Proximal Tubular Epithelial Cell Line Research Articles

Synthesis, anticancer activity and mechanism of action of Fe(III) complexes.

To inhibit the growth and metastasis of triple-negative breast cancer (TNBC), two Fe(III) thiosemicarbazone complexes (Fe1 and Fe2) were designed and synthesized. The structures of the Fe(III) complexes were characterized by single crystal X-ray diffraction. The antiproliferative activity of Fe1 and Fe2 against four cancer lines (MDA-MB-231, T98G, HepG2, 143B) and human renal proximal tubular epithelial cell line (HK-2) was evaluated by MTT assay. Among all cells, Fe2 showed significant cytotoxicity to TNBC cells (MDA-MB-231), with an IC50 value of 12.38 μM. Furthermore, Fe2 showed less toxicity to HK-2 cells. The two Fe(III) complexes can produce excess of reactive oxygen species, decrease of mitochondrial membrane potential, and induce DNA damage, then lead to apoptosis of MDA-MB-231 cells. In addition, Fe1 and Fe2 can also inhibit migration and invasion of MDA-MB-231 cells. This study provides guidance for the development of metal complexes that inhibit the growth and metastasis of TNBC.

Hypoxia activates FGF-23-ERK / MAPK signaling pathway in ischemia-reperfusion induced acute kidney injury.

Both hypoxia and fibroblast growth factor-23 (FGF-23) are key factors in ischemia-reperfusion (I/R)-induced acute kidney injury (AKI). This study aimed to explore the relationship between hypoxia and FGF-23 in AKI. An I/R-AKI animal model was established using male BALB/c mice. HK-2 cells, a part of the human proximal tubular epithelial cell line, were subjected to hypoxia/reoxygenation (H/R). qPCR was used to measure FGF-23 and HIF-1α, ELISA was used to measure inflammatory and oxidative stress cytokines. Western blotting used to measure the phosphorylation of ERK level. In I/R mice, the levels of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), malondialdehyde (MDA), and the phosphorylation of extracellular signal-regulated kinase (ERK) were increased, whereas the levels of interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione peroxidase (GPx), and klotho were decreased, compared to the sham operated mice. Silencing the FGF-23 expression in I/R mice normalized the levels of IL-6, IL-10, TNF-α, MDA, SOD, Gpx, and ERK phosphorylation (p-ERK). In HK-2 cells, hypoxia-reperfusion (H/R) elevated the levels of IL-6, TNF-α, MDA, and ERK phosphorylation, but reduced IL-10, SOD, GPx, and klotho levels. Hypoxia induced apoptosis in HK-2 cells but silencing of FGF-23 expression blocked the effects of hypoxia on cell apoptosis, proinflammatory factors levels, oxidative stress response, and p-ERK levels. FGF-23 is a key molecule in AKI, and hypoxia plays a crucial role in AKI by inducing cell apoptosis; however, its role is regulated by FGF-23. FGF-23 affects oxidative stress and the inflammatory response of kidney tissues by activating the ERK/mitogen-activated protein kinase (MAPK) signaling pathway.

Silencing LncRNA SNHG14 alleviates renal tubular injury via the miR-483-5p/HDAC4 axis in diabetic kidney disease.

