658
CORRESPONDENCE
HEPATOLOGY, August 2015
In conclusion, our observation extends the findings by Liu et al. by demonstrating that autosis occurs in patients with a severe liver disease. This is the first observation of autosis in humans. Acknowledgment: The authors thank Adel Hammoutene and Alain Schmitt (Cochin electron microscopy core facility, Paris) for their help in electron microscopy analyses. MAROUANE KHELOUFI, M.SC.1 CHANTAL M. BOULANGER, PH.D.1 PATRICE CODOGNO, PH.D.2 PIERRE-EMMANUEL RAUTOU, M.D., PH.D.1,3 1 INSERM UMR-S970 Universite Paris Descartes Sorbonne Paris Cit e Paris, France 2 INSERM U1151 Institut Necker Enfants-Malades-INEM Paris, France 3 DHU Unity Service d’He patologie H^ opital Beaujon Assistance Publique-H^ opitaux de Paris Clichy, France
Fig. 1. Hepatocytes from patients with severe anorexia nervosa contain features typical of autosis. On the lower right panel, the low amount of material in the perinuclear space suggests that this cell may be in transition from phase Ib to phase 2. White arrows point to autophagic vacuoles, black arrows show dilated endoplasmic reticulum, and arrowheads indicate swollen perinuclear space. N, nucleus; M, mitochondria. endoplasmic reticulum, and numerous autophagosomes, autolysosomes, and empty vacuoles. Phase 1b morphological features were also present in all patients. Indeed, the perinuclear space of hepatocytes was swollen at discrete regions surrounding the inner nuclear membrane, and these swollen areas contained membrane-bound regions with a density and granularity resembling the cytosol. However, we did not see phase 2 autosis features in our specimens. This could be owing to the fact that phase 2 is short-lasting (15-20 minutes), whereas phase 1 is more sustained (lasting a few hours). The probability of observing phase 2 features is thus low. Transaminases are intracellular enzymes mainly contained in hepatocytes. The fact that autosis is associated with plasma membrane permeability could account for the marked increase in serum transaminases we observed in our patients. Liu et al. found that cardiac glycosides (i.e., antagonists of Na1,K1-ATPase) inhibited autotic cell death both in vitro and in vivo. Impaired cardiac function can occur in patients with severe anorexia nervosa and lead to heart failure.2 If autosis occurs not only in the liver, but also in the heart of these patients, we can speculate that cardiac glycosides might be particularly useful in this setting.
References 1. Liu Y, Shoji-Kawata S, Sumpter RM, Jr., Wei Y, Ginet V, Zhang L, et al. Autosis is a Na1,K1-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proc Natl Acad Sci U S A 2013;110:20364-20371. 2. Rautou PE, Cazals-Hatem D, Moreau R, Francoz C, Feldmann G, Lebrec D, et al. Acute liver cell damage in patients with anorexia nervosa: a possible role of starvation-induced hepatocyte autophagy. Gastroenterology 2008;135:840-848. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27597 Financial Support: This work was supported by the Fondation pour la Recherche M e dicale (DPC20111122979) and the Agence Nationale pour la Recherche (ANR-12-EMMA-0012-03) Potential conflict of interest: Nothing to report.
