Lysophosphatidylcholine mediates fast decline in kidney function in diabetic kidney disease
Authors: Kentaro Yoshioka, Yosuke Hirakawa, Makoto Kurano, Yuko Ube, Yoko Ono, Kensuke Kojima, Taiga Iwama, Kuniyuki Kano, Sho Hasegawa, Tsuyoshi Inoue, Takashi Shimada, Junken Aoki, Yutaka Yatomi, Masaomi Nangaku, Reiko Inagi
Journal: Kidney International (2021)

Some patients with diabetic kidney disease (DKD) show a fast progression of kidney dysfunction and are known as a "fast decliner" (FD). Therefore, it is critical to understand pathomechanisms specific for fast decline. Here, we performed a comprehensive metabolomic analysis of patients with stage G3 DKD and identified increased urinary lysophosphatidylcholine (LPC) in fast decline. This was confirmed by quantification of urinary LPC using mass spectrometry and identified urinary LPC containing saturated fatty acids palmitic (16:0) and stearic (18:0) acids was increased in FDs. The upsurge in urinary LPC levels was correlated with a decline in estimated glomerular filtration rate after 2.5 years. To clarify a pathogenic role of LPC in FD, we studied an accelerated rat model of DKD and observed an increase in LPC (16:0) and (18:0) levels in the urine and kidney tubulointerstitium as the disease progressed. These findings suggested that local dysregulation of lipid metabolism resulted in excessive accumulation of this LPC species in the kidney. Our in vitro studies also confirmed LPC-mediated lipotoxicity in cultured proximal tubular cells. LPC induced accumulation of lipid droplets via activation of peroxisome proliferator-activated receptor-δ followed by upregulation of the lipid droplet membrane protein perilipin 2 and decreased autophagic flux, thereby inducing organelle stress and subsequent apoptosis. Thus, LPC (16:0) and (18:0) may mediate a fast progression of DKD and may serve as a target for novel therapeutic approaches. Some patients with diabetic kidney disease (DKD) show a fast progression of kidney dysfunction and are known as a "fast decliner" (FD). Therefore, it is critical to understand pathomechanisms specific for fast decline. Here, we performed a comprehensive metabolomic analysis of patients with stage G3 DKD and identified increased urinary lysophosphatidylcholine (LPC) in fast decline. This was confirmed by quantification of urinary LPC using mass spectrometry and identified urinary LPC containing saturated fatty acids palmitic (16:0) and stearic (18:0) acids was increased in FDs. The upsurge in urinary LPC levels was correlated with a decline in estimated glomerular filtration rate after 2.5 years. To clarify a pathogenic role of LPC in FD, we studied an accelerated rat model of DKD and observed an increase in LPC (16:0) and (18:0) levels in the urine and kidney tubulointerstitium as the disease progressed. These findings suggested that local dysregulation of lipid metabolism resulted in excessive accumulation of this LPC species in the kidney. Our in vitro studies also confirmed LPC-mediated lipotoxicity in cultured proximal tubular cells. LPC induced accumulation of lipid droplets via activation of peroxisome proliferator-activated receptor-δ followed by upregulation of the lipid droplet membrane protein perilipin 2 and decreased autophagic flux, thereby inducing organelle stress and subsequent apoptosis. Thus, LPC (16:0) and (18:0) may mediate a fast progression of DKD and may serve as a target for novel therapeutic approaches. see commentary on page 454Translational StatementOur metabolomic analysis of a prospective cohort of patients with diabetic kidney disease (DKD) and animal studies utilizing an accelerated model of DKD revealed changes of urinary lysophosphatidylcholine (LPC) levels in a fast progression of DKD. Imaging mass spectrometry further revealed dysregulated metabolism of LPC species in the tubulointerstitium. LPC-treated cultured tubular cells showed abnormal lipid metabolism, followed by impaired autophagic flux and organelle stress through the activation of the peroxisome proliferator–activated receptor δ–perilipin 2 axis, demonstrating a novel pathophysiological role of LPC. Our results provide a foundation for elucidating