Renal Proximal Tubule Epithelial Cells Research Articles

Oridonin attenuates diabetes‑induced renal fibrosis via the inhibition of TXNIP/NLRP3 and NF‑κB pathways by activating PPARγ in rats.

Oridonin possesses remarkable anti-inflammatory, immunoregulatory properties. However, the renoprotective effects of Oridonin and the underlying molecular mechanisms have not been explored in Diabetic Nephropathy (DN). We hypothesized that Oridonin could ameliorate diabetes‑induced renal fibrosis. We used streptozocin (STZ)-induced diabetic rats combined high-fat diet to establish a type 2 diabetes mellitus (T2DM) animal model, and then treated with Oridonin (10,20mg/kg/day) for two weeks. Kidney function and renal fibrosis were assessed. We also treated high glucose-induced human renal proximal tubule epithelial cells (HK-2) with Oridonin. In addition, the expression of inflammatory factors and fibrotic markers were analyzed. Oridonin treatment preserved kidney function and markedly limited the renal fibrosis size in diabetic rats. The renal fibrotic markers were inhibited in the 10mg/kg/day group and 20mg/kg/day group compared to the T2DM group. Moreover, the expression levels of TXNIP/NLRP3 and NF‑κB pathway were decreased and the level of PPARγ were increased in the Oridonin treatment group compared to non-treated group. In vitro, intervention of PPARγ could significantly regulate the effect of Oridonin on the high glucose-induced inflammatory changes in HK-2. Oridonin reduces renal fibrosis and preserves kidney function via the inhibition of TXNIP/NLRP3 and NF‑κB pathway by activating PPARγ in T2DM rat model, which indicates potential therapeutic effect of Oridonin on DN.

Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.

Comparative Analysis of the Physiological and Transport Functions of Various Sources of Renal Proximal Tubule Cells Under Static and Fluidic Conditions in PhysioMimix{trade mark, serif} T12 Platform.

In vitro models that can faithfully replicate critical aspects of kidney tubule function such as directional drug transport are in high demand in pharmacology and toxicology. Accordingly, development and validation of new models is underway. The objective of this study was to characterize physiological and transport functions of various sources of human renal proximal tubule epithelial cells (RPTECs). We tested TERT1-immortalized RPTEC, including OAT1-, OCT2- or OAT3-overexpressing variants, and primary RPTECs. Cells were cultured on transwell membranes in static (24-well transwells) and fluidic (transwells in PhysioMimix{trade mark, serif} T12 organ-on-chip with 2 mL/s flow) conditions. Barrier formation, transport, and gene expression were evaluated. We show that two commercially available primary RPTECs were not suitable for studies of directional transport on transwells because they formed a substandard barrier even though they exhibited higher expression of transporters, especially under flow. TERT1-parent, -OAT1 and -OAT3 cells formed robust barriers, but were unaffected by flow. TERT1-OAT1 cells exhibited inhibitable para-aminohippurate transport, it was enhanced by flow. However, efficient tenofovir secretion and perfluorooctanoic acid reabsorption by TERT1-OAT1 cells were not modulated by flow. Gene expression showed that TERT1 and TERT1-OAT1 cells were most correlated with human kidney than other cell lines, but that flow did not have noticeable effects. Overall, our data show that addition of flow to in vitro studies of the renal proximal tubule may afford benefits in some aspects of modeling kidney function, but that careful consideration of the impact such adaptations would have on the cost and throughput of the experiments is needed. Significance Statement The topic of reproducibility and robustness of the complex microphysiological systems is looming large in the field of biomedical research; therefore, the uptake of these new models by the end-users is slow. This study systematically compared various RPTEC sources and experimental conditions, aiming to identify the level of model complexity needed for testing renal tubule transport. We demonstrate that while tissue chips may afford some benefits, their throughput and complexity need careful consideration in each context of use.

Carboxymethylated Rhizoma alismatis polysaccharides reduces the risk of calcium oxalate stone formation by reducing cellular inflammation and oxidative stress.