PNPLA3 rs738409 and TM6SF2 rs58542926 Gene Variants Affect Renal Disease and Function in Nonalcoholic Fatty Liver Disease To the Editor: We read with interest the article by Dongiovanni et al.1 reporting on the impact of the rs58542926 C>T variant of the transmembrane 6 superfamily member 2 gene (TM6SF2) on liver histology and cardiovascular disease (CVD) risk in NAFLD. Elucidating mechanisms connecting NAFLD to CVD would have important preventive and therapeutic implications in these patients. Chronic kidney disease (CKD) is a frequent, underappreciated condition and an established risk factor for CVD.2 In a recent meta-analysis of observational studies, the presence and severity of NAFLD at baseline were associated with an increased incidence and stage of CKD, independently of traditional risk factors.3
CKD might be a mediator or a marker of an increased CVD risk in NAFLD. Thus, we explored the impact of the two polymorphisms in PNPLA3 and TM6SF2 genes, both modulating liver disease severity and CVD risk in NAFLD, on renal function in a cohort of 202 nonobese nondiabetic subjects (61 with biopsyproven noncirrhotic NAFLD; Table 1). CKD, estimated glomerular filtration rate (eGFR), and microalbuminuria were defined according to current Kidney Disease: Improving Global Outcomes (KDIGO) guidelines (see legend to Table 1).2 We found that the presence of PNPLA3 G allele was associated with lower eGFR and with a higher prevalence of microalbuminuria and CKD (Table 1). Conversely, the TM6SF2 T allele was associated with higher eGFR and with a lower prevalence of albuminuria and
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CORRESPONDENCE
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Table 1. Main Characteristics of Patients With NAFLD and Controls NAFLD*
Age, years Sex, %males BMI, kg/m2 Systolic BP, mmHg Diastolic BP, mmHg Waist, cm Tg, mg/dL LDL-C, mg/dL HDL-C, mg/dL Total C, mg/dL Glucose, mg/dL Insulin, mU/mL HOMA-IR AST, U/L ALT, U/L C-reactive Protein, ng/mL Metabolic syndrome, % Histological steatosis, % hepatocytes* Necroinflammatory grade* Fibrosis stage* NASH, %* TM6SF2 CC, % CT/TT, % eGFR, mL/min/1.73 m2) Microalbuminuria, % CKD, %
Controls
PNPLA3 CC (n 5 30)
PNPLA3 CG/GG (n 5 31)
P NAFLD
PNPLA3 CC (n 5 60)
PNPLA3 CG/GG (n 5 81)
P Controls
49 6 3 63 25.7 6 1.5 130 6 5 86 6 6† 90 6 4 100 6 32 111 6 8 51 6 3† 178 6 10 97 6 3 13.3 6 2.6† 3.40 6 1.88† 42 6 3 71 6 5 1.6 6 0.2 37† 16 6 10
48 6 4 64 25.9 6 1.4 131 6 6 88 6 6† 92 6 4 102 6 34 119 6 7 48 6 2† 186 6 10 99 6 4 17.4 6 3.7† 4.68 6 2.6† 59 6 4 89 6 6 2.8 6 0.2† 44† 32 6 7
0.892 0.713 0.785 0.926 0.262 0.218 0.389 0.562 0.139 0.569 0.598 0.318 0.118 0.001 0.025 0.006 0.412 0.022
49 6 3 68 25.8 6 1.7 130 6 6 81 6 5 91 6 4 105 6 41 129 6 9 59 6 2 199 6 12 97 6 5 7.6 6 1.6 1.68 6 1.11 15 6 3 16 6 4 1.1 6 0.3 12 —
39 6 4 70 26.2 6 1.3 131 6 8 82 6 6 90 6 4 107 6 42 137 6 9 60 6 2 193 6 13 98 6 6 7.5 6 1.7 1.76 6 0.98 16 6 4 17 6 3 1.8 6 0.2 11 —
0.889 0.815 0.314 0.813 0.887 0.235 0.964 0.317 0.911 0.587 0.713 0.713 0.369 0.815 0.613 0.042 0.835 —
1.7 6 0.3 1.0 6 0.4 33
2.2 6 0.4 1.9 6 0.4 64
0.028 0.038 0.039
— — —
— — —
— — —
82 18 79 6 11 0 0
83 17 66 6 8‡ 26† 29†
0.771 0.816 0.001 0.009 0.005
85 15 96 6 10 0 0
84 16 69 6 9 10 10
0.812 0.913 0.041 0.032 0.032
