolished by the presence of MPP+ in the trans-compartment. In the presence of 7-ketocholesterol, both Kt and Vmax of MPP+ influx decreased. Molecular docking using OCT2 structure in outward occluded conformation showed overlapping poses and similar binding energies between cholesterol and 7-ketocholesterol. The thermal stability of OCT2 was not changed when cholesterol was replaced with 7-ketocholesterol. We conclude that 7-ketocholesterol confers a higher rigidity to the carrier by reducing its conformational entropy, arguably as a result of changes in plasma membrane physical properties, thereby facilitating the achievement of a higher affinity state at the expense of the mobility and overall cycling rate of the transporter.

Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

IntroductionBK Polyomavirus (BKPyV) infection is a common complication in kidney transplant recipients and can result in poor outcomes and graft failure. Currently, there is no known effective antiviral agent. This study investigated the possible antiviral effects of Interferon alpha (IFNα) and its induced protein, MxA, against BKPyV. MethodsIn vitro cell culture experiments were conducted using human primary renal proximal tubular epithelial cells (HRPTECs). We also did animal studies using Balb/c mice with unilateral kidney ischemic reperfusion injury. ResultsOur results demonstrated that IFNα effectively inhibited BKPyV in vitro and murine polyomavirus in animal models. Additionally, IFNα and MxA were found to suppress BKPyV TAg and VP1 production. Silencing MxA attenuated the antiviral efficacy of IFNα. We observed that MxA interacted with BKPyV TAg, causing it to remain in the cytosol and preventing its nuclear translocation. To determine MxA's essential domain for its antiviral activities, different mutant MxA constructs were generated. The MxA mutant K83A retained its interaction with BKPyV TAg, and its antiviral effects were intact. The MxA T103A mutant, on the other hand, abolished GTPase activity, lost its protein-protein interaction with BKPyV TAg, and lost its antiviral effect. ConclusionIFNα and its downstream protein, MxA, have potent antiviral properties against BKPyV. Furthermore, our findings indicate that the interaction between MxA and BKVPyV TAg plays a crucial role in determining the anti-BKPyV effects of MxA.

Toxicity induced by orellanine from the mushroom Cortinarius orellanus in primary renal tubular proximal epithelial cells (RPTEC): Novel mechanisms of action

The toxicity of Orellanine (OR), a significant factor in mushroom poisoning, has severe effects on the kidneys, particularly the proximal tubules. This study investigated the acute toxicity of OR from the Cortinarius orellanus mushroom in human Primary Renal Tubular Proximal Epithelial Cells (RPTEC). Additionally, the half maximal inhibitory concentration (IC50) of OR in MCF-7 cells was established. RPTEC were subjected to a 6.25 μg/ml dose of orellanine for 24 h, while Control cells were exposed to 0.05% DMSO (vehicle). The RT2 Profiler™ PCR Array Human Nephrotoxicity was utilized to identify genes that were upregulated or downregulated. Western blotting confirmed the protein product of some significantly regulated genes compared to control cells. The IC50 of OR was found to be 319.2 μg/ml. The mechanism of OR toxicity involved several pathways including apoptosis, metal ion binding, cell proliferation, tissue remodeling, xenobiotic metabolism, transporters, extracellular matrix molecules, and cytoskeleton pathways. Other genes from non-specific pathways were also identified. These findings enhance our understanding of OR nephrotoxicity and pave the way for future research into potential treatments or antidotes for natural mushroom poisoning.

Role of PLC/IP3 /IP3 R axis in excess molybdenum exposure induced apoptosis in duck renal tubular epithelial cells.

Excess molybdenum (Mo) is harmful to animals, but its nephrotoxicity has not been comprehensively explained. To appraise the influences of excess Mo on Ca homeostasis and apoptosis via PLC/IP3 /IP3 R axis, primary duck renal tubular epithelial cells were exposed to 480 μM and 960 μM Mo, and joint of 960 μM Mo and 10 μM 2-APB or 0.125 μM U-73122 for 12 h (U-73122 pretreated for 1 h), respectively. The data revealed that the increment of [Ca2+ ]c induced by Mo mainly originated from intracellular Ca storage. Mo exposure reduced [Ca2+ ]ER , elevated [Ca2+ ]mit , [Ca2+ ]c , and the expression of Ca homeostasis-related factors (Calpain, CaN, CRT, GRP94, GRP78 and CaMKII). 2-APB could effectively reverse subcellular Ca2+ redistribution by inhibiting IP3 R, which confirmed that [Ca2+ ]c overload induced by Mo originated from ER. Additionally, PLC inhibitor U-73122 remarkably mitigated the change, and dramatically reduced the number of apoptotic cells, the expression of Bak-1, Bax, cleaved-Caspase-3/Caspase-3, and notably increased the expression of Bcl-xL, Bcl-2, and Bcl-2/Bax ratio. Overall, the results confirmed that the Ca2+ liberation of ER via PLC/IP3 /IP3 R axis was the main cause of [Ca2+ ]c overload, and then stimulated apoptosis in duck renal tubular epithelial cells.

