The Human PNPLA3 I148M Polymorphism Alters the Renal Kinome and Transporters to Cause Hyperfiltration and Hypertension

Figures

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  FIGURE 1. Pnpla3I148M polymorphism leads to renal hyperfiltration and injury. Direct glomerular filtration rate (GFR) assessment by FITC‐Sinistrin in (A) males (D) females. Indirect GFR assessment by plasma cystatin C in (B) males (E) females. Urinary albumin to creatinine ratio (UACr) assessment in (C) males (F) females. Data are expressed as mean ± SEM of 10–15 mice/group. A two‐way ANOVA was performed to detect differences.

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  FIGURE 2. Hyperfiltration is due to increased expression of sodium transporters in renal cortex. Representative western blots and quantification of sodium‐hydrogen exchanger 3 (NHE3) in (A) males and (D) females. Representative western blots and quantification of sodium potassium ATPase (Na+/K+‐ATPase) in (B) males and (E) females. Representative western blots and quantification of the alpha subunit of epithelial sodium channel (α‐ENaC) in (C) males and (F) females. Data are expressed as mean ± SEM of 8–15 mice/group. A two‐way ANOVA was performed to detect differences.

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  FIGURE 3. Pnpla3I148M mice show alterations in cardiac function. Echocardiographic analysis of the heart in B and M mode was used to determine Ejection fraction in (A) males (I) females. Fractional shortening in (B) males (J) Females. Stroke volume in (C) males (K) females. Cardiac output in (D) males (L) Females. Data from B and M modes were averaged for each mouse and then for each group. Echocardiographic analysis of the heart in M mode was used to determine End systolic volume in (E) males (M) females. End diastolic volume in (F) males (N) females. Left ventricular internal dimension (LVID) at systole in (G) males (O) females. LVID at diastole in (H) males (P) females. Data are expressed as mean ± SEM of 10–15 mice/group. A two‐way ANOVA was performed to detect differences.

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  FIGURE 4. Pnpla3I148M is hypertensive irrespective of diet. Blood pressure measured in (A) males and (B) females. Blood pressure was measured by fluid‐filled catheter in conscious, unrestrained mice for 4 h on two consecutive days and then averaged. Data are expressed as mean ± SEM of 5–9 mice/group. A two‐way ANOVA was performed to detect differences.

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  FIGURE 5. HSD Pnpla3I148M mice have increased renal lipid storage and injury. Renal cortical triglyceride levels in (A) male and (C) female mice measured by ELISA. Representative image (20× magnification) and quantification of periodic acid–Schiff staining in kidney of (B) male and (C) female mice using the Mantra 2 quantitative pathology imaging microscope. Data are expressed as mean ± SEM of 5–7 mice/group. A two‐way ANOVA was performed to detect differences.

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  FIGURE 6. Pnpla3I148M polymorphism alters serine–threonine kinases in a sex and diet‐dependent manner. Volcano plot of top 15 altered serine/threonine kinases (STK) in renal cortex of male (A, B) and female (G, H) Pnpla3I148M mice compared to controls. Data are expressed as fold change compared to wild‐type (CTL) mice. n = 5/group. Quantification of STK by MEOW plots comparing male (C, D) and female (G, H) Pnpla3I148M mice compared to control. The blue line represents the average of the activity for each subfamily of kinases (E). Representative western blot and quantification of phosphorylated PKCα (Th638) to total PKCα in male mice. (F) Representative western blot and quantification of phosphorylated PKCα (Th497) to total PKCα in male mice. Data are expressed as mean ± SEM of 10–12 mice/group. A two‐way ANOVA was performed to detect differences.