This study aims to elucidate the mechanism and potential of Rhizoma alismatis polysaccharides (RAPs) in preventing oxidative damage to human renal proximal tubule epithelial cells. The experimental approach involved incubating HK-2 cells with 100nm calcium oxalate monohydrate for 24h to establish a cellular injury model. Protection was provided by RAPs with varying carboxyl group contents: 3.57%, 7.79%, 10.84%, and 15.33%. The safeguarding effect of RAPs was evaluated by analyzing relevant cellular biochemical indicators.Findings demonstrate that RAPs exhibit notable antioxidative properties. They effectively diminish the release of reactive oxygen species, lactate dehydrogenase, and malondialdehyde, a lipid oxidation byproduct. Moreover, RAPs enhance superoxide dismutase activity and mitochondrial membrane potential while attenuating the permeability of the mitochondrial permeability transition pore. Additionally, RAPs significantly reduce levels of inflammatory factors, including NLRP3, TNF-α, IL-6, and NO. This reduction corresponds to the inhibition of overproduced pro-inflammatory mediator nitric oxide and the caspase 3 enzyme, leading to a reduction in cellular apoptosis. RAPs also display the ability to suppress the expression of the HK-2 cell surface adhesion molecule CD44.The observed results collectively underscore the substantial anti-inflammatory and anti-apoptotic potential of all four RAPs. Moreover, their capacity to modulate the expression of cell surface adhesion molecules highlights their potential in inhibiting the formation of kidney stones. Notably, RAP3, boasting the highest carboxyl group content, emerges as the most potent agent in this regard.

Redox homeostasis in human renal cells that had been treated with amphotericin B in combination with selected 1,3,4-thiadiazole derivatives.

The use of amphotericin B (AmB) in the therapy of systemic mycosis is associated with strong side effects, including nephrotoxicity, and hepatotoxicity. Therefore, agents that can reduce the toxic effects of AmB while acting synergistically as antifungal agents are currently being sought. 1,3,4-thiadiazole derivatives are promising compounds that have an antifungal activity and act synergically with AmB. Such combinations might allow the dose of AmB, which is essential for preventing patients from having serious side effects, to be decreased. This might result from the antioxidant properties of 1,3,4-thiadiazoles. Thus, the aim of the study was to investigate redox homeostasis in human renal proximal tubule epithelial cells (RPTEC) after they had been treated with AmB in combination with 1,3,4-thiadiazole derivatives. Cellular redox homeostasis was assessed by investigating the total antioxidant capacity (TAC) of cells, the malondialdehyde (MDA) concentration, and the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). TAC was measured using an ABTS method. The MDA concentration, and the activity of SOD, GPX, and CAT were determined spectrophotometrically using commercially available assays. Additionally, the antioxidant defense system-related gene expression profile was determined using oligonucleotide microarrays (HG-U133A 2.0). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm the microarray results. Amphotericin B and selected 1,3,4-thiadiazole derivatives had a significant effect on the total antioxidant capacity of the RPTEC cells, and the activity of the antioxidant enzymes. We also revealed that the effect of thiadiazoles on the SOD and CAT activities is dependent on the treatment of RPTEC cells with AmB. At the transcriptional level, the expression of several genes was affected by the studied compounds and their combinations. The results confirmed that thiadiazoles can stimulate the RPTEC cells to defend against the oxidative stress that is generated by AmB. In addition, together with the previously demonstrated synergistic antifungal activity, and low nephrotoxicity, these compounds have the potential to be used in new therapeutic strategies in the treatment of fungal infections.