This study explored the clinical value of long non-coding RNA small nucleolar RNA host gene 14 (SNHG14) in diabetic kidney disease (DKD) and the mechanism of renal tubular injury. Patients with DKD, type 2 diabetes mellitus (T2DM) and healthy individuals (HVs) were included, as well as the human proximal tubular epithelial cell line (HK-2) induced by high glucose was also included. The mRNA levels of SNHG14 in the serum and cells were detected using RT-qPCR. Diagnostic significance was examined using receiver operating characteristic (ROC) analysis. A commercial test kit, flow cytometry, and enzyme-linked immunosorbent assays were employed to assess reactive oxygen species (ROS) production, apoptosis, inflammatory factor secretion, and extracellular matrix protein levels in HK-2 cells. The dual-luciferase reporter assay and RNA immunoprecipitation were used to validate miR-483-5p concerning SNHG14 or histone deacetylase 4 (HDAC4). SNHG14 and HDAC4 levels were elevated in the serum of DKD patients and HG-induced HK-2 cells, while miR-483-5p levels were decreased (P < 0.001). SNHG14 increased HDAC4 levels by sponging miR-483-5p. Elevated SNHG14 levels significantly differentiated DKD patients from HVs (AUC = 0.944) and T2DM (AUC = 0.867). Silencing of SNHG14 alleviated HG-induced ROS production and apoptosis as well as the over-secretion of inflammatory factors and extracellular matrix proteins; however, this alleviation was typically suppressed by low expression of miR-483-5p (P < 0.001). Elevated miR-483-5p alleviates HG-induced renal tubular injury, but this alleviation is suppressed by HDAC4 overexpression. In summary, suppression of SNHG14 has been shown in our study to mitigate renal tubular injury in DKD by regulating apoptosis, oxidative stress, inflammation, and fibrosis through the miR-483-5p/HDAC4 axis.

Nrf2/FSP1/CoQ10 axis-mediated ferroptosis is involved in sodium aescinate-induced nephrotoxicity

Sodium aescinate (SA), an active compound found in horse chestnut seeds, is widely used in clinical practice. Recently, the incidence of SA-induced adverse events, particularly renal impairment, has increased. Our previous work demonstrated that SA causes severe nephrotoxicity via nephrocyte ferroptosis; however, the underlying mechanism remains to be fully elucidated. In the current study, we investigated additional molecular pathways involved in SA-induced nephrotoxicity. Our results showed that SA inhibited cell viability, disrupted cellular membrane integrity, and enhanced reactive oxygen species (ROS), ferrous iron (Fe2+), and malondialdehyde (MDA) levels, as well as lipid peroxidation in rat proximal renal tubular epithelial cell line (NRK-52E) cells. SA also depleted coenzyme Q10 (CoQ10, ubiquinone) and nicotinamide adenine dinucleotide (NADH) and reduced ferroptosis suppressor protein 1 (FSP1) and polyprenyltransferase (coenzyme Q2, COQ2) activity, triggering lipid peroxidation and ROS accumulation in mouse kidneys and NRK-52E cells. The overexpression of COQ2, FSP1, or CoQ10 (ubiquinone) supplementation effectively attenuated SA-induced ferroptosis, whereas iFSP1 or 4-formylbenzoic acid (4-CBA) pretreatment exacerbated SA-induced nephrotoxicity. Additionally, SA decreased nuclear factor-erythroid-2-related factor 2 (Nrf2) levels and inhibited Nrf2 binding to the −1170/-1180 bp ARE site in FSP1 promoter, resulting in FSP1 suppression. Overexpression of Nrf2 or its agonist dimethyl fumarate (DMF) promoted FSP1 expression, thereby improving cellular antioxidant capacity and alleviating SA-induced ferroptosis. These results suggest that SA-triggers renal injury through oxidative stress and ferroptosis, driven by the suppression of the Nrf2/FSP1/CoQ10 axis.

Gemigliptin mitigates TGF-β-induced renal fibrosis through FGF21-mediated inhibition of the TGF-β/Smad3 signaling pathway

Fibroblast growth factor 21 (FGF21), a well-known regulator of metabolic disorders, exhibits the potential to prevent renal fibrosis by negatively regulating the transforming growth factor β (TGF-β)/Smad3 signaling pathway. Gemigliptin and other dipeptidyl peptidase-4 inhibitors are frequently used for the management of patients with type 2 diabetes. However, the protective effect of gemigliptin against renal fibrosis, particularly its potential to upregulate the expression of FGF21, remains incompletely understood. This study assessed the renoprotective effects of gemigliptin against TGF-β-induced renal fibrosis by enhancing the expression of FGF21 in the cultured human proximal tubular epithelial cell line HK-2. Treatment with FGF21 effectively prevented TGF-β-induced renal fibrosis by attenuating the TGF-β/Smad3 signaling pathway. Similarly, gemigliptin exhibited protective effects against TGF-β-induced renal fibrosis by mitigating TGF-β/Smad3 signaling through the upregulation of FGF21 expression. However, the protective effects of gemigliptin were blocked when FGF21 expression was knocked down in TGF-β-treated HK-2 cells. These results indicate that gemegliptin has the potential to exhibit protective effects against TGF-β-induced renal fibrosis by elevating FGF21 expression levels in cultured human proximal tubular epithelial cells.