the pathogenesis of DKD and for developing new therapies for preventing a fast progression of DKD. see commentary on page 454 Our metabolomic analysis of a prospective cohort of patients with diabetic kidney disease (DKD) and animal studies utilizing an accelerated model of DKD revealed changes of urinary lysophosphatidylcholine (LPC) levels in a fast progression of DKD. Imaging mass spectrometry further revealed dysregulated metabolism of LPC species in the tubulointerstitium. LPC-treated cultured tubular cells showed abnormal lipid metabolism, followed by impaired autophagic flux and organelle stress through the activation of the peroxisome proliferator–activated receptor δ–perilipin 2 axis, demonstrating a novel pathophysiological role of LPC. Our results provide a foundation for elucidating the pathogenesis of DKD and for developing new therapies for preventing a fast progression of DKD. The prevalence of diabetic kidney disease (DKD) is increasing worldwide.1Cho N.H. Shaw J.E. Karuranga S. et al.IDF Diabetes Atlas: global estimates of diabetes prevalence for 2017 and projections for 2045.Diabetes Res Clin Pract. 2018; 138: 271-281Google Scholar Despite improvements in glycemic control and blood pressure management with renin-angiotensin system blockade, the current therapy cannot completely halt DKD progression to end-stage kidney disease. Analysis of the Global Burden of Disease study data showed that the global incidence of chronic kidney disease increased by 89%, prevalence increased by 87%, death due to chronic kidney disease increased by 98%, and disability-adjusted life years increased by 62% from 1990 to 2016.2Xie Y. Bowe B. Mokdad A.H. et al.Analysis of the Global Burden of Disease study highlights the global, regional, and national trends of chronic kidney disease epidemiology from 1990 to 2016.Kidney Int. 2018; 94: 567-581Google Scholar Decomposition analyses showed that approximately one-half of the increase in chronic kidney disease disability-adjusted life years was driven by the burden of DKD. Pathogenesis of DKD is multifactorial and includes both metabolic and hemodynamic factors. Glomerular hyperfiltration, oxidative stress, hypoxia of the kidney, advanced glycation end products, activation of intracellular signaling pathways, dysregulated autophagy, and epigenetic changes can result in kidney inflammation and fibrosis in DKD.3Sugahara M. Pak W.L.W. Tanaka T. et al.Update on diagnosis, pathophysiology, and management of diabetic kidney disease.Nephrology. 2021; : 491-500Google Scholar Previous studies focused on glomerular damages in the pathogenesis of DKD. However, accumulating evidence of kidney protection by sodium glucose co-transporter 2 (SGLT2) inhibition emphasizes a crucial role of tubular damage because SGLT2 is exclusively localized in proximal tubules.4Tanaka S. Sugiura Y. Saito H. et al.Sodium–glucose cotransporter 2 inhibition normalizes glucose metabolism and suppresses oxidative stress in the kidneys of diabetic mice.Kidney Int. 2018; 94: 912-925Google Scholar Metabolic derangements result in tubular damage in DKD. Particularly, dysregulation of mitochondrial metabolism in the kidney leads to the development and progression of DKD.5Galvan D.L. Long J. Green N. et al.Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice.J Clin Invest. 2019; 129: 2807-2823Google Scholar, 6Wang W. Wang Y. Long J. et al.Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells.Cell Metab. 2012; 15: 186-200Google Scholar, 7Qi W. Keenan H.A. Li Q. et al.Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.Nat Med. 2017; 23: 753-762Google Scholar Energy supply from the kidney mitochondria majorly depends on lipid metabolism, with programmed enzyme systems in fatty acid β-oxidation and Krebs cycle. On the other hand, abnormal mitochondrial metabolism causes aberrant glucose and lipid metabolism, leading to the production of metabolites that are toxic to the kidney. In diabetic patients, in addition to blood glucose levels, dyslipidemia is often observed and is currently thought to contribute to proximal tubular cell damage and DKD.8Kume S. Maegawa H. Lipotoxicity, nutrient-sensing signals, and autophagy in diabetic nephropathy.JMA J. 2020; 3: 87-94Google Scholar Furthermore, lipotoxicity, which occurs because of intracellular accumulation of cholesterol or sphingolipids, highlights the pathophysiological impact of abnormalities in lipid metabolism on the progression of DKD.9Mitrofanova A. Sosa M.A. Fornoni A. Lipid mediators of insulin signaling in diabetic kidney disease.Am J Physiol Ren Physiol. 