Abstract 627: IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is a top ten malignancy in the U.S. Overexpression of the proviral integration site for moloney murine leukemia virus 1 (PIM1) kinase is associated with poor clinical outcomes in RCC patients. PIM1 is a constitutively active serine/threonine kinase promoting cell proliferation, apoptosis resistance, invasion, and migration. The mechanisms underlying PIM1 expression and its function in RCC are not fully delineated. IL-6 is a pleiotropic cytokine that activates the JAK/STAT signaling cascade. High serum IL-6 levels are associated with the poor prognosis of RCC patients and may contribute to RCC invasion and metastasis. STAT3/5 binds directly to the PIM1 promoter inducing PIM1 expression. An IL-6/STAT3/PIM1 axis exists in pancreatic and breast cancer. We previously reported that PIM1 is overexpressed in a panel of human RCC cell lines relative to normal and immortalized renal proximal tubule epithelial cells. We also identified that RCC cells secrete IL-6. Our prior studies suggest that differential expression of PIM1 may be linked to autocrine IL-6 signaling. We thus hypothesize that an IL-6/JAK/STAT pathway regulates the expression of PIM1 in RCC. To understand how IL-6 signaling through the JAK/STAT pathway may regulate PIM1 expression in RCC cells, we examined whether IL-6 blockade using anti-IL-6 antibody or tocilizumab, would modulate PIM1 expression. Similarly, we assessed whether ruxolitinib, and LLL12, a STAT3 inhibitor could regulate PIM1 expression. We then evaluated the effect of ruxolitinib treatment on cell viability. In RCC cell lines, IL-6 blockade through either anti-IL-6 antibody or tocilizumab was sufficient to decrease PIM1 protein levels. Treatment with ruxolitinib leads to a dose and time-dependent decrease in PIM1 levels. Incubation with a STAT3 inhibitor also resulted in decreased PIM1 levels in RCC cells. Treatment with ruxolitinib also appears to decrease cell viability in a dose-dependent manner. These results suggest that differential expression of PIM1 in RCC may be linked to autocrine IL-6 signaling via a JAK/STAT/PIM1 axis. Multiple FDA-approved agents are available that target this pathway. Further investigation is required to determine the efficacy of these agents in pre-clinical models and even clinical trials. Citation Format: Kimberly S. Meza, Kimberly Seymour, Sheldon L. Holder. IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 627.

Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.

TNIK depletion induces inflammation and apoptosis in injured renal proximal tubule epithelial cells.

In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase (PARP) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI.NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of siTNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.

Renal proximal tubule-on-a-chip in PDMS: fabrication, functionalization, and RPTEC:HUVEC co-culture evaluation

‘On-a-chip’ technology advances the development of physiologically relevant organ-mimicking architecture by integrating human cells into three-dimensional microfluidic devices. This method also establishes discrete functional units, faciliting focused research on specific organ components. In this study, we detail the development and assessment of a convoluted renal proximal tubule-on-a-chip (PT-on-a-chip). This platform involves co-culturing Renal Proximal Tubule Epithelial Cells (RPTEC) and Human Umbilical Vein Endothelial Cells (HUVEC) within a polydimethylsiloxane microfluidic device, crafted through a combination of 3D printing and molding techniques. Our PT-on-a-chip significantly reduced high glucose level, exhibited albumin uptake, and simulated tubulopathy induced by amphotericin B. Remarkably, the RPTEC:HUVEC co-culture exhibited efficient cell adhesion within 30 min on microchannels functionalized with plasma, 3-aminopropyltriethoxysilane, and type-I collagen. This approach significantly reduced the required incubation time for medium perfusion. In comparison, alternative methods such as plasma and plasma plus polyvinyl alcohol were only effective in promoting cell attachment to flat surfaces. The PT-on-a-chip holds great promise as a valuable tool for assessing the nephrotoxic potential of new drug candidates, enhancing our understanding of drug interactions with co-cultured renal cells, and reducing the need for animal experimentation, promoting the safe and ethical development of new pharmaceuticals.

Continuous genetic monitoring of transient mesenchymal gene activities in distal tubule and collecting duct epithelial cells during renal fibrosis.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-β, a major inducer of EMT.

Predicting proximal tubule failed repair drivers through regularized regression analysis of single cell multiomic sequencing

Renal proximal tubule epithelial cells have considerable intrinsic repair capacity following injury. However, a fraction of injured proximal tubule cells fails to undergo normal repair and assumes a proinflammatory and profibrotic phenotype that may promote fibrosis and chronic kidney disease. The healthy to failed repair change is marked by cell state-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq sequencing offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We develop a regularized regression approach to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generate a single nucleus multiomic dataset from seven adult human kidney samples and apply our method to study drivers of a failed injury response associated with kidney disease. We demonstrate that our approach is a highly effective tool for predicting key cis- and trans-regulatory elements underpinning the healthy to failed repair transition and use it to identify NFAT5 as a driver of the maladaptive proximal tubule state.