MiR-92a-3p Knockdown Attenuates Transforming Growth Factor-β1-induced Tubulointerstitial Fibrosis by Targeting LIN28A-mediated EMT Pathway.

The role of microRNAs in regulating tubulointerstitial fibrosis, a key feature of progressive chronic kidney disease, is of significant importance. LIN28A has been reported to attenuate renal fibrosis in obstructive nephropathy. Here, our objective was to investigate the precise biological function of the miR-92a-3p/LIN28A axis in tubulointerstitial fibrosis. The human renal proximal tubular epithelial (HK-2) cell line was exposed to transforming growth factor (TGF)-β1, establishing an in vitro model mimicking tubulointerstitial fibrosis. Luciferase reporter assay was utilized to investigate the relationship between miR-92a-3p and LIN28A. Cell transfection techniques were employed to modify the expression of miR-92a-3p and LIN28A. An in vivo model of tubulointerstitial fibrosis was created by inducing unilateral ureteral obstruction (UUO) in C57BL/6N mice. Our initial observations showed that TGF-β1 treatment of HK-2 cells and the UUO mice model led to an increase in miR-92a-3p expression and a decrease in LIN28A expression. We confirmed that miR-92a-3p directly targeted LIN28A in HK-2 cells. In TGF-β1-stimulated HK-2 cells, knocking down miR-92a-3p notably reduced the levels of alpha smooth muscle actin and vimentin and concurrently enhanced the expression of E-cadherin. These changes were counteracted upon transfection with si-LIN28A. Thus, directing interventions toward miR-92a-3p holds the potential to emerge as a viable therapeutic approach for addressing tubulointerstitial fibrosis.

Sleeve gastrectomy links the attenuation of diabetic kidney disease to the inhibition of renal tubular ferroptosis through down-regulating TGF-β1/Smad3 signaling pathway

PurposeTo investigate how sleeve gastrectomy (SG), a typical operation of bariatric surgery, attenuated symptom, and progression of diabetic kidney disease (DKD).MethodsDKD model was induced by high-fat diet (HFD) combined with streptozocin in Wistar rats. SG was performed, and the group subjected to sham surgery served as control. The animals were euthanized 12 weeks after surgery, followed by sample collection for the subsequent experiment. The HK-2, a renal proximal tubular epithelial cell line derived from human, was utilized to investigate the potential mechanisms.ResultsSG improved metabolic parameters and glucose homeostasis, and could alleviate DKD in terms of renal function indices as well as histological and morphological structures in DM rats, accompanied with a significant reduction in renal tubular injury. Compared with sham group, SG reduced the renal tubular ferroptosis. To further clarify the mechanism involved, in vitro experiments were performed. In the presence of high glucose, renal tubular TGF-β1 secretion was significantly increased in HK-2 cell line, which led to activation of ferroptosis through TGF-β1/Smad3 signaling pathway. Inhibition of TGF-β1 receptor and phosphorylation of Smad3 significantly ameliorated TGF-β1-mediated ferroptosis. In vivo experiments also found that SG improved the hyperglycemic environment, reduced renal TGF-β1 concentrations, and down-regulated the TGF-β1/Smad3 signaling pathway.ConclusionsWith the capacity to lower the glucose, SG could attenuate the ferroptosis by inhibiting TGF-β1/Smad3 signaling pathway in DKD rats, and eventually attenuated DKD.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury

BackgroundThe mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.MethodsA 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein–Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.ResultsA total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.ConclusionsThe proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Inhibition of PFKP in renal tubular epithelial cell restrains TGF-β induced glycolysis and renal fibrosis