2019; 317: F1241-F1252Google Scholar Some patients with DKD show a fast progression of decline in kidney function, and these patients are known as "fast decliners" (FDs).10Yoshida Y. Kashiwabara K. Hirakawa Y. et al.Conditions, pathogenesis, and progression of diabetic kidney disease and early decliner in Japan.BMJ Open Diabetes Res Care. 2020; 8: 1-9Google Scholar It is important to understand the pathogenesis of FD as the mediator of the fast progression of DKD should be a good therapeutic target to individualize medical approach in high-risk patients.11Tofte N. Persson F. Rossing P. Omics research in diabetic kidney disease: new biomarker dimensions and new understandings?.J Nephrol. 2020; 33: 931-948Google Scholar,12Tuttle K.R. Bakris G.L. Bilous R.W. et al.Diabetic kidney disease: a report from an ADA consensus conference.Diabetes Care. 2014; 37: 2864-2883Google Scholar In this study, we performed comprehensive metabolomic analysis of the plasma and urine of stage G3 DKD patients to identify novel metabolites to mediate FD. Our animal experiments and in vitro studies revealed the role of the identified metabolites in the pathogenesis of the fast progression of DKD. The detailed methods are described in the Supplementary Methods. The clinical cohort study was approved by the Institutional Review Board at the University of Tokyo Graduate School of Medicine, Tokyo, Japan (approval number 10660) and adhered to the Declaration of Helsinki. All patients provided written informed consent to participate before inclusion in the study. Animal experiments were conducted at Ina Research, Inc. (Nagano, Japan), in accordance with the National Institutes of Health guidelines and the Standards for Proper Conduct of Animal Experiments, with approval from the Kyowa Kirin Co., Ltd., Institutional Animal Care and Use Committee (approval number 20J0034). We prospectively recruited 150 Asian patients between January 2015 and August 2017 from the University of Tokyo Hospital with stage G3 DKD, whose estimated glomerular filtration rate (eGFR) values were 30 to 60 ml/min per 1.73 m2 at the time of informed consent. Most patients were those with type 2 diabetes. Comorbidity, medical history, and laboratory data were collected from electronic medical records. The baseline inclusion criteria were as follows: subjects aged 20 to 80 years with no evidence of coexistence of nondiabetic kidney disease or cirrhosis, no therapy for systemic cancer within the past 1 year, organ transplantation, or ingestion of steroids within 1 month, and controlled blood pressure and glucose (systolic blood pressure < 170 mm Hg and hemoglobin A1c < 9.5%). Plasma and urine samples from participants were collected at baseline and after 10 months. Kidney function of the participants was evaluated by measuring eGFR over 2.5 years based on the equation for Japanese populations: eGFR = 194 × serum creatinine−1.094 × age−0.287 (for men) or eGFR = 194 × serum creatinine−1.094 × age−0.287 × 0.739 (for women). A threshold of a median eGFR decline rate of 10% per year from baseline was employed to define FD. The median eGFR decline rate was calculated as the average change from baseline to 4 time points every 10 months using least square method. Participants who matched the following criteria were excluded from our analysis: insufficient collected samples (n = 2) and missed appearances in the follow-up period because of hospital transfer or withdrawal of agreement (n = 15). We validated the pathophysiological association between altered lysophosphatidylcholine (LPC) metabolism and DKD progression by comparing the following 3 groups of rats: unilateral nephrectomized spontaneously diabetic Torii fatty (SDTF) rats (SDT fatty/Jcl, male; CLEA Japan, Inc.) fed with 