Disulfideptosis-associated lncRNAs reveal features of prognostic, immune escape, tumor mutation, and tumor malignant progression in renal clear cell carcinoma.

Investigating the role of lncRNAs associated with the latest cell death mode (Disulfideptosis) in renal clear cell carcinoma, as well as their correlation with tumor prognosis, immune escape, immune checkpoints, tumor mutational burden, and malignant tumor progression. Searching for potential biomarkers and targets for renal clear cell carcinoma. Downloaded the expression profile data and clinical data of 533 cases of renal clear cell carcinoma from the TCGA database, and randomly divided them into a test set (267 cases) and a validation set (266 cases). Based on previous research, 13 genes associated with Disulfideptosis were obtained. Using R software, lncRNAs with a differential expression that is related to the prognosis of renal clear cell carcinoma and associated with Disulfideptosis were screened out. After univariate Cox regression analysis, Lasso regression analysis, and multivariate Cox regression analysis, lncRNAs with independent predictive ability were obtained. A predictive risk model was established based on the risk scores. Verification was carried out between the obtained high-risk and low-risk groups and their subgroups (including Age, Gender, tumor mutational burden (TMB), tumor grading, and staging). Subsequently, a nomogram was established, and a calibration curve was generated for verification. Performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) functional enrichment analyses. Downloaded the values of Tumor Immune Dysfunction and Exclusion (TIDE) for all samples and calculated the difference between the high and low-risk groups. Selected human renal tumor cell lines (786-O, OS-RC-2, A-498, ACHN) and human renal cortex proximal tubule epithelial cell line (HK-2). The RNA expression levels of the above lncRNAs in each cell line were analyzed using RT-qPCR (Real-time Quantitative PCR Detecting System). Used siRNA (small interfering RNA) to knock down FAM225B in 786-O and OS-RC-2 cell lines, and then performed in vitro cell experiments to validate the functional characteristics of FAM225B. Our constructed predictive model includes 5 lncRNAs with an independent predictive ability (FAM225B, ZNF503-AS1, SPINT1-AS1, WWC2-AS2, LINC01338), which can effectively distinguish between patients in high and low-risk groups and their subgroups. The 1, 3, and 5-year AUC (Area Under the ROC Curve) values of the established nomogram are 0.756, 0.752, and 0.781, respectively. The 5-year AUC value is higher compared to other clinical characteristics (Age: 0.598, Gender: 0.488, Grade: 0.680, Stage: 0.717). After the knockdown of FAM225B, the proliferation, migration, and invasion abilities of renal cancer cell lines OS-RC-2 and 786-O all decreased. We have constructed and validated a prognostic model based on Disulfideptosis-associated lncRNAs. This model can effectively predict the high or low risk of patient prognosis and can distinguish the tumor cell mutational burden and immune escape capabilities among high-risk and low-risk patients. This predictive model can serve as an independent prognostic factor for renal clear cell carcinoma, providing a new direction for personalized treatment of patients with renal clear cell carcinoma.

Silica-coated LiYF4:Yb3+, Tm3+ upconverting nanoparticles are non-toxic and activate minor stress responses in mammalian cells.

Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. The interaction of nanomaterials with biological systems determines whether they are suitable for use in living cells. In-depth knowledge of the nano-bio interactions is therefore a pre-requisite for the development of biomedical applications. The current study evaluates fundamental aspects of the NP-cell interface for square bipyramidal UCNPs containing a LiYF4:Yb3+, Tm3+ core and two different silica surface coatings. Given their importance for mammalian physiology, fibroblast and renal proximal tubule epithelial cells were selected as cellular model systems. We have assessed the toxicity of the UCNPs and measured their impact on the homeostasis of living non-malignant cells. Rigorous analyses were conducted to identify possible toxic and sub-lethal effects of the UCNPs. To this end, we examined biomarkers that reveal if UCNPs induce cell killing or stress. Quantitative measurements demonstrate that short-term exposure to the UCNPs had no profound effects on cell viability, cell size or morphology. Indicators of oxidative, endoplasmic reticulum, or nucleolar stress, and the production of molecular chaperones varied with the surface modification of the UCNPs and the cell type analyzed. These differences emphasize the importance of evaluating cells of diverse origin that are relevant to the intended use of the nanomaterials. Taken together, we established that short-term, our square bipyramidal UCNPs are not toxic to non-malignant fibroblast and proximal renal epithelial cells. Compared with established inducers of cellular stress, these UCNPs have minor effects on cellular homeostasis. Our results build the foundation to explore square bipyramidal UCNPs for future in vivo applications.