Metabolic reprogramming to glycolysis is closely associated with the development of chronic kidney disease (CKD). Although it has been reported that phosphofructokinase 1 (PFK) is a rate-limiting enzyme in glycolysis, the role of the platelet isoform of PFK (PFKP) in kidney fibrosis initiation and progression is as yet poorly understood. Here, we investigated whether PFKP could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells (PTECs). We induced PFKP overexpression or knockdown in renal tubules via an adeno-associated virus (AAV) vector in the kidneys of mice following unilateral ureteral occlusion. Our results show that the dilated tubules, the area of interstitial fibrosis, and renal glycolysis were promoted by proximal tubule-specific overexpression of PFKP, and repressed by knockdown of PFKP. Furthermore, knockdown of PFKP expression restrained, while PFKP overexpression promoted TGF-β1-induced glycolysis in the human PTECs line. Mechanistically, Chip-qPCR revealed that TGF-β1 recruited the small mothers against decapentaplegic (SMAD) family member 3-SP1 complex to the PFKP promoter to enhance its expression. Treatment of mice with isorhamnetin notably ameliorated PTEC-elevated glycolysis and kidney fibrosis. Hence, our results suggest that PFKP mediates the progression of kidney interstitial fibrosis by regulating glycolysis in PTECs.

Knockdown of ADAM8 inhibits the proliferation, migration, invasion, and tumorigenesis of renal clear cell carcinoma cells to enhance the immunotherapy efficacy

The current study performed bioinformatics and in vitro and in vivo experiments to explore the effects of ADAM8 on the malignant behaviors and immunotherapeutic efficacy of renal clear cell carcinoma (ccRCC) Cells. The modular genes most associated with immune cells were screened. Then, prognostic risk models were constructed by univariate COX analysis, LASSO regression analysis and multivariate COX analysis, and their diagnostic value was determined. The correlation between tumor mutation load (TMB) scores and the prognosis of ccRCC patients was clarified. Finally, six key genes (ABI3, ADAM8, APOL3, MX2, CCDC69, and STAC3) were analyzed for immunotherapy efficacy. Human and mouse ccRCC cell lines and human proximal tubular epithelial cell lines were used for in vitro cell experiments. The effect of ADAM8 overexpression or knockdown on tumor formation and survival in ccRCC cells was examined by constructing subcutaneous transplanted tumor model. Totally, 636 Black module genes were screened as being most associated with immune cell infiltration. Six genes were subsequently confirmed for the construction of prognostic risk models, of which ABI3, APOL3 and CCDC69 were low-risk factors, while ADAM8, MX2 and STAC3 were high-risk factors. The constructed risk model based on the identified six genes could accurately predict the prognosis of ccRCC patients. Besides, TMB was significantly associated with the prognosis of ccRCC patients. Furthermore, ABI3, ADAM8, APOL3, MX2, CCDC69 and STAC3 might play important roles in treatment concerning CTLA4 inhibitors or PD-1 inhibitors or combined inhibitors. Finally, we confirmed that ADAM8 could promote the proliferation, migration and invasion of ccRCC cells through in vitro experiments, and further found that in in vivo experiments, ADAM8 knockdown could inhibit tumor formation in ccRCC cells, improve the therapeutic effect of anti-PD1, and prolong the survival of mice. Our study highlighted the alleviative role of silencing ADAM8 in ccRCC patients.

HK-2 cell response to TGF-β highly depends on cell culture medium formulations

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-β) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-β-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-β1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-β1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

Limonin mitigates cisplatin-induced acute kidney injury through metabolic reprogramming

