Renal Epithelial Cells In Culture Research Articles

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.

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.

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.

FRI163 Central Role Of Mirnas During Kidney Development And Establishment Of The Mineralocorticoid Signaling Pathway Establishment Of The Mineralocorticoid Signaling Pathway

Abstract Disclosure: I. Hani: None. T. Vu: None. R. Brillet: None. J. Perrot: None. J. Bouligand: None. J. Guegan: None. N. Cherradi: None. P. Kamenicky: None. M. Lombes: None. L. Martinerie: None. S. Viengchareun: None. In tight epithelia, aldosterone controls sodium and water homeostasis by binding to the Mineralocorticoid Receptor (MR), a steroid nuclear receptor. We have identified a specific temporal window during renal development in which the mineralocorticoid signaling pathway is ineffective due to downregulation of MR expression, explaining physiological sodium wasting observed in human newborns during first days of life. However, underlying molecular mechanisms controlling MR expression remain unknown. Recently, we demonstrated in adult renal KC3AC1 cell line that MR expression is controlled by microRNAs (miR-30c-2-3p and miR-324-5p) in response to variations of tonicity. These regulators bind to 3'-untranslated region (3'-UTR) of MR transcripts to modulate their stability and translation. Therefore, we hypothesized that these miRNAs may be responsible for low expression level of MR at birth, where variations in tonicity are observed due to the transition from intra-amniotic to extra-uterine life. Using primary cultures of renal epithelial cells from neonatal mouse kidneys, harvested at birth (D0) and at Day 8 postnatal (D8), we showed that only miR-30c-2-3p regulates MR expression at D8. For this reason, we performed a complete transcriptomic analysis (miRNA-Seq and RNA-Seq) of kidneys collected at D0 and D8 to identify other miRNAs specifically involved in regulation of MR expression in perinatal period. Bioinformatics analysis allowed us to identify deregulated miRNAs and transcripts between D0 and D8 and to specify their involvement in biological processes and/or signaling pathways. We mainly focused on deregulated miRNAs that may modulate MR expression and affect mineralocorticoid signaling. miR-Seq identified 221 differentially expressed miRNAs. First, we selected 3 over- (miR-431-5p, miR-409-3p, and miR-92a-1-5p) and 3 under-expressed (miR-30a-5p, miR-30e-5p, and miR-802-5p) candidate miRNAs (FDR<0.05 and log2FC>1) having putative binding sites in MR 3'-UTR. Paradoxically, we showed in primary kidney cultures that overexpression of miR-30a-5p, miR-30e-5p, or miR-802-5p increased MR expression (from 50 to 100%, P<0.05) at D0, suggesting that these miRNAs may either positively modulate MR expression or may involve an intermediate regulator. On the opposite, over-expression of miR-92a-1-5p, miR-431-5p, and miR-409-3p at D8 led to a decrease (from 30-40%, P<0.05) in MR expression, suggesting that these miRNAs may modulate MR expression in postnatal period. Currently, we are evaluating whether they could be used as prognostic or severity biomarkers of water loss in neonates by quantifying these candidate miRNAs in urine exosomes of preterm and term infants. A better understanding of mechanisms regulating MR expression in the perinatal period will undoubtedly have a major impact on the management of premature infants with sodium balance maladjustment at birth. Presentation Date: Friday, June 16, 2023

Activated SOX9+ renal epithelial cells promote kidney repair through secreting factors.

A broad spectrum of lethal kidney diseases involves the irreversible destruction of the tubular structures, leading to renal function loss. Following injury, a spectrum of tissue-resident epithelial stem/progenitor cells are known to be activated and then differentiate into mature renal cells to replace the damaged renal epithelium. Here, however, we reported an alternative way that tissue-resident cells could be activated to secrete multiple factors to promote organ repair. At single-cell resolution, we showed that the resident SOX9+ renal epithelial cells (RECs) could expand in the acutely injured kidney of both mouse and human. Compared to other cells, the SOX9+ RECs overexpressed much more secretion related genes, whose functions were linked to kidney repair pathways. We also obtained long-term, feeder-free cultured SOX9+ RECs from human urine and analysed their secretory profile at both transcriptional and proteomic levels. Engraftment of cultured human SOX9+ RECs or injection of its conditional medium facilitated the regeneration of renal tubular and glomerular epithelium, probably through stimulating endogenous REC self-activation and mediating crosstalk with other renal cells. We also identified S100A9 as one of the key factors in the SOX9+ REC secretome. Altogether, the abilities to extensively propagate SOX9+ RECs in culture whilst concomitantly maintaining their intrinsic secretory capacity suggest their future application in cell-free therapies and regeneration medicine.

