PCSK9Q?-003 Vaccine Ameliorates Renal Fibrosis And The Mechanistic Study

Part?The effectiveness experiment of PCSK9Q?-003 vaccine in renal fibrosis mice Objective: Recently,our group has developed a therapeutic vaccine against Proprotein convertase subtilisin/kexin type 9(PCSK9)named PCSK9Q?-003.To explore the possibility of PCSK9Q?-003 vaccine in ameliorating renal fibrosis,the LDLR+/-mice were used to establish renal fibrosis model similar to human chronic kidney disease by using unilateral ureteral obstruction(UUO)and N-nitro-L-arginine methyl ester(L-NAME).Method: The PCSK9-003 peptide was covalently conjugated to Q?-2aa VLPs using the Sulfo-SMCC cross-linker to produce the PCSK9Q?-003 vaccine.The LDLR+/-male mice fed with a high-cholesterol(1%)western diet were randomly assigned into four groups: the sham group,the phosphate-buffer saline group(PBS),the Q? virus-like particles group(VLP)and the PCSK9Q?-003 vaccine group.Mice of the PCSK9Q?-003 group were injected with the PCSK9Q?-003 vaccine(100 ?g/time)every two or four weeks.The UUO mice model was established for 2 weeks at week 20.The mice were weighed each week.PCSK9-003-specific antibody titers were detected every two weeks.At week 22,urine samples were collected using metabolic cages and the supernatant was used forexamination of the 24-hour urinary protein.At the end of the experiment,the plasma samples were used for the measurement of creatinine(Cr),blood urea nitrogen(BUN)and renal tissues for histopathology detection.Furthermore,the other model of renal fibrosis induced by N-nitro-L-arginine methyl ester(L-NAME)was established.The LDLR+/-mice were randomly assigned into four groups: the control group,the PBS group,the VLP group and the PCSK9Q?-003 vaccine group.And the mice were administered with L-NAME(50 mg/kg/day)for six weeks to establish renal fibrosis model.At week 26 and 28,the 24-hour urinary protein was determined.The remaining test items were the same as before.Result: The LDLR+/-mice generated high-titer antibody against PCSK9-003 peptide in the study.Compared to other three groups,the PCSK9Q?-003 vaccine obviously decreased total cholesterol and low-density lipoprotein cholesterol in LDLR+/-mice with hypercholesterolemia.Compared with the PBS and VLP groups,the PCSK9Q?-003 vaccine improved hepatic steatosis and renal function.Histology analysis showed that the PCSK9Q?-003 vaccine significantly ameliorated renal lipid accumulation and renal fibrosis.The mice treated with PCSK9Q?-003 vaccine reduced 24 h urine protein excretion,blood urea nitrogen and creatinine levels compared to PBS and VLP groups.Compared with the PBS and VLP groups,the PCSK9Q?-003 vaccine significantly ameliorated tubular dilatation,edema,inflammatory cell infiltration,and interstitial fibrosis of kidney.Conclusions: This study suggested that the PCSK9Q?-003 vaccine attenuated the progression of UUO or L-NAME-induced renal fibrosis.In addition to lower blood lipid,PCSK9Q?-003 vaccination reduced biochemical parameters of renal dysfunction and ameliorated renal pathological changes.Part?The mechanistic study in renoprotection effect of PCSK9Q?-003 vaccineObjective: The potential mechanisms of renal protection of PCSK9Q?-003 vaccine in the mice induced with renal fibrosis was investigated in our study from two aspects including inflammatory fibrosis and regulation of fatty acid oxidation pathway.Method: The mice experimental model and grouping were the same as before.Oil red O staining was used to observe lipid deposition in the kidney.The fatty acid metabolism(PPARs,PGC-1?,Cpt1/ 2,ACOX1/2,CD36)and cholesterol transport molecules(ABCA1?ABCG1?ACAT1)were further tested by western blot and quantitative real-time PCR(q RT-PCR).The extend of interstitial expansion was quantified by Masson's trichrome-staining.Also,we detected transforming growth factor-?1(TGF-?1)expression.The m RNA expressions of profibrotic and p Smad3 in kidney cortex were measured by q RT-PCR and western blot.Result: Both in two different renal fibrosis models,compared to PBS and VLP groups,PCSK9Q?-003 vaccine significantly ameliorated renal lipid deposition locating in renal tubular.Compared with other three groups,the PCSK9Q?-003 vaccine increased the expression of LDLR,VLDLR and SREBP2.The PCSK9Q?-003 vaccine remarkably up-regulated the levels of fatty acid metabolism-related factors,including PPAR?,PPAR?,PGC1,ACOX1,CD36 and LPL in contrast to other three groups.In addition,compared to the PBS and VLP groups,the PCSK9Q?-003 vaccine administration obviously reduced the level of TGF-? and p Smad3,which indicated the anti-fibrosis effect of the PCSK9Q?-003 vaccine.Conclusion: The modulation of FAO and inhibition of fibrosis contribute to the renoprotection effect of PCSK9Q?-003 vaccine in UUO or L-NAME induced renal fibrosis.