BackgroundAcute kidney injury (AKI) is a known complication of cisplatin administration; currently, there are no effective ways to prevent it. Therefore, it largely limited the use of cisplatin in chemotherapy in the clinic. In this study, we reported that Limonin, a triterpenoid compound extracted from citrus, alleviated cisplatin-induced AKI through metabolic reprogramming in the diseased kidneys. MethodsCisplatin was employed to induce AKI in mice. Three groups were set up: Sham, cisplatin + vehicle, and cisplatin + Limonin. Using UHPLC-TOF/MS, we conducted metabolomics to profile the kidneys' endogenous metabolites and metabolic pathways. A network pharmacological method was performed to identify the targets of Limonin on AKI. The human proximal tubular epithelial cell line (HK-2) was applied for in vitro studies. ResultsLimonin preserved serum creatinine and blood urea nitrogen levels after cisplatin-induced AKI. Employing metabolomics, we identified 33 endogenous differentially expressed metabolites and 7 significantly disturbed metabolic pathways in the diseased kidneys within three groups. After AKI, Limonin significantly reduced linoleic acid and its downstream product, arachidonic acid, thus exerting a protective effect on the kidney. The network pharmacological method identified CYP3A4 as a key target of Limonin in treating AKI, while CYP3A4 also serve as a mediator of arachidonic acid metabolism. In vitro, Limonin markedly reduced the level of arachidonic acid and HK-2 cell apoptosis triggered by cisplatin, mainly related to the targeted inhibition of CYP3A4-mediated arachidonic acid metabolism. ConclusionLimonin ameliorates cisplatin-induced AKI by inhibiting CYP3A4 activity to regulate arachidonic acid metabolism, ultimately preserving kidney function.

Proteasomes Are Critical for Maintenance of CD133+CD24+ Kidney Progenitor Cells.

Kidney progenitor cells, although rare and dispersed, play a key role in the repair of renal tubules after acute kidney damage. However, understanding these cells has been challenging due to the limited access to primary renal tissues and the absence of immortalized cells to model kidney progenitors. Previously, our laboratory utilized the renal proximal tubular epithelial cell line, RPTEC/TERT1, and the flow cytometry technique to sort and establish a kidney progenitor cell model called Human Renal Tubular Precursor TERT (HRTPT) which expresses CD133 and CD24 and exhibits the characteristics of kidney progenitors, such as self-renewal capacity and multi-potential differentiation. In addition, a separate cell line was established, named Human Renal Epithelial Cell 24 TERT (HREC24T), which lacks CD133 expression and shows no progenitor features. To further characterize HRTPT CD133+CD24+ progenitor cells, we performed proteomic profiling which showed high proteasomal expression in HRTPT kidney progenitor cells. RT-qPCR, Western blot, and flow cytometry analysis showed that HRTPT cells possess higher proteasomal expression and activity compared to HREC24T non-progenitor cells. Importantly, inhibition of the proteasomes with bortezomib reduced the expression of progenitor markers and obliterated the potential for self-renewal and differentiation of HRTPT progenitor cells. In conclusion, proteasomes are critical in preserving progenitor markers expression and self-renewal capacity in HRTPT kidney progenitors.

Urinary Modified C-Reactive Protein is Closely Associated with Tubulointerstitial Lesions in Lupus Nephritis.

Modified C-reactive protein (mCRP) is known to be involved in the upregulation and amplification of the local inflammatory response. This study investigated the circulating and local levels of mCRP and their relevance to clinicopathological features in patients with lupus nephritis. Ninety-five patients with renal biopsy-proven lupus nephritis and 30 normal controls were enrolled in this study. Plasma and urinary mCRP were screened by enzyme-linked immunosorbent assay (ELISA). The renal deposition of mCRP was detected by immunohistochemistry and immunofluorescence staining. A human proximal tubular epithelial cell line (HK2 cells) was incubated with purified IgG from lupus nephritis, and the production of CRP by HK2 cells was further evaluated. Plasma and urinary levels of mCRP increased significantly in patients with lupus nephritis compared with normal controls (P = 0.013, P < 0.001, respectively). The urinary mCRP levels were associated with interstitial inflammatory cell infiltration (r = 0.514, P < 0.001) and interstitial fibrosis (r = 0.270, P = 0.008). The ROC-AUC of the urinary mCRP levels for diagnosing tubulointerstitial lesions was 0.766. The urinary mCRP levels were closely associated with poor outcomes (HR: 1.204, 95% CI: 1.029-1.409, P = 0.020). However, no correlations were found of the plasma mCRP levels with clinicopathological data or the prognosis of lupus nephritis. CRP was mostly deposited in the renal tubules in patients with lupus nephritis, and the expression of CRP was significantly correlated with tubulointerstitial lesion indices. Immunofluorescence staining showed that mCRP could colocalize with IgG in tubules. Lupus nephritis-derived IgG could induce CRP production by HK2 cells. Urinary mCRP levels were significantly increased, and urinary mCRP might be a biomarker for tubulointerstitial lesions in patients with lupus nephritis. Renal CRP could be produced by tubular epithelial cells after stimulation by lupus nephritis-derived IgG, and the local presence of mCRP might play a critical role in the development of tubulointerstitial lesions.