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.

Higher Concentrations of Folic Acid Cause Oxidative Stress, Acute Cytotoxicity, and Long-Term Fibrogenic Changes in Kidney Epithelial Cells.

Kidney fibrosis is a common step during chronic kidney disease (CKD), and its incidence has been increasing worldwide. Aberrant recovery after repeated acute kidney injury leads to fibrosis. The mechanism of fibrogenic changes in the kidney is not fully understood. Folic acid-induced kidney fibrosis in mice is an established in vivo model to study kidney fibrosis, but the mechanism is poorly understood. Moreover, the effect of higher concentrations of folic acid on kidney epithelial cells in vitro has not yet been studied. Oxidative stress is a common property of nephrotoxicants. Therefore, this study evaluated the role of folic acid-induced oxidative stress in fibrogenic changes by using the in vitro renal proximal tubular epithelial cell culture model. To obtain comprehensive and robust data, three different cell lines derived from human and mouse kidney epithelium were treated with higher concentrations of folic acid for both acute and long-term durations, and the effects were determined at the cellular and molecular levels. The result of cell viability by the MTT assay and the measurement of reactive oxygen species (ROS) levels by the DCF assay revealed that folic acid caused cytotoxicity and increased levels of ROS in acute exposure. The cotreatment with antioxidant N-acetyl cysteine (NAC) protected the cytotoxic effect, suggesting the role of folic acid-induced oxidative stress in cytotoxicity. In contrast, the long-term exposure to folic acid caused increased growth, DNA damage, and changes in the expression of marker genes for EMT, fibrosis, oxidative stress, and oxidative DNA damage. Some of these changes, particularly the acute effects, were abrogated by cotreatment with antioxidant NAC. In summary, the novel findings of this study suggest that higher concentrations of folic acid-induced oxidative stress act as the driver of cytotoxicity as an acute effect and of fibrotic changes as a long-term effect in kidney epithelial cells.

Bisphenol A impairs renal function by reducing Na+/K+-ATPase and F-actin expression, kidney tubule formation in vitro and in vivo

The kidney proximal tubule is responsible for reabsorbing water and NaCl to maintain the homeostasis of the body fluids, electrolytes, and nutrients. Thus, abnormal functioning of the renal proximal tubule can lead to life-threatening imbalances. Bisphenol A (BPA) has been used for decades as a representative chemical in household plastic products, but studies on its effects on the kidney proximal tubule are insufficient. In this study, immunocytochemical and cytotoxicity tests were performed using two- and three-dimensional human renal proximal tubular epithelial cell (hRPTEC) cultures to investigate the impact of low-dose BPA (1–10 μM) exposure. BPA was found to interfere with straight tubule formation as observed by low filamentous actin formation and reduced Na+/K+-ATPase expression in the tubules of hRPTEC 3D cultures. Similar results were observed in rat pup kidneys following oral administration of 250 mg/kg BPA. Moreover, the expression of HO-1 and 8-OHdG, key markers for oxidative stress, was increased in vitro and in vivo following BPA administration, whereas that of OAT1 and OAT, important transporters of the renal proximal tubules, was not altered. Overall, no-observed-adverse-effect-level (NOAEL)-dose BPA exposure can decrease renal function by promoting abnormal tubular formation both in vitro and in vivo. Therefore, we propose that although it does not exhibit life-threatening toxicity, exposure to low levels of BPA can negatively affect homeostasis in the body by means of long-term deterioration of renal proximal tubular function in humans.

Inhibition of Malate Dehydrogenase-2 Protects Renal Tubular Epithelial Cells from Anoxia-Reoxygenation-Induced Death or Senescence.