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

The Influence of Betulin Derivatives EB5 and ECH147 on the Expression of Selected TGFβ Superfamily Genes, TGFβ1, GDF15 and BMP2, in Renal Proximal Tubule Epithelial Cells.

Betulin derivatives are proposed to serve as an alternative to the drugs already established in oncologic treatment. Drug-induced nephrotoxicity leading to acute kidney injury frequently accompanies cancer treatment, and thus there is a need to research the effects of betulin derivatives on renal cells. The objective of our study was to assess the influence of the betulin derivatives 28-propynylobetulin (EB5) and 29-diethoxyphosphoryl-28-propynylobetulin (ECH147) on the expression of TGFβ1, BMP2 and GDF15 in renal proximal tubule epithelial cells (RPTECs) cultured in vitro. The changes in mRNA expression and copy numbers were assessed using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and the standard curve method, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the effect of the betulin derivatives on the protein concentration in the culture media's supernatant. The assessment of the betulin derivatives' influence on gene expression demonstrated that the mRNA level and protein concentration did not always correlate with each other. Each of the tested compounds affected the mRNA expression. The RT-qPCR analyses showed that EB5 and ECH147 induced effects similar to those of betulin or cisplatin and resulted in a decrease in the mRNA copy number of all the analyzed genes. The ELISA demonstrated that EB5 and ECH147 elevated the protein concentration of TGFβ1 and GDF15, while the level of BMP2 decreased. The concentration of the derivatives used in the treatment was crucial, but the effects did not always exhibit a simple linear dose-dependent relationship. Betulin and its derivatives, EB5 and ECH147, influenced the gene expression of TGFβ1, BMP2 and GDF15 in the renal proximal tubule epithelial cells. The observed effects raise the question of whether treatment with these compounds could promote the development of renal fibrosis.

Expired Platelet Concentrate Up-Cycling: Growth Factor-Rich Bioproduct Preparation for FBS Substitute

Due to the short storage period, large quantities of platelet concentrate (PC) are expiring. The expired PC cannot be injected into a blood vessel, but the activity of bioactive molecules, especially growth factors, is still preserved. In this paper, we organized a process to obtain a growth factor-rich bioproduct for use as a supplement in human cell culture by optimizing freezing, thawing, and sterilization conditions. Each unit of PC displayed visual differences, diverse biochemical values, and growth factor concentrations. To minimize lot-to-lot variation, we pooled a minimum of 10 PC units. The concentrations of growth factors were maximized through five freeze–thaw cycles for 12 h at −80 °C for freezing and for 5 min at 36 °C for thawing. We used a cell strainer with 40 µm pores, followed by a 0.45 μm filter and a 0.22 μm filter sequentially to sterilize the bioproduct with minimizing loss. The obtained growth factors remained stable for 4–6 h at room temperature (23 °C), 24 h at 4 °C, and 12 months at −80 °C. Cellular responses to the growth factor-rich bioproduct were tested with primary human renal proximal tubule epithelial cells. The cells exhibited a significantly increased growth rate, compared to the fetal bovine serum (FBS)-treated control group. The cells maintained their characteristic cuboidal shape, and stem cells and renal progenitor cells also preserved their genetic characteristics during culture. Therefore, the growth factor-rich bioproduct isolated from expired PC through our process can be used as a medium supplement to replace FBS in human cell culture for clinical application.