Data are presented as mean 6 SEM. Differences between groups were analyzed by ANOVA for normal variables; otherwise, Mann-Whitney’s test was used for nonparametric variables. Normality was evaluated by Shapiro-Wilk’s test. Fisher’s or chi square test were used to compare categorical variables, as appropriate. Differences were considered statistically significant at P < 0.05. Analysis of different parameters and of genetic polymorphisms was made using Spearman’s correlation test. Genetic polymorphisms were modeled as an additive effect, that is, quantitative predictor variables reflecting the number of risk alleles (0, 1, or 2). When a relation was found on univariate analysis, multiple linear regression and logistic regression analyses were used to estimate relationships between different variables, after log transformation of skewed data. Microalbuminuria was defined by an albumin-creatinine ratio of 30-300 mg/mg on fresh morning urine sample. *40 NAFLD patients with liver biopsy. † P < 0.05 versus controls. ‡ P < 0.05 versus controls with PNPLA3 CC genotype. Abbreviations: BP, blood pressure; C, cholesterol; CKD, chronic kidney disease, defined by persistent (>3 months) albuminuria and/or eGFR
CORRESPONDENCE
HEPATOLOGY, August 2015
In conclusion, our observation extends the findings by Liu et al. by demonstrating that autosis occurs in patients with a severe liver disease. This is the first observation of autosis in humans. Acknowledgment: The authors thank Adel Hammoutene and Alain Schmitt (Cochin electron microscopy core facility, Paris) for their help in electron microscopy analyses. MAROUANE KHELOUFI, M.SC.1 CHANTAL M. BOULANGER, PH.D.1 PATRICE CODOGNO, PH.D.2 PIERRE-EMMANUEL RAUTOU, M.D., PH.D.1,3 1 INSERM UMR-S970 Universite Paris Descartes Sorbonne Paris Cit e Paris, France 2 INSERM U1151 Institut Necker Enfants-Malades-INEM Paris, France 3 DHU Unity Service d’He patologie H^ opital Beaujon Assistance Publique-H^ opitaux de Paris Clichy, France
Fig. 1. Hepatocytes from patients with severe anorexia nervosa contain features typical of autosis. On the lower right panel, the low amount of material in the perinuclear space suggests that this cell may be in transition from phase Ib to phase 2. White arrows point to autophagic vacuoles, black arrows show dilated endoplasmic reticulum, and arrowheads indicate swollen perinuclear space. N, nucleus; M, mitochondria. endoplasmic reticulum, and numerous autophagosomes, autolysosomes, and empty vacuoles. Phase 1b morphological features were also present in all patients. Indeed, the perinuclear space of hepatocytes was swollen at discrete regions surrounding the inner nuclear membrane, and these swollen areas contained membrane-bound regions with a density and granularity resembling the cytosol. However, we did not see phase 2 autosis features in our specimens. This could be owing to the fact that phase 2 is short-lasting (15-20 minutes), whereas phase 1 is more sustained (lasting a few hours). The probability of observing phase 2 features is thus low. Transaminases are intracellular enzymes mainly contained in hepatocytes. The fact that autosis is associated with plasma membrane permeability could account for the marked increase in serum transaminases we observed in our patients. Liu et al. found that cardiac glycosides (i.e., antagonists of Na1,K1-ATPase) inhibited autotic cell death both in vitro and in vivo. Impaired cardiac function can occur in patients with severe anorexia nervosa and lead to heart failure.2 If autosis occurs not only in the liver, but also in the heart of these patients, we can speculate that cardiac glycosides might be particularly useful in this setting.
References 1. Liu Y, Shoji-Kawata S, Sumpter RM, Jr., Wei Y, Ginet V, Zhang L, et al. Autosis is a Na1,K1-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proc Natl Acad Sci U S A 2013;110:20364-20371. 2. Rautou PE, Cazals-Hatem D, Moreau R, Francoz C, Feldmann G, Lebrec D, et al. Acute liver cell damage in patients with anorexia nervosa: a possible role of starvation-induced hepatocyte autophagy. Gastroenterology 2008;135:840-848. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27597 Financial Support: This work was supported by the Fondation pour la Recherche M e dicale (DPC20111122979) and the Agence Nationale pour la Recherche (ANR-12-EMMA-0012-03) Potential conflict of interest: Nothing to report.