Ischemia-reperfusion injury is the leading cause of acute kidney injury. Reactive oxygen species (ROS) production causes cell death or senescence. In cultures of primary human renal tubular epithelial cells (RPTECs) subjected to anoxia-reoxygenation, inhibition of the Krebs cycle at the level of malate dehydrogenase-2 (MDH-2) decreases hypoxia-inducible factor-1α and oxidative stress and protects from apoptotic or ferroptotic cell death. Inhibition of MDH-2 decreased reoxygenation-induced upregulation of p53 and p21, restored the levels of the proliferation marker Ki-67, and prevented the upregulation of the senescence marker beta-galactosidase and interleukin-1β production. MDH-2 inhibition reduced the reoxygenation-induced upregulation of ATP, but the alterations of critical cell metabolism enzymes allowed enough ATP production to prevent cell energy collapse. Thus, inhibition of the Krebs cycle at the level of MDH-2 protects RPTECs from anoxia-reoxygenation-induced death or senescence. MDH-2 may be a promising pharmaceutical target against ischemia-reperfusion injury.

Measurement of oxygen consumption rates of human renal proximal tubule cells in an array of organ-on-chip devices to monitor drug-induced metabolic shifts

Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.

Ang II (Angiotensin II)–Induced FGFR1 (Fibroblast Growth Factor Receptor 1) Activation in Tubular Epithelial Cells Promotes Hypertensive Kidney Fibrosis and Injury

Elevated Ang II (angiotensin II) level leads to a range of conditions, including hypertensive kidney disease. Recent evidences indicate that FGFR1 (fibroblast growth factor receptor 1) signaling may be involved in kidney injuries. In this study, we determined whether Ang II alters FGFR1 signaling to mediate renal dysfunction. Human archival kidney samples from patients with or without hypertension were examined. Multiple genetic and pharmacological approaches were used to investigate FGFR1-mediated signaling in tubular epithelial NRK-52E cells in response to Ang II stimulation. C57BL/6 mice were infused with Ang II for 28 days to develop hypertensive kidney disease. Mice were treated with either adeno-associated virus expressing FGFR1 shRNA or FGFR1 inhibitor AZD4547. Kidney specimens from subjects with hypertension and mice challenged with Ang II have increased FGFR1 activity in renal epithelial cells. Renal epithelial cells in culture initiate extracellular matrix programming in response to Ang II, through the activation of FGFR1, which is independent of both AT1R (angiotensin II receptor type 1) and AT2R (angiotensin II receptor type 2). The RNA sequencing analysis indicated that disrupting FGFR1 suppresses Ang II-induced fibrogenic responses in epithelial cells. Mechanistically, Ang II-activated FGFR1 leads to STAT3 (signal transducer and activator of transcription 3) activation, which is responsible for fibrogenic factor expression in kidneys. In the mouse model of hypertensive kidney disease, genetic knockdown of FGFR1 or pharmacological inhibition of its activity protected kidneys from dysfunction and fibrosis upon Ang II challenge. Our studies uncover a novel mechanism causing renal fibrosis in hypertension and indicate FGFR1 as a potential target to preserve renal function and integrity.

Tubular IL-1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages.

Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. Thirty-four-week-old db/db mice display significantly higher levels of IL-1β in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1β in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1β when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1β promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1β in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. Renal tubular epithelial cell-derived IL-1β polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1β synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