Molecular mechanism of NR4A1/MDM2/P53 signaling pathway regulation inducing ferroptosis in renal tubular epithelial cells involved in the progression of renal ischemia-reperfusion injury

Revealing the possible molecular mechanism of the NR4A1 (nuclear receptor subfamily 4 group A member 1)-MDM2 (MDM2 proto-oncogene)-P53 (tumor protein p53) signaling pathway that induces ferroptosis in renal tubular epithelial cells. Renal ischemia-reperfusion injury (RIRI) -related datasets were obtained from the GEO database. Differentially expressed genes in RIRI were analyzed using R language, intersected with RIRI-related genes in the GeneCard database, and retrieved from the literature to finally obtain differential ferroptosis-related genes. An in vitro cell model of RIRI was constructed using mouse renal cortical proximal tubule epithelial cells (mRTEC cells) treated with hypoxia-reoxygenation (H/R). Bioinformatic analysis showed that NR4A1 may be involved in RIRI through the induction of ferroptosis; in addition, we predicted through online databases that the downstream target gene of NR4A1, MDM2, could be targeted and regulated by ChIP and dual luciferase assays, and that NR4A1 could prevent MDM2 by inhibiting it, and NR4A1 was able to promote ferroptosis by inhibiting the ubiquitinated degradation of P53. NR4A1 expression was significantly increased in mRTEC cells in the hypoxia/reoxygenation model, and the expression of ferroptosis-related genes was increased in vitro experiments. NR4A1 reduces the ubiquitinated degradation of P53 by targeting the inhibition of MDM2 expression, thereby inducing ferroptosis and ultimately exacerbating RIRI by affecting the oxidative respiration process in mitochondria and producing oxidized lipids. This study presents a novel therapeutic approach for the clinical treatment of renal ischemia-reperfusion injury by developing drugs that inhibit NR4A1 to alleviate kidney damage caused by renal ischemia-reperfusion.

Identification of Novel and Early Biomarkers for Cisplatin-induced Nephrotoxicity and the Nephroprotective Role of Cimetidine using a Pharmacometabolomic-based Approach Coupled with In Vitro Toxicodynamic Modeling and Simulation

Cisplatin is widely used for the treatment of various types of cancer. However, cisplatin-induced nephrotoxicity (CIN) is frequently observed in patients receiving cisplatin therapy which poses a challenge in its clinical utility. Currently used clinical biomarkers for CIN are not adequate for early detection of nephrotoxicity, hence there is a need to identify potential early biomarkers in predicting CIN. In the current study, a combination of in vitro toxicodynamic (TD) modeling and untargeted global metabolomics approach was used to identify novel potential metabolite biomarkers for early detection of CIN. In addition, we investigated the protective role of cimetidine (CIM), an inhibitor of the organic cation transporter 2 (OCT2), in suppressing CIN. We first characterized the time-course of nephrotoxic effects of cisplatin (CIS) and the protective effects of CIM in a human pseudo-immortalized renal proximal tubule epithelial cell line (RPTEC), SA7K cell line. Secondly, we used a mathematical cell-level, in vitro TD modeling approach to quantitatively characterize the time-course effects of CIS and CIM as single agents and combination in SA7K cells. Based on the experimental and modeling results, we selected relevant concentrations of CIS and CIM for our metabolomics study. With the help of PCA (Principal Component Analysis) and PLS-DA (Projection to Latent Structure – Discriminate Analysis) analyses, we confirmed global metabolome changes for different groups (CIS, CIM, CIS+CIM vs control) in SA7K cells. Based on the criterion of a p-value ≤ 0.05 and a fold change ≥ 2 or ≤ 0.5, we identified 20 top metabolites that were significantly changed during the early phase i.e. within first 12 h of CIS treatment. Finally, pathway analysis was conducted that revealed the key metabolic pathways that were most impacted in CIN.

Significance of Organic Anion Transporter 2 and Organic Cation Transporter 2 in Creatinine Clearance: Mechanistic Evaluation Using Freshly Prepared Human Primary Renal Proximal Tubule Cells.