0.2% v/w saline as an accelerated DKD model (SDTF uNx; n = 20); nonnephrectomized SDTF rats as a general progression DKD model (SDTF; n = 10); and Sprague-Dawley rats (Jcl:SD, male; CLEA Japan, Inc.), which are genetically originated from SDTF rats, as a healthy control group (SD; n = 8). All rats had free access to Purina 5008 (Charles River Laboratories Japan) throughout the study. At 6 weeks of age, 30 SDTF rats were randomly divided into 2 groups (20 + 10), and the right kidney was removed from 20 SDTF rats after inducing anesthesia with isoflurane (Mylan, Inc.). One week later, 20 unilateral nephrectomized SDTF rats were continuously fed with 0.2% v/w saline for free intake, and all groups were evaluated for their pathophysiological status 11 weeks after the operation (17 weeks of age). No animals died during the observation period. We obtained HK-2 cells from the American Type Culture Collection. The cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture (D8062; Sigma-Aldrich) containing 10% fetal bovine serum (F7524; Sigma-Aldrich), 50 U/ml penicillin, and 50 mg/mL streptomycin at 37 °C in an atmosphere containing 5% CO2. Human renal proximal tubule epithelial cells (RPTECs; CC-2553; Lonza) were cultured in Renal Epithelial Cell Growth Medium (CC-3191; Lonza) containing 0.5% fetal bovine serum. For LPC treatment on HK-2 cells, the cells were preincubated in the assay medium, which contained Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture with 0.1% fatty acid–free bovine serum albumin (017-15141; Wako), overnight at 37 °C in an atmosphere containing 5% CO2. LPC (16:0) or (18:0) (855675C or 855775C; Avanti Polar Lipids) was preconjugated to fatty acid–free bovine serum albumin at 37 °C for 5 minutes. We applied a metabolomic approach to identify metabolites related to a fast progression of DKD using a prospective cohort from the COI-STREAM project (Figure 1a). A total of 10.5% of subjects were considered as FD patients, as they showed a >10% decline in the eGFR per year (Figure 1b). The baseline characteristics of the subjects are shown in Table 1. Metabolomic analysis of plasma and urine samples at baseline revealed 260 and 198 known metabolites in the plasma and urine, respectively. Among these metabolites, 4 metabolites each in plasma and urine showed significant differences between the FD and non-FD groups (fold ratio > 2 or < 0.5; P < 0.05; Supplementary Table S1). To clarify the association of these metabolites with the fast progression of DKD, we calculated Spearman correlation coefficients. Among the 4 urinary metabolites, only LPC (16:0) in the urine showed the significant correlation with the rate of eGFR decline (Figure 1c). Thus, we further analyzed urinary LPC in subsequent experiments.Table 1Baseline characteristics of recruited patients with DKDCharacteristicsValueNo.Age, yr70.3 ± 6.9133Male sex, %79.7133Systolic blood pressure, mm Hg139.6 ± 15.9133Diastolic blood pressure, mm Hg71.2 ± 10.3133BMI, kg/m225.5 ± 3.6133HDL, mg/dl56.4 ± 16.3129TG, mg/dl153.8 ± 123.5133Fasting glucose, mg/dl131.7 ± 32.6133HbA1c, %6.9 ± 0.6131Hemoglobin, g/dl13.9 ± 1.4133eGFR, ml/min per 1.73 m247.4 ± 7.9133Serum creatinine, mg/dl1.2 ± 0.2133ACR, mg/g creatinine68.7 ± 126.996Urinary protein, mg/g creatinine1130.1 ± 2049.252ACR, albumin-to-creatinine ratio; BMI, body mass index; DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; TG, triglyceride.Values are expressed as mean ± SD, unless otherwise indicated. Open table in a new tab ACR, albumin-to-creatinine ratio; BMI, body mass index; DKD, diabetic kidney disease; eGFR, estimated glomerular filtration rate; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; TG, triglyceride. Values are expressed as mean ± SD, unless otherwise indicated. LPC species are bioactive lipids associated with cardiovascular diseases and diabetes.13Schmitz G. Ruebsaamen K. Metabolism and atherogenic disease association of lysophosphatidylcholine.Atherosclerosis. 