Renal Tubular IL‐1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages

We recently showed that interleukin (IL)‐6 induces salt‐sensitivity in diabetic mice by dysregulating the epithelial sodium channel (ENaC). After 4 weeks on a high salt diet, male db/db mice displayed salt sensitivity, the accumulation of proinflammatory (CD80+) macrophages, and higher levels of IL‐6 and IL‐1β in the kidney compared to db/+ controls. However, the origin of this inflammatory response is unknown. We hypothesize that, during diabetes, renal tubular epithelial cells release IL‐1β which activates renal macrophages to produce IL‐6 and impair kidney function. Immunofluorescence analysis showed that the higher IL‐1β expression of db/db mice is exclusively localized in the renal tubules. Flow cytometry analysis of renal cells showed that db/db mice have higher IL‐1β expression in tubular (E‐cadherin+) cells (21 ± 2 vs. 8 ± 3 % of E‐cadherin+ cells, P<0.001) compared to db/+ controls. To evaluate whether high glucose triggers IL‐1β synthesis in tubular cells, a primary culture of renal tubular epithelial cells from wild‐type (WT) mice was exposed to either a low (LG, 5mM) or high (HG, 15mM) glucose media. HG‐treated cells released higher levels of IL‐1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and displayed higher expression of NLRP3 (4 ± 1‐fold increase, P<0.001) and Caspase‐1 (2.1 ± 0.2‐fold increase, P<0.001) compared to LG‐treated cells. These are major components of the NLRP3 inflammasome that synthesizes IL‐1β. No equivalent effect was observed using mannitol. To evaluate whether tubular cells can modify macrophage phenotype through IL‐1β, we co‐cultured tubular cells with bone marrow‐derived macrophages. Flow cytometry revealed that HG‐treated tubular cells induced a pro‐inflammatory polarization of macrophages not observed with LG‐treated cells (50 ± 4 vs. 15 ± 2 CD80+ macrophages, P<0.001). This effect was blunted when macrophages were obtained from IL‐1 receptor KO mice (IL1RKO) confirming a role of IL‐1β. To evaluate whether IL‐1β‐mediated macrophage activation plays a role in vivo, 2‐mo‐old diabetic db/db mice (n=6) were transplanted with a bone marrow of IL1RKO mice. These mice, named db/db‐IL1RKO, cannot respond to IL‐1β. db/db receiving wild‐type (WT) bone marrow (db/db‐WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to high salt diet for 4 weeks. Compared to db/db‐WT, db/db‐IL1RKO did not develop salt‐sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal CD80+ macrophages, (21 ± 4 vs. 54 ± 5 CD80+ macrophages, P<0.01), less renal IL‐6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05), and lower ENaC activity (65% decrease by amiloride test, P<0.05) despite an accumulation of tubular IL‐1β. In conclusion, our data show that tubular epithelial cells are a major source of IL‐1β under high glucose conditions. This cytokine promotes a pro‐inflammatory phenotype of macrophages resulting in higher renal IL‐6 levels which increases ENaC activity leading to salt sensitivity.

The Role of Indoleamine 2,3-Dioxygenase in Renal Tubular Epithelial Cells Senescence under Anoxia or Reoxygenation.

Ischemia-reperfusion injury is the commonest form of acute kidney injury (AKI). Tubular epithelial cell senescence contributes to incomplete recovery from AKI and predisposes to subsequent chronic kidney disease. In cultures of primary proximal renal tubular epithelial cells (RPTECs) subjected to anoxia or reoxygenation, we evaluated the role of indoleamine 2,3-dioxygenase 1 (IDO) in cellular senescence. Proteins of interest were assessed with Western blotting or enzyme-linked immunosorbent assay or histochemically. Under anoxia or reoxygenation, IDO expression and activity were increased. Moreover, the two IDO-derived pathways, the general control nonderepressible 2 kinase (GCN2K) pathway and the aryl-hydrocarbon receptor (AhR) pathway, were also activated. A DNA damage response (DDR) took place and led to increased levels of the cell-cycle inhibitors p21 and p16, and senescence-associated β-galactosidase (SA-β-Gal) activity. Cell proliferation was inhibited, and more IL-6 was produced. The IDO inhibitor 1-DL-methyl-tryptophan ameliorated the DDR; decreased p21, p16, and SA-β-Gal activity; restored cell proliferation; and decreased IL-6 production. The AhR inhibitor CH223191 did not affect the above parameters. In conclusion, anoxia and the subsequent reoxygenation upregulate IDO. IDO depletes tryptophan and activates GCN2K. The latter enhances the anoxia- or reoxygenation-induced DDR, resulting in increased p21 and p16 expression and eventually leading to RPTEC senescence. Since cellular senescence affects AKI outcome, the role of IDO in cellular senescence and the possible therapeutic role of IDO inhibitors deserve further investigation.