REST contributes to AKI-to-CKD transition through inducing ferroptosis in renal tubular epithelial cells.

Ischemic-reperfusion injury (IRI) is a major pathogenic factor in acute kidney injury (AKI), which directly leads to the hypoxic injury of renal tubular epithelial cells (RTECs). Although emerging studies suggest repressor element 1-silencing transcription factor (REST) as a master regulator of gene repression under hypoxia, its role in AKI remains elusive. Here, we found that REST was upregulated in AKI patients, mice, and RTECs, which was positively associated with the degree of kidney injury, while renal tubule-specific knockout of Rest significantly alleviated AKI and its progression to chronic kidney disease (CKD). Subsequent mechanistic studies indicated that suppression of ferroptosis was responsible for REST-knockdown-induced amelioration of hypoxia-reoxygenation injury, during which process Cre-expressing adenovirus-mediated REST downregulation attenuated ferroptosis through upregulating glutamate-cysteine ligase modifier subunit (GCLM) in primary RTECs. Further, REST transcriptionally repressed GCLM expression via directly binding to its promoter region. In conclusion, our findings revealed the involvement of REST, a hypoxia regulatory factor, in AKI-to-CKD transition and identified the ferroptosis-inducing effect of REST, which may serve as a promising therapeutic target for ameliorating AKI and its progression to CKD.

A Study on the Role of Wip1 in Renal Fibrosis by Modulating Macrophage Phenotype

Renal fibrosis is the result of chronic kidney diseases, the exploration of the pathogenesis of renal fibrosis and the development of effective treatment methods have become major challenges. To investigate the effect of wild-type p53-induced phosphatase 1 (Wip1) on macrophage phenotype regulation and the role played in renal fibrosis. RAW264.7 macrophages were stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) or interleukin 4 (IL-4) to differentiate into M1 or M2 macrophages. Lentivirus vectors were transduced into RAW264.7 macrophages to construct the cell lines that overexpressed or silenced Wip1, respectively. Furthermore, E-cadherin, Vimentin, and α-SMA levels of primary renal tubular epithelial cells (RTECs) were measured after co-culture with macrophages overexpressed or silenced by Wip1. Macrophages stimulated by LPS plus IFN-γ differentiated into M1 macrophages with high expression of iNOS and TNF-α, while those stimulated by IL-4 differentiated into M2 macrophages with high expression of Arg-1 and CD206. Increased expression of iNOS and TNF-α was observed in macrophages transduced with Wip1 RNA interference, while an increased expression of Arg-1 and CD206 was observed in macrophages transduced with Wip1 overexpressed vector, indicating that RAW264.7 macrophages could be transformed into M2 macrophages after Wip1 overexpression, and transformed into M1 macrophages by down-regulating Wip1. In addition, the E-cadherin mRNA level decreased and Vimentin and α-SMA increased in RTECs co-cultured with Wip1 overexpressed macrophages compared to the control group. Wip1 may participate in the pathophysiological process of renal tubulointerstitial fibrosis by transforming macrophages into the M2 phenotype.

Oxy210, a Semi-Synthetic Oxysterol, Inhibits Profibrotic Signaling in Cellular Models of Lung and Kidney Fibrosis.

