GRK2 knockdown in mice exacerbates kidney injury and alters renal mechanisms of blood pressure regulation

Figures

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  Figure 1. GRK2 knockdown alters kidney morphometry and reduces glomerular filtration rate (GFR). (a) Representative images of right and left kidneys from 1.5-, 3- and 6-month-old shGRK2 and age-matched C57Bl/6 mice. (b) Mean ± S.E.M of kidney mass (right & left) relative to tibia length of shGRK2 and C57Bl/6 mice at 1.5, 3 and 6 months of age (n = 6). (c) Mean ± S.E.M of organ mass (heart, lungs, kidneys, and liver) relative to body weight in 3-month-old shGRK2 and age-matched C57Bl/6 mice (n = 6). (d) Mean ± S.E.M of glomerular filtration rate (GFR) measured by FITC-inulin clearance in 6-month-old shGRK2 and age-matched control C57Bl/6 mice (n = 6). * denotes significantly different at (p < 0.05, unpaired Student’s t test).

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  Figure 2. GRK2 knockdown alters glomerular maturation and count. (a) Representative images of Periodic-Acid Schiff stained histological kidney sections and mean ± S.E.M of the number of mature glomeruli in low power field (LPF) from postnatal day 7 (P7) shGRK2 and age-matched C57Bl/6 mice. Scale bar is 20 µm (n = 6). (b) Representative images of H&E stained histological kidney sections and mean ± S.E.M of the number of mature glomeruli/mm2 from 6-month-old shGRK2 and age-matched C57Bl/6 mice. Scale bar is 100 µm (n = 6). * denotes significantly different at (p < 0.05, unpaired Student’s t test).

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  Figure 3. Expression of GRK3, 4, 5 and 6 is not changed in shGRK2 kidneys. Representative western blots and mean ± S.E.M for the densiometric analysis of (a) GRK2, (b) GRK3, (c) GRK4, (d) GRK5, and (e) GRK6 expression with the corresponding loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or actin in renal cortex of 6-month-old shGKR2 and age-matched C57Bl/6 mice (n = 5–6). GRK band intensity was normalized to corresponding actin (for GRK2) or GAPDH (for GRK3, 4, 5, 6) band and expressed as a fraction of the C57Bl/6 value. * denotes significantly different at (p < 0.05, unpaired Student’s t test).

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  Figure 4. GRK2 knockdown increases renal and plasma renin expression. Mean ± S.E.M showing folds change in (a) renal angiotensinogen and (b) renin mRNA expression from 6-month-old shGRK2 over age-matched C57Bl/6 mice. Expressions levels are normalized to β-actin (n = 6). (c) Representative western blots and mean ± S.E.M for the densiometric analysis of renin protein expression with the corresponding loading control Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in renal cortex of 6-month-old shGKR2 and age-matched C57Bl/6 mice (n = 6). Renin band intensity was normalized to corresponding GAPDH band intensity and expressed as a fraction of the C57Bl/6 value (n = 6). (d) Mean ± S.E.M of plasma renin levels in 6-month-old shGRK2 and age-matched C57Bl/6 mice measured by Mouse renin ELISA kit as per manufactures’ instructions (n = 6). * denotes significantly different at (p < 0.05, unpaired Student’s t test).

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  Figure 5. ROS production and glomerular injury are exacerbated in shGRK2 mice. Mean ± S.E.M for renal cortex levels (nmol/µmol) of (a) ethidium (Eth) and (b) 2-hydroxydihydroethidium (2OH-Eth) after incubation with dihydroethidium (DHE) and separation by high-performance liquid chromatography (HPLC). Eth and 2OH-Eth levels were normalized to dihydroethidium (DHE) levels. (c) Representative images of Periodic-Acid Schiff stained glomerulus from 6-month-old shGRK2 and age-matched C57Bl/6.Scale bar is 20 µm. Mean ± S.E.M of the glomerular injury score based on mesangial matrix area done by trained pathologist in blinded manner (n = 6). * denotes significantly different at (p < 0.05, unpaired Student’s t test).

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  Figure 6. AT1R affinity and density are not changed in shGRK2 kidneys. (a) Representative saturation binding curves and (b) mean ± S.E.M of binding affinities (Kd, nM) and maximal binding capacities (Bmax, fmol/mg protein) for [125I]-Ang II in renal cortex of 6-month-old shGRK2 mice and age-matched C57Bl/6 mice (n = 4 independent experiments run in duplicates). Lack of significant difference was verified using unpaired Student’s t test.

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  Figure 7. Losartan mitigates the hypertensive phenotype and impaired GFR in shGRK2 mice. Mean ± S.E.M of systolic (left) and diastolic blood pressure (right) from 6-month-old C57Bl/6 (a) and age-matched shGRK2 (b) using the non-invasive CODA tail-cuff blood pressure system. Blood pressure values were measured before treatment start (baseline), weekly during the 4-week period of treatment (with either plain drinking water or 100 mg/kg/day Losartan in drinking water), and 3 weeks after washout ended (washout) (n = 3–6). * denotes significantly different from corresponding plain water value at (p < 0.05, two-way ANOVA). (c) Mean ± S.E.M of mean arterial blood pressure for 6-month-old shGRK2 and age-matched C57Bl/6 mice before (baseline) and 4 weeks after treatment with either plain drinking water or Losartan (n = 3–6). # denotes significantly different at (p < 0.05, two-way ANOVA) from baseline C57Bl/6 receiving plain water. * denotes significantly different from corresponding plain water value at (p < 0.05, two-way ANOVA). (d) Mean ± S.E.M of glomerular filtration rate (GFR) measured by FITC-inulin clearance in 6-month-old shGRK2 and age-matched control C57Bl/6 mice after 4 weeks of…