RhMYDGF Alleviates I/R-induced Kidney Injury by Inhibiting Inflammation and Apoptosis via the Akt Pathway.

Renal ischemia/reperfusion (I/R) injury is one of the crucial factors affecting the outcome of renal transplantation. In recent years, myeloid-derived growth factor (MYDGF) has received a lot of attention for its extensive beneficial effects on cardiac repair and protection of cardiomyocytes from cell death. Therefore, we hypothesized that the recombinant human MYDGF (rhMYDGF) protein might play an essential role in safeguarding renal I/R injury. In vivo experiments were conducted using a mouse unilateral I/R model. Mice were pretreated with rhMYDGF by intraperitoneal injection to study the potential mechanism of renal protection. In vitro, we established hypoxia/reoxygenation and H 2 O 2 treatment models to pretreat cells with rhMYDGF. The expression levels of oxidative stress, inflammation, and apoptosis-related factors in tissues and cells were detected. Finally, we explored the role of the protein kinase B (Akt) pathway in the renal protective mechanism of rhMYDGF. In this study, we found that intraperitoneal injection of 1.25 μg rhMYDGF could significantly improve renal function of I/R mice, and reduce oxidative stress, inflammation, and apoptosis. For the human proximal tubular epithelial cell line and human kidney cell line, pretreatment with 0.3 μg/mL rhMYDGF for 24 h significantly downregulated oxidative stress, inflammation, and apoptosis via the phosphorylation of Akt, which could be ameliorated by LY294002. rhMYDGF protects kidney from I/R injury by attenuating oxidative stress, inflammation, and apoptosis through the activation of the Akt pathway.

Human umbilical cord mesenchymal stem cell exosomes alleviate acute kidney injury by inhibiting pyroptosis in rats and NRK-52E cells

Human umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia–reperfusion injury (IRI) group, and IRI + hucMSC-Ex group. In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 μg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1β) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis.

Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9

BackgroundSepsis-related acute kidney injury (AKI) is an inflammatory disease associated with extremely high mortality and health burden. This study explored the possibility of exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs) serving as a carrier for microRNA (miR)-342-5p to alleviate sepsis-related AKI and investigated the possible mechanism.MethodsSerum was obtained from 30 patients with sepsis-associated AKI and 30 healthy volunteers for the measurement of miR-342-5p, blood urea nitrogen (BUN), and serum creatinine (SCr) levels. For in vitro experiments, AMSCs were transfected with LV-miR-342-5p or LV-miR-67 to acquire miR-342-5p-modified AMSCs and miR-67-modified AMSCs, from which the exosomes (AMSC-Exo-342 and AMSC-Exo-67) were isolated. The human renal proximal tubular epithelial cell line HK-2 was induced by lipopolysaccharide (LPS) to construct a cellular model of sepsis. The expression of Toll-like receptor 9 (TLR9) was also detected in AKI cells and mouse models. The interaction between miR-342-5p and TLR9 was predicted by dual luciferase reporter gene assay.ResultsDetection on clinical serum samples showed that BUN, SCr, and TLR9 were elevated and miR-342-5p level was suppressed in the serum of patients with sepsis-associated AKI. Transfection with LV-miR-342-5p reinforced miR-342-5p expression in AMSCs and AMSC-secreted exosomes. miR-342-5p negatively targeted TLR9. LPS treatment enhanced TLR9 expression, reduced miR-342-5p levels, suppressed autophagy, and increased inflammation in HK-2 cells, while the opposite trends were observed in LPS-induced HK-2 cells exposed to AMSC-Exo-342, Rapa, miR-342-5p mimic, or si-TLR9. Additionally, the effects of AMSC-Exo-342 on autophagy and inflammation in LPS-induced cells could be weakened by 3-MA or pcDNA3.1-TLR9 treatment. Injection of AMSC-Exo-342 enhanced autophagy, mitigated kidney injury, suppressed inflammation, and reduced BUN and SCr levels in sepsis-related AKI mouse models.ConclusionmiR-342-5p transferred by exosomes from miR-342-5p-modified AMSCs ameliorated AKI by inhibiting TLR9 to accelerate autophagy.Graphical

Vitamin D-VDR (vitamin D receptor) alleviates glucose metabolism reprogramming in lipopolysaccharide-induced acute kidney injury.