2010; 208: 10-18Google Scholar The major species of LPC in mammalian systems, such as 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 20:3, 20:4, 20:5, and 20:6, were quantitatively measured and compared with an internal standard. LPC (16:0) and (18:0) were highly abundant in the baseline urine of >95% of patients with DKD, whereas other LPC species were detected in <50% of subjects (Figure 2a and Supplementary Figure S1A). The concentration of LPC (16:0) and LPC (18:0) in the urine was higher in the FD group (P = 0.019 and P = 0.152, respectively; Figure 2b). More important, both LPC (16:0) and (18:0) were correlated with the rate of eGFR decline after 2.5 years (Figure 2c), but not with eGFR at baseline (Figure 2d). Multiple regression analyses revealed significant association of eGFR change rate with LPC (16:0) or LPC (18:0), even after adjusting with baseline eGFR (Supplementary Table S2). In addition, both LPC (16:0) and (18:0) were positively correlated with baseline serum triglycerides and negatively correlated with baseline serum high-density lipoprotein (Supplementary Table S3). The correlation between increased urinary LPC (16:0) or (18:0) and eGFR decline persisted at 10 months (Supplementary Figure S1B and C). Logistic regression analysis revealed that a high concentration of urinary LPC (16:0), (18:0), or both at baseline predicted fast decline in eGFR (Figure 2e and f and Supplementary Table S4). These results suggest that LPC (16:0) and (18:0) in urine may mediate the fast progression of DKD. Next, we investigated whether equivalent changes of LPC species occurred in a rat model of DKD: wild type (SD), DKD (SD with type 2 diabetes: SDTF), and accelerated DKD rats (unilateral nephrectomized SDTF: SDTF uNx). SDTF rats showed hyperglycemia and dyslipidemia, with particularly increased levels of total cholesterol and triglycerides (Figure 3a–c). In SDTF uNx rats, kidney injury involving decreased creatinine clearance led to an increase in urinary albumin-to-creatinine ratio, tubular injury, and increase in kidney weight. These effects were more severe than that observed in SDTF rats; however, SDTF uNx rats did not exhibit exacerbated hyperglycemia (Figure 3a and d–f and Supplementary Figure S2A and B). Measurement of urinary concentrations of LPC species showed elevated levels of LPC (16:0) and (18:0) in SDTF uNx rats (Figure 3g) compared with other LPC species. Notably, LPC (16:0) and (18:0) were inversely correlated with creatinine clearance (Figure 3h). Taken together, the elevation of urinary LPC, particularly (16:0) and (18:0), indicated fast progression of DKD in rats, which was consistent with our clinical observations in patients with DKD. Next, we investigated whether urinary LPC (16:0) and (18:0) play causative roles in the fast progression of DKD. Increased urinary LPC (16:0) and (18:0), without corresponding changes in the blood, suggested that urinary LPC (16:0) and (18:0) were derived from abnormal lipid metabolism in the kidney. Therefore, we quantified LPC species in the kidney tissue of DKD model rats. Both kidney LPC (16:0) and (18:0) were significantly elevated in SDTF uNx rats (Figure 4a). The levels of these LPC species in the kidney were significantly correlated with those in the urine (Figure 4b). In contrast, the levels of these LPC species in the plasma or liver did not substantially elevate in SDTF uNx rats compared with SDTF rats (Supplementary Figure S2C and D). The proportion of LPC (16:0) and (18:0) components relative to the total LPC species of SDTF uNx rats was higher in the kidney (both >30%) compared with that in the plasma or liver tissue (Figure 4c). Our mass spectrometry imaging analysis revealed increased levels of LPC (16:0) and (18:0) around the tubular interstitial area in SDTF uNx rats compared with SDTF or wild-type SD rats, whereas the levels of these LPC species in the glomerulus did not change among the 3 groups (Figure 4d). These results indicated that LPC (16:0) and (18:0) were markedly elevated in the urine and kidney of SDTF uNx rats because of changes in the local kidney metabolism rather than being a systemic consequence.Figure 4Lysophosphatidylcholine (LPC) (16:0) and (18:0) are ectopically accumulated in kidney tissues of spontaneously diabetic Torii fatty (SDTF) unilateral nephrectomy (uNx) rats. (a) Concentration of kidney LPC species in SD and