Oxy210, a semi-synthetic oxysterol derivative, displays cell-selective inhibition of Hedgehog (Hh) and transforming growth factor beta (TGF-β) signaling in epithelial cells, fibroblasts, and macrophages as well as antifibrotic and anti-inflammatory efficacy in models of liver fibrosis. In the present report, we examine the effects of Oxy210 in cellular models of lung and kidney fibrosis, such as human lung fibroblast cell lines IMR-90, derived from healthy lung tissue, and LL97A, derived from an idiopathic pulmonary fibrosis (IPF) patient. In addition, we examine the effects of Oxy210 in primary human renal fibroblasts, pericytes, mesangial cells, and renal tubular epithelial cells, known for their involvement in chronic kidney disease (CKD) and kidney fibrosis. We demonstrate in fibroblasts that the expression of several profibrotic TGF-β target genes, including fibronectin (FN), collagen 1A1 (COL1A1), and connective tissue growth factor (CTGF) are inhibited by Oxy210, both at the basal level and following TGF-β stimulation in a statistically significant manner. The inhibition of COL1A1 gene expression translated directly to significantly reduced COL1A1 protein expression. In human primary small airway epithelial cells (HSAECs) and renal tubular epithelial cells, Oxy210 significantly inhibited TGF-β target gene expression associated with epithelial-mesenchymal transition (EMT). Oxy210 also inhibited the proliferation of fibroblasts, pericytes, and mesangial cells in a dose-dependent and statistically significant manner.

Formononetin ameliorates ferroptosis-associated fibrosis in renal tubular epithelial cells and in mice with chronic kidney disease by suppressing the Smad3/ATF3/SLC7A11 signaling

AimsChronic kidney disease (CKD) is characterized by interstitial fibrosis, while limited treatment drugs are available. Ferroptosis is a newly identified process that can trigger tubular atrophy and fibrosis. The aim of this study is to investigate the effects of formononetin (FN), a bioflavonoid, on ferroptosis and renal fibrosis. Main methodsIn vivo experiments, unilateral ureteral obstruction (UUO)- and folic acid (FA, 250 mg/kg)-induced CKD models were constructed in C57BL/6 mice of 6–8 weeks old, followed by the administration with 40 mg/kg/day FN by gavage. For in vitro experiments, ferroptosis was induced with RSL3 or erastin in primary mouse renal tubular epithelial cells (TECs), followed by the addition of indicated concentrations of FN. Then, the levels of ferroptosis and fibrosis were analyzed. The translocation of Smad3, ATF3, and Nrf2 from the cytoplasm to the nucleus was checked by western blotting. The interaction of Smad3 and ATF3 was detected by Co-immunoprecipitation. Key findingsFN dramatically ameliorated tubular injury along with reduced expression of the profibrotic genes including α-SMA, Col1a1, and fibronectin in both two CKD mouse models and RSL3/erastin-treated TECs. Furthermore, FN administration also significantly suppressed ferroptosis, as evidenced by increased expression of SLC7A11 and GPX4, and decreased levels of 4-HNE. In mechanism, FN disrupted the interaction between Smad3 and ATF3, resulting in the blocking of their translocation from the cytoplasm to the nucleus. In addition, FN also promoted the separation of the Nrf2/Keap1 complex and enhanced Nrf2 nuclear accumulation. SignificanceFN alleviates CKD by impeding ferroptosis-associated fibrosis by suppressing the Smad3/ATF3/SLC7A11 signaling and could serve as a candidate therapeutic drug for CKD.

Dapagliflozin Prevents High-Glucose-Induced Cellular Senescence in Renal Tubular Epithelial Cells.

Gliflozins are a new class of antidiabetic drugs with renoprotective properties. In cultures of primary human renal tubular epithelial cells (RPTECs) subjected to high-glucose conditions in the presence or absence of dapagliflozin, we evaluated cellular senescence pathways. High glucose increased sodium-glucose cotransporter-2 (SGLT-2) expression and glucose consumption, enhancing reactive oxygen species production. The latter induced DNA damage, ataxia telangiectasia mutated kinase (ATM), and p53 phosphorylation. Stabilized p53 increased the cell cycle inhibitor p21, resulting in cell cycle arrest and increasing the cellular senescence marker beta-galactosidase (GLB-1). RPTECs under high glucose acquired a senescence-associated secretory phenotype, which was detected by the production of IL-1β, IL-8, and TGF-β1. By decreasing SGLT-2 expression and glucose consumption, dapagliflozin inhibited the above pathway and prevented RPTEC senescence. In addition, dapagliflozin reduced the cell cycle inhibitor p16 independently of the glucose conditions. Neither glucose concentration nor dapagliflozin affected the epithelial-to-mesenchymal transition when assessed with α-smooth muscle actin (α-SMA). Thus, high glucose induces p21-dependent RPTEC senescence, whereas dapagliflozin prevents it. Since cellular senescence contributes to the pathogenesis of diabetic nephropathy, delineating the related molecular mechanisms and the effects of the widely used gliflozins on them is of particular interest and may lead to novel therapeutic approaches.