Background: Our previous study showed that vitamin D (VD)-vitamin D receptor (VDR) plays a nephroprotective role in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Recently, glucose metabolism reprogramming was reported to be involved in the pathogenesis of AKI. Objective: To investigate the role of VD-VDR in glucose metabolism reprogramming in LPS-induced AKI. Methods: We established a model of LPS-induced AKI in VDR knockout (VDR-KO) mice, renal proximal tubular-specific VDR-overexpressing (VDR-OE) mice and wild-type C57BL/6 mice. In vitro, human proximal tubular epithelial cells (HK-2 cells), VDR knockout and VDR overexpression HK-2 cell lines were used. Results: Paricalcitol (an active vitamin D analog) or VDR-OE reduced lactate concentration, hexokinase activity and PDHA1 phosphorylation (a key step in inhibiting aerobic oxidation) and simultaneously ameliorated renal inflammation, apoptosis and kidney injury in LPS-induced AKI mice, which were more severe in VDR-KO mice. In in vitro experiments, glucose metabolism reprogramming, inflammation and apoptosis induced by LPS were alleviated by treatment with paricalcitol or dichloroacetate (DCA, an inhibitor of p-PDHA1). Moreover, paricalcitol activated the phosphorylation of AMP-activated protein kinase (AMPK), and an AMPK inhibitor partially abolished the protective effect of paricalcitol in LPS-treated HK-2 cells. Conclusion: VD-VDR alleviated LPS-induced metabolic reprogramming in the kidneys of AKI mice, which may be attributed to the inactivation of PDHA1 phosphorylation via the AMPK pathway.

Canagliflozin ameliorates epithelial-mesenchymal transition in high-salt diet-induced hypertensive renal injury through restoration of sirtuin 3 expression and the reduction of oxidative stress

Hypertensive nephropathy is characterized by long-term damage to renal tissues by chronic uncontrolled hypertension, and ultimately leads to the development of renal fibrosis. The epithelial-mesenchymal transition (EMT) potentially contributes to the promotion of renal fibrosis in chronic kidney disease (CKD). In this study, we investigated the potential roles of canagliflozin (Cana) on renal EMT and oxidative stress through its effects on sirtuin 3 (SIRT3) expression. High-salt diet (HSD)-induced Dahl salt-sensitive rats hypertensive renal injury led to decreased SIRT3 expression and an increase in EMT and oxidative stress. In contrast, Cana administration rescued SIRT3 expression, decreased both EMT and levels of oxidative stress, and ameliorated renal injury. Furthermore, we compared the antihypertensive and renoprotective properties of Cana when combined with irbesartan (Irb), a renin-angiotensin system (RAS) blocker. We concluded that administration of Cana in combination with Irb had a significantly greater effect in lowering systolic blood pressure when compared to Cana monotherapy. However, no statistical differences were observed between combined therapy and monotherapy groups with regards to the lowering of diastolic blood pressure and renoprotection. Utilizing the human renal proximal tubular epithelial cell line (HK-2), Angiotensin II (AngⅡ) induced HK-2 negatively regulated the expression of SIRT3, FOXO3a, catalase, and promoted EMT, all of which were reversed by Cana. Furthermore, SIRT3 silencing abolished Cana-mediated rescue of forkhead box O3a (FOXO3a) and catalase expression and Cana-mediated suppression of EMT in AngⅡ induced HK-2. Taken together, Cana acts as a renoprotective agent by suppressing EMT in the pathology of renal fibrosis via interaction with the SIRT3-FOXO3a pathway.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.