The Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Nephrotic Syndrome-Associated Hypercholesterolemia

Abstract

Background: In nephrotic syndrome, damage to the podocytes of the kidney produces severe hypercholesterolemia for which novel treatments are urgently needed. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as an important regulator of plasma cholesterol levels and therapeutic target. Here, we tested the role of PCSK9 in mediating the hypercholesterolemia of nephrotic syndrome.

Methods: PCSK9 and plasma lipids were studied in nephrotic syndrome patients before and after remission of disease, mice with genetic ablation of the podocyte (Podocyte Apoptosis Through Targeted Activation of Caspase-8, Pod-ATTAC mice) and mice treated with nephrotoxic serum (NTS), which triggers immune-mediated podocyte damage. In addition, mice with hepatic deletion of Pcsk9 were treated with NTS to determine the contribution of PCSK9 to the dyslipidemia of nephrotic syndrome.

Results: Patients with nephrotic syndrome showed a decrease in plasma cholesterol and plasma PCSK9 on remission of their disease (P<0.05, n=47-50). Conversely, Pod-ATTAC mice and NTS-treated mice showed hypercholesterolemia and a 7- to 24-fold induction in plasma PCSK9. The induction of plasma PCSK9 appeared to be attributable to increased secretion of PCSK9 from the hepatocyte coupled with decreased clearance. Interestingly, knockout of Pcsk9ameliorated the effects of NTS on plasma lipids. Thus, in the presence of NTS, mice lacking hepatic Pcsk9 showed a 40% to 50% decrease in plasma cholesterol and triglycerides. Moreover, the ability of NTS treatment to increase the percentage of low-density lipoprotein-associated cholesterol (from 9% in vehicle-treated Flox mice to 47% after NTS treatment), was lost in mice with hepatic deletion of Pcsk9 (5% in both the presence and absence of NTS).

Conclusions: Podocyte damage triggers marked inductions in plasma PCSK9, and knockout of Pcsk9 ameliorates dyslipidemia in a mouse model of nephrotic syndrome. These data suggest that PCSK9 inhibitors may be beneficial in patients with nephrotic syndrome-associated hypercholesterolemia.

Keywords: PCSK9 protein, human; PCSK9 protein, mouse; cholesterol; hypercholesterolemia; kidney; nephrotic syndrome; podocytes.

Figure 1. Plasma PCSK9 in nephrotic patients

PCSK9 levels in nephrotic patients at baseline (348.0 ± 139.5 ng/mL) and remission (300.5 ± 130.3 ng/mL). Boxes show 25^th-75^th percentiles, whiskers represent 5% - 95% confidence intervals. n = 50, paired two-sided t-test.

Figure 2. Nephrotoxic serum increases plasma PCSK9

Six- to eight-week-old male B6 mice were injected with nephrotoxic serum (NTS) or normal sheep serum (Vehicle) and were sacrificed four days after the initial injection. 2 μL of spot urine collected the morning of sacrifice was subjected to SDS-PAGE and Coomassie Blue staining (A). Plasma taken at the time of sacrifice was used to measure triglycerides (B), total cholesterol (C), and PCSK9 (G), or subjected to size exclusion chromatography for lipoprotein analysis. Brackets indicate the percentage of total cholesterol found in ApoB-containing lipoproteins (VLDL + LDL) (D). Hepatic protein was measured by western blotting liver whole-cell lysates (E, I). Hepatic gene expression was measured by real-time PCR (F, H). n=3-8. For lipoprotein analysis, equal amounts of plasma from 4-6 mice were pooled from each group.

Figure 3. Podocyte apoptosis increases plasma PCSK9

Five- to eleven-week-old mice with (Pod-ATTAC) or without (Control) the Pod-ATTAC transgene were injected with dimerizer AP20187 and sacrificed seven days after injection. 2 μL of spot urine collected the morning of sacrifice was subjected to SDS-PAGE and Coomassie Blue staining (A). Plasma taken at the time of sacrifice was used to measure triglycerides (B), total cholesterol (C) and PCSK9 (G), or subjected to size exclusion chromatography for lipoprotein analysis. Brackets indicate the percentage of total cholesterol found in ApoB-containing lipoproteins (VLDL + LDL) (D). Hepatic protein was measured by western blotting liver whole-cell lysates (E, J). Hepatic gene expression was measured by real-time PCR (F, H). PCSK9 clearance was measured as described in Methods (I). n=8-10. For lipoprotein analysis, equal amounts of plasma from 8-9 mice were pooled from each group.

Figure 4. Knockout of Pcsk9 reduces plasma cholesterol in the presence and absence of NTS

Five- to eight-week-old wild type (WT) or Pcsk9 global knockout (PCSK9-KO) mice were injected with nephrotoxic serum (NTS) or normal sheep serum (Vehicle) and were sacrificed three to four days after the initial injection. Plasma taken at the time of sacrifice was used to measure triglycerides (A), cholesterol (B) and PCSK9 (C) or subjected to size exclusion chromatography for lipoprotein analysis. Brackets indicate the percentage of total cholesterol found in ApoB-containing lipoproteins (VLDL + LDL) (E). Hepatic gene expression was measured by real-time PCR (D, H). Protein levels were measured by western blotting plasma (F) or liver whole-cell lysates (G). n=4-7; * p < 0.05. N.M. not measured. For lipoprotein analysis, equal amounts of plasma from 4-7 mice were pooled from each group.

Figure 5. Liver-specific knockout of Pcsk9 prevents increased LDL cholesterol after NTS treatment

Five- to eight-week-old male Pcsk9^flox/flox (Flox) and liver-specific knockout (L-KO) mice were injected with nephrotoxic serum (NTS) or normal sheep serum (Vehicle) and sacrificed four days after the initial NTS injection. Hepatic gene expression was measured by real-time PCR (A). Plasma collected at the time of sacrifice was used to measure PCSK9 (B), triglycerides (C), and cholesterol (D), or subjected to size exclusion chromatography for lipoprotein analysis. Brackets indicate the percentage of total cholesterol found in ApoB-containing lipoproteins (VLDL + LDL) (E). n= 4-8; * p < 0.05. For lipoprotein analysis, equal amounts of plasma from 4-8 mice were pooled from each group.