Abstract MP32: Renal Tubular Epithelial Cell-derived IL-1β Triggers The Inflammatory Response That Induces Salt Sensitivity In Diabetes

Renal inflammation, and specifically interleukin (IL)-6, induces salt-sensitivity in diabetic mice by upregulating the epithelial sodium channel (ENaC). However, the origin of this inflammatory response is still unknown. We hypothesize that, during diabetes, tubular epithelial cells initiate renal inflammation by releasing IL-1β that further activates renal macrophages to produce IL-6 and impair kidney function. A primary culture of renal tubular epithelial cells from wild-type (WT) mice was exposed to either low (LG, 5mM) or high (HG, 15mM) glucose media. HG-treated cells released higher levels of IL-1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and have higher expression of NLRP3 (4 ± 1 fold increase, P<0.001) and Caspase-1 (2.1 ± 0.2 fold increase, P<0.001) compared to LG-treated cells. No effect was observed using mannitol as an osmotic control. Tubular cells were also co-cultured with bone marrow-derived macrophages. Flow cytometry analysis revealed that HG-treated tubular cells induced an M1-type polarization of macrophages. This effect was blunted when macrophages were obtained from IL-1 receptor KO mice (IL1RKO) confirming a role of IL-1β. Here, the percentage of M1 macrophages (CD45 + F4/80 + CD80 + ) of total macrophages (CD45 + F4/80 + ) decreased from 31 ± 4% to 10 ± 3% (P<0.01). To evaluate whether IL-1β-mediated macrophage activation plays a role in vivo, 2-mo-old diabetic db/db mice (n=6) were transplanted with bone marrow from IL1RKO mice. In these mice, named db/db-IL1RKO, immune cells cannot respond to IL-1β. Db/db receiving wild-type (WT) bone marrow (db/db-WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to a high salt diet (NaCl 4% w/w) for 4 weeks. Db/db-IL1RKO did not develop salt-sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal IL-6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05) and lower ENaC activity (0.4-fold decrease by amiloride test, P<0.05) compared to db/db-WT. In conclusion, our data show that tubular epithelial cells are a major source of IL-1β under high glucose conditions. This cytokine promotes M1 macrophage polarization resulting in higher renal IL-6 levels which increases ENaC activity leading to salt sensitivity.

A New Method of Isolation of Mouse Renal Primary Tubular Epithelial Cells.

Kidney diseases are becoming an emerging public health problem. In order to further explore the etiology of various kidney diseases, we improved the methods of isolation of primary cultures of mouse renal tubular epithelial cells. At the first stage, the kidneys were perfused with collagenase solution. To this end, the superior mesenteric artery, celiac artery and thoracic aorta were ligated and perfusion was performed through the abdominal aorta. Then, the cells were isolated ex vivo and their integrity, purity, viability, and concentration were evaluated. The proposed cost-effective and simple method provides high purity and high concentration of primary renal epithelial cells for molecular biology studies of the kidneys.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

Increased Expression of Circulating NLRP3 Inflammasome in Canines With Chronic Kidney Disease

ObjectivesInflammation is a key component in the initiation and progression of chronic kidney disease (CKD) and can contribute to renal fibrosis and a gradual loss of renal function. Inflammasomes are multiprotein immune complexes and their activation contributes to inflammation. While inflammasome-related protein including NOD-like receptor family, pyrin domain-containing-3 (NLRP3) is important in acute and chronic kidney disorders in rodent models, the role of inflammasomes in renal disorders in canines is not clear. MethodsWe assessed gene expression levels of inflammatory markers, in the blood of dogs clinically diagnosed with CKD, post-mortem. Pathology reports indicated interstitial inflammation, fibrosis, and thickening of the Bowman’s capsule. RNA was extracted from blood collected at necropsy from dogs with CKD (n = 6; 11–15 yr) and controls (n = 6; 10–14 yr). Gene expression was investigated using the NanoString nCounter® platform, and results analyzed using the nSolver software. ResultsThere was a significant up-regulation in the level of NLRP3 in dogs with CKD when compared to controls (1.36 fold, P < 0.05). Consistent with this, there was also an increase in IL1b, one of the cytokines activated by the NLRP3 pathway, in CKD dogs (1.44 fold, ns). However, there was a decrease in IL18 (1.28 fold, ns), another cytokine activated downstream of NLRP3 pathway. Further, IL17 and IFNg, two cytokines activated by IL18, were barely undetectable in CKD dogs and in controls. These results indicate that the NLRP3-IL1b pathway may play a more important role in CKD in canines than the NLRP3-IL18 pathway. There was also an increase in the intercellular adhesion molecule-3 (ICAM3), a T-cell activator, in CKD dogs (1.76 fold, ns). In contrast, there was a significant decrease in IL7 and CD19 in CKD dogs (1.99 and 2.26 fold respectively, both P < 0.05 vs con). While IL7 is a growth factor for T and B-cells, it inhibits fibrosis-like response in renal epithelial cells in culture. Whether a decline in IL7 may also promote renal fibrosis in canines is not clear. Moreover, increased NLRP3 is also implicated in renal fibrosis. ConclusionsOur results indicate that the NLRP3 plays an important role in kidney disease. Nutritional interventions targeting the NLRP3-IL-1b pathway may be important in mitigating the inflammasome-mediated deleterious effects in CKD Funding SourcesHills PNC.