PNPLA3 rs738409 and TM6SF2 rs58542926 Gene Variants Affect Renal Disease and Function in Nonalcoholic Fatty Liver Disease To the Editor: We read with interest the article by Dongiovanni et al.1 reporting on the impact of the rs58542926 C>T variant of the transmembrane 6 superfamily member 2 gene (TM6SF2) on liver histology and cardiovascular disease (CVD) risk in NAFLD. Elucidating mechanisms connecting NAFLD to CVD would have important preventive and therapeutic implications in these patients. Chronic kidney disease (CKD) is a frequent, underappreciated condition and an established risk factor for CVD.2 In a recent meta-analysis of observational studies, the presence and severity of NAFLD at baseline were associated with an increased incidence and stage of CKD, independently of traditional risk factors.3
CKD might be a mediator or a marker of an increased CVD risk in NAFLD. Thus, we explored the impact of the two polymorphisms in PNPLA3 and TM6SF2 genes, both modulating liver disease severity and CVD risk in NAFLD, on renal function in a cohort of 202 nonobese nondiabetic subjects (61 with biopsyproven noncirrhotic NAFLD; Table 1). CKD, estimated glomerular filtration rate (eGFR), and microalbuminuria were defined according to current Kidney Disease: Improving Global Outcomes (KDIGO) guidelines (see legend to Table 1).2 We found that the presence of PNPLA3 G allele was associated with lower eGFR and with a higher prevalence of microalbuminuria and CKD (Table 1). Conversely, the TM6SF2 T allele was associated with higher eGFR and with a lower prevalence of albuminuria and
HEPATOLOGY, Vol. 62, No. 2, 2015
CORRESPONDENCE
659
Table 1. Main Characteristics of Patients With NAFLD and Controls NAFLD*
Age, years Sex, %males BMI, kg/m2 Systolic BP, mmHg Diastolic BP, mmHg Waist, cm Tg, mg/dL LDL-C, mg/dL HDL-C, mg/dL Total C, mg/dL Glucose, mg/dL Insulin, mU/mL HOMA-IR AST, U/L ALT, U/L C-reactive Protein, ng/mL Metabolic syndrome, % Histological steatosis, % hepatocytes* Necroinflammatory grade* Fibrosis stage* NASH, %* TM6SF2 CC, % CT/TT, % eGFR, mL/min/1.73 m2) Microalbuminuria, % CKD, %
Controls
PNPLA3 CC (n 5 30)
PNPLA3 CG/GG (n 5 31)
P NAFLD
PNPLA3 CC (n 5 60)
PNPLA3 CG/GG (n 5 81)
P Controls
49 6 3 63 25.7 6 1.5 130 6 5 86 6 6† 90 6 4 100 6 32 111 6 8 51 6 3† 178 6 10 97 6 3 13.3 6 2.6† 3.40 6 1.88† 42 6 3 71 6 5 1.6 6 0.2 37† 16 6 10
48 6 4 64 25.9 6 1.4 131 6 6 88 6 6† 92 6 4 102 6 34 119 6 7 48 6 2† 186 6 10 99 6 4 17.4 6 3.7† 4.68 6 2.6† 59 6 4 89 6 6 2.8 6 0.2† 44† 32 6 7
0.892 0.713 0.785 0.926 0.262 0.218 0.389 0.562 0.139 0.569 0.598 0.318 0.118 0.001 0.025 0.006 0.412 0.022
49 6 3 68 25.8 6 1.7 130 6 6 81 6 5 91 6 4 105 6 41 129 6 9 59 6 2 199 6 12 97 6 5 7.6 6 1.6 1.68 6 1.11 15 6 3 16 6 4 1.1 6 0.3 12 —
39 6 4 70 26.2 6 1.3 131 6 8 82 6 6 90 6 4 107 6 42 137 6 9 60 6 2 193 6 13 98 6 6 7.5 6 1.7 1.76 6 0.98 16 6 4 17 6 3 1.8 6 0.2 11 —
0.889 0.815 0.314 0.813 0.887 0.235 0.964 0.317 0.911 0.587 0.713 0.713 0.369 0.815 0.613 0.042 0.835 —
1.7 6 0.3 1.0 6 0.4 33
2.2 6 0.4 1.9 6 0.4 64
0.028 0.038 0.039
— — —
— — —
— — —
82 18 79 6 11 0 0
83 17 66 6 8‡ 26† 29†
0.771 0.816 0.001 0.009 0.005
85 15 96 6 10 0 0
84 16 69 6 9 10 10
0.812 0.913 0.041 0.032 0.032
Data are presented as mean 6 SEM. Differences between groups were analyzed by ANOVA for normal variables; otherwise, Mann-Whitney’s test was used for nonparametric variables. Normality was evaluated by Shapiro-Wilk’s test. Fisher’s or chi square test were used to compare categorical variables, as appropriate. Differences were considered statistically significant at P < 0.05. Analysis of different parameters and of genetic polymorphisms was made using Spearman’s correlation test. Genetic polymorphisms were modeled as an additive effect, that is, quantitative predictor variables reflecting the number of risk alleles (0, 1, or 2). When a relation was found on univariate analysis, multiple linear regression and logistic regression analyses were used to estimate relationships between different variables, after log transformation of skewed data. Microalbuminuria was defined by an albumin-creatinine ratio of 30-300 mg/mg on fresh morning urine sample. *40 NAFLD patients with liver biopsy. † P < 0.05 versus controls. ‡ P < 0.05 versus controls with PNPLA3 CC genotype. Abbreviations: BP, blood pressure; C, cholesterol; CKD, chronic kidney disease, defined by persistent (>3 months) albuminuria and/or eGFR