SDTF rats with or without uNx. P values are only expressed to compare SDTF and SDTF uNx rats of the representative LPC species accounting for >10% of the total LPC species. (b) Spearman correlation coefficients between urinary and kidney LPC (LPC [16:0] and [18:0]) in all SDTF with or without uNx rats. (c) Proportion of LPC (16:0) or (18:0) compared with total LPC species in SDTF uNx rats (significant differences indicate comparisons to kidney). (d) Mass spectrometry (MS) imaging of LPC in the renal cortex of SD and SDTF with or without uNx rats (left, hematoxylin and eosin [HE] staining; middle, LPC [16:0]; right, LPC [18:0]). Black angles indicate the glomerulus. Bar =200 μm, and color bars indicate MS intensity of LPC normalized by the total ion chromatogram. (e–g) Relative mRNA expression levels of LDLR and LOX-1 (e), β-oxidation related genes (f), and PLIN2 (g). (h) Immunohistochemistry of kidney cortex in SD and SDTF with or without uNx rats labeled with anti-PLIN2 antibody. Bar = 50 μm. One-way analysis of variance, followed by the Sidak post hoc test, was used. Values are presented as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005. NS, not significant. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the molecular mechanisms associated with lipid metabolism in the kidney, we evaluated the expression of genes related to lipid metabolism in DKD rat kidney tissues. The expression of low-density lipoprotein receptor (LDLR) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which are responsible for normal and oxidized low-density lipoprotein uptake, respectively, was significantly upregulated in SDTF uNx rats (Figure 4e). Moreover, mitochondrial β-oxidation–related genes, which are important for fatty acid oxidation and maintaining lipid homeostasis, were downregulated in SDTF uNx rats (Figure 4f). We also confirmed that perilipin 2 (PLIN2), the main component of intracellular lipid droplets,14Heid H.W. Moll R. Schwetlick I. et al.Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.Cell Tissue Res. 1998; 294: 309-321Google Scholar was highly upregulated in SDTF uNx rats (Figure 4g). Immunohistologic staining revealed upregulation of PLIN2 in tubular epithelial cells in the cortex (Figure 4h). These results demonstrated that tubular cells were highly susceptible to abnormal lipid metabolism (collectively termed as "lipotoxicity"), which may mediate a fast progression of DKD. To address the pathophysiological implications of elevated LPC species in the kidney, we evaluated the effect of exposing cultured proximal tubular cells to LPC (16:0) or (18:0). We used both RPTECs, primary cultured tubular cells, and HK-2 cells, an immortalized human proximal tubular cell line, and the results were consistent. Cultured proximal tubular cells treated with LPC (16:0) or (18:0) showed intracellular accumulation of these LPC species but not of other LPC species (Figure 5a). Notably, intracellular lipid droplets significantly accumulated only in the cells treated with LPC (16:0) and (18:0) (Figure 5b). When cultured proximal tubular cells were treated with human oxidized low-density lipoprotein or an inhibitor of lysophospholipid acyltransferase (inhibits lysophospholipid metabolism), CI-976,15Chambers K. Judson B. Brown W.J. A unique lysophospholipid acyltransferase (LPAT) antagonist, CI-976, affects secretory and endocytic membrane trafficking pathways.J Cell Sci. 2005; 118: 3061-3071Google Scholar intracellular LPC (16:0) and (18:0) levels also increased (Supplementary Figure S3A and B). These results supported the hypothesis that exposure to exogenous LPC or oxidized low-density lipoprotein, or altered lysophospholipid metabolism, causes intracellular accumulation of LPC and lipotoxicity, as demonstrated in our animal model of accelerated DKD.Figure 5Exposure to lysophosphatidylcholine (LPC) leads to intracellular accumulation and apoptosis. (a) Concentration of intracellular LPC in HK-2 cells. Cells were treated with 60 μM LPC (16:0) and (18:0) for 6 hours (n = 5, each group). (b) Fluorescence images of lipid droplet formation in HK-2 cells treated with 90 μM LPC (16:0) or (18:0) for 24 hours (n = 6). The