Creatinine, a clinical marker for kidney function, is predominantly cleared by glomerular filtration, with active tubular secretion contributing to about 30% of its renal clearance. Recent studies suggested the potential involvement of organic anion transporter (OAT)2, in addition to the previously known organic cation transporter (OCT)2-mediated basolateral uptake, in creatinine active secretion. Here we characterized the transport mechanisms of creatinine using transfected human embryonic kidney (HEK)293 cells and freshly prepared human primary renal proximal tubule epithelial cells (hPTCs). Creatinine showed transport by OAT2 in transfected HEK293 cells. In addition, both creatinine and metformin showed transport by OCT2 and multidrug and toxin extrusion pump (MATE)1 and MATE2K, while penciclovir was selective for OAT2. Time-dependent cell accumulation was observed for creatinine and metformin in hPTCs. Their accumulation was increased by pyrimethamine but inhibited by decynium-22, likely due to differential inhibition of OCT2 versus MATEs. Additionally, indomethacin (an OAT2 inhibitor) reduced penciclovir uptake (∼75%) in hPTCs illustrating functional OAT2 activity. However, no modulation of creatinine and metformin cell accumulation was apparent with indomethacin. Creatinine transport characteristics in the presence of inhibitors approached those of metformin, an OCT2/MATE substrate, but were distinct from those of penciclovir, an OAT2-selective substrate. Moreover, indomethacin showed no significant effect on the basolateral-to-apical transport and net secretion of creatinine across hPTC monolayers. Collectively, the functional studies suggest OCT2 as the primary basolateral uptake mechanism and that OAT2 has a minimal role, in creatinine renal secretion. Our results highlight the utility of hPTCs to enable the functional assessment of renal transport mechanisms. SIGNIFICANCE STATEMENT: Our results obtained with primary hPTCs indicate that OCT2/MATE (vs. OAT2) play a major role in the active renal secretion of creatinine. Quantitative pharmacokinetic models should therefore focus on OCT2/MATE when describing serum creatinine and creatinine clearance modulation by inhibitor drugs and genotype- or disease-related activity changes. The present study highlights the utility of freshly isolated hPTCs to support solute carrier phenotyping to enable the functional assessment of renal transport mechanisms.

Abstract 13109: Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration

Empagliflozin, an FDA-approved anti-hyperglycemic agent, is a potent and highly selective inhibitor of sodium glucose co-transporter-2 (SGLT-2) in renal proximal tubule epithelial cells. It blocks glucose reabsorption and promotes glucose excretion via urine. Intriguingly, several clinical trials have reported the pleiotropic beneficial effects of SGLT-2 inhibitors in cardiovascular diseases, including antioxidant and anti-inflammatory effects. Here we investigated whether empagliflozin reverses oxidatively modified LDL (OxLDL)-induced matrix metalloproteinase activation and smooth muscle cell (SMC) migration and proliferation. Quiesced human aortic SMCs (Lonza) were treated with empagliflozin (1 uM) followed by human medium OxLDL (40 ug/mL; Kalen Biomedical). OxLDL stimulated SMC migration (1.8-fold, P<0.05, n=4) and proliferation (1.5-fold, P<0.05, n=4), and empagliflozin attenuated both by 67% (P<0.05, n=4) and 61% (P<0.05, n=4), respectively. Increased migration and proliferation by OxLDL were accompanied by elevated MMP-2 (3.2-fold, P<0.05, n=5) and 9 (3.1-fold, P<0.05, n=5) activities, which were also suppressed by empagliflozin by 65% and 67%, respectively (both P<0.05, n=5). RECK (reversion inducing cysteine rich protein with Kazal motifs) is a membrane anchored MMP inhibitor, and we found that OxLDL dose-dependently downregulated RECK in SMCs (by 60%, P<0.05, n=3) via NF-kB-dependent miR-30b induction. Intriguingly, empagliflozin restored the OxLDL downregulation of RECK to 88% of the control (n=3), as it inhibited miR-30b induction (by 49%, P<0.05, n=5). Consistent with RECK-mediated effects of empagliflozin, adenoviral overexpression of RECK inhibited OxLDL-induced SMC migration (by 67%, P<0.05, n=5) and proliferation (by 50%, P<0.05, n=5). Together, these results suggest that the vasculoprotective effects of empagliflozin are at least in part mediated by restoring RECK, thereby attenuating SMC migration and proliferation under elevated oxidative stress condition. Our results also suggest therapeutic potential of empagliflozin or RECK overexpression in vascular proliferative diseases.