Chemokine Profiles Are Affected in Serum of Patients with Acute Rejection of Kidney Allograft.

Kidney allograft transplantation improved the prognosis and quality of life of patients with end-stage renal diseases but the occurrence of acute rejection represents a limitation of the final outcome. Noninvasive biomarkers are needed as well as further advancements in the understanding of immune mechanisms of reaction to the allograft. Our study of 138 patients focused on one-year monitoring of serum concentrations of 12 chemokines regulating the recruitment of different immune cells into transplanted allograft and on in vitro regulation of the same chemokines release by interactions of renal proximal epithelial cells with monocyte/macrophage cell line stimulated with TNF alpha. In a group of 44 patients with acute rejection, higher serum pretransplant levels of CXCL1, CXCL5, CXCL6, CCL2, CCL21, and particularly CXCL10 and CX3CL1(both p < 0.001) were found suggesting their higher proinflammatory status as compared to subjects with the uncomplicated outcome. In samples collected at the day of biopsy positive for acute rejection, chemokines CXCL9 and CXCL11 attracting preferentially Th1 lymphocytes were found to be upregulated. In our in vitro model with TNF alpha induction, renal proximal epithelial cells seemed to be a more potent source of chemokines attracting neutrophils as compared to monocyte/macrophage cell line but the coculture of these cells potentiated release of neutrophilic chemokines CXCL5 and CXCL6. Similar augmentation of chemokine production was found also in the case of CCL2. On the other hand, adding of monocytes/macrophages to a culture of renal epithelial cells suppressed the release of CXCL10 and CXCL11 attracting T lymphocytes. We assume from our data that in kidney allograft transplantation, chemokines attracting neutrophils, T lymphocytes, and monocytes are induced simultaneously and measurement some of them in combination might be used as biomarkers of acute rejection. Mutual cell-cell interactions of immune cells with renal parenchyma seem to be important for fine regulation of chemokine release.

The long noncoding RNA Ptprd-IR is a novel molecular target for TGF-β1-mediated nephritis.

The role of microRNAs (miRNAs) in chronic kidney disease (CKD) is relatively well established, but much less is known about the role(s) of long noncoding RNAs (lncRNAs). Transforming growth factor β1 (TGF-β1) mediates inflammatory and fibrogenic signaling in CKD via the transcription factor Smad3; however, the extent of lncRNAs-based regulation of TGF-β1 signaling in CKD remains unknown. Herein, we identified np_4334, a lncRNA we named Ptprd-IR, whose promoter contains a highly-conserved site for Smad3 binding. Smad3 knockout (KO) eliminated Ptprd-IR upregulation in a murine model of obstructive nephropathy. Furthermore, Ptprd-IR KO in renal tubular epithelial cell cultures blocked TGF-β1- and interleukin-1β (IL-1β)-mediated NF-κB inflammatory signaling but did not impact TGF-β1-triggered Smad3 pathway activity and fibrosis. Accordingly, Ptprd-IR overexpression (OE) upregulated TGF-β1- and IL-1β-mediated NF-κB pathway activation and production of pro-inflammatory cytokines but did not influence TGF-β1-mediated fibrogenic signaling. Additionally, transfection of obstructed kidneys with Ptprd-IR-directed shRNA attenuated the inflammatory response via NF-κB but did not impact TGF-β1/Smad3-mediated fibrogenesis. Overall, our findings demonstrate that the lncRNA Ptprd-IR stimulates the inflammatory response in kidneys and advocate Ptprd-IR as a possible therapeutic target for CKD.