fluorescence area of lipid droplets was calculated after normalization with that of nucleus. Bar = 20 μm. (c) Cell viability assay of HK-2 cells treated with LPC (18:0) stimulus for 24 hours (n = 4). (d) Caspase 3/7 activity of HK-2 cells treated with 90 μM LPC (16:0) or (18:0) for 24 hours. Caspase activity was measured using the luminescent assay (n = 6). (e) Percentage of apoptotic cells (annexin V+ and 7-amino-actinomycin D [7-AAD]+/–; n = 6). HK-2 cells were treated with 90 μM LPC (18:0) for 24 hours. (f) Relative mRNA levels of endoplasmic reticulum stress-related genes on treatment with LPC (16:0) or LPC (18:0) for 24 hours (n = 4). (g) Immunoblots of c-Jun N-terminal kinase (JNK) phosphorylation on treatment with 90 μM LPC (18:0) for 6 hours (n = 3). (h) Relative mRNA expression of FASL and HRK (n = 4). (i) Relative mRNA levels of CHOP and HRK in renal proximal tubule epithelial cells treated with LPC (18:0) for 24 hours (n = 4). Comparisons were performed by unpaired Student t test or Student t test followed by the Sidak post hoc test. Data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005. BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; GADD34, growth arrest and DNA damage gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, 78-kDa glucose-regulated protein; LD, lipid droplet; p-JNK, phosphorylated JNK. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furthermore, exposure of cultured proximal tubular cells to LPC (16:0) or (18:0) caused severe cytotoxicity in a dose-dependent manner (Figure 5c). Both LPC species also increased caspase 3/7 activity (Figure 5d) and resulted in enhanced cell apoptosis (Figure 5e). An increased level of cytosolic Ca2+ induced by LPC (16:0) or (18:0) may contribute to endoplasmic reticulum (ER) stress-mediated apoptosis16Orrenius S. Zhivotovsky B. Nicotera P. Regulation of cell death: the calcium-apoptosis link.Nat Rev Mol Cell Biol. 2003; 4: 552-565Google Scholar (Supplementary Figure S3C). Further evidence supporting LPC-induced ER stress was confirmed by the upregulation of genes related to unfolded protein response, such as C/EBP homologous protein (CHOP), growth arrest and DNA damage gene (GADD34), and ER luminal binding protein (BiP/GRP78; Figure 5f). LPC (16:0) and (18:0) are structurally similar, and both bind long-chain saturated fatty acids. LPC (16:0) and (18:0) showed similar pathophysiological effects; however, LPC (18:0) showed more potent effects. Therefore, we further examined the molecular mechanism of LPC-mediated lipotoxicity using LPC (18:0) as a representative lipid species. Treatment with LPC (18:0) activated phosphorylation of c-Jun N-terminal kinase protein, which functions downstream of the ER stress response (Figure 5g). Activation of c-Jun N-terminal kinase was associated with upregulation of Fas ligand (FASL) and harakiri (HRK), both of which are c-Jun N-terminal kinase target genes and activate proapoptotic signaling (Figure 5h). The apoptotic signal induction by LPC (18:0) was confirmed in RPTECs, estimated by the increase in CHOP and HRK expressions (Figure 5i). Considering that HRK initiates mitochondria-dependent apoptosis17Cunha D.A. Igoillo-Esteve M. Gurzov E.N. et al.Death protein 5 and p53-upregulated modulator of apoptosis mediate the endoplasmic reticulum stress-mitochondrial dialog triggering lipotoxic rodent and human β-cell apoptosis.Diabetes. 2012; 61: 2763-2775Google Scholar and ER stress is linked to mitochondrial dysfunction, we evaluated the effect of intracellular LPC on the mitochondrial function of tubular cells. LPC (18:0) treatment increased mitochondrial reactive oxygen species in a dose-dependent manner (Figure 6a). Energy metabolism studies were performed with RPTECs because of concerns regarding abnormal cellular metabolism in immortalized cells and revealed that LPC (18:0) treatment severely impaired the mitochondrial oxygen consumption ratio in cultured proximal tubular cells (Figure 6b); moreover, adenosine triphosphate production and the calculated basal and maximal respiration rates were also decreased by LPC treatment. Mitochondrial membrane permeability also increased on LPC (18:0) treatme