The Role Research Of PCSK9-Mediated Inflammatory To Promote Diabetic Renal Tubular Injury

Background: The global prevalence of Diabetes mellitus(DM)has led to Diabetic nephropathy(DN)emerging as the primary cause of endstage renal disease,imposing a significant burden on both diabetic patients and healthcare systems.The progression of DN is heavily influenced by renal tubule injury,which disrupts the permeability of the glomerular filtration membrane.Consequently,a substantial quantity of protein is excreted in the urine,leading to proteinuria and exacerbating the burden of kidney injury.The role of inflammation in the progression of DN is significant,with various inflammatory factors,including IL-1β,TNF-α,and MCP-1,actively contributing to the development of this condition.Consequently,investigating novel pathogenic mechanisms underlying DN holds immense importance in the pursuit of developing targeted pharmaceutical interventions for this disease.The proprotein convertase subtilisin/kexin type 9(PCSK9)is primarily synthesized by the liver but is expressed in various organs.Its significance has been demonstrated in nephrotic syndrome,acute kidney injury,and chronic kidney disease.Furthermore,PCSK9 is implicated in lipid metabolism as well as cardiovascular diseases,sepsis,and other inflammatory reactions.Nevertheless,the precise involvement of PCSK9 in diabetic nephropathy(DN)remains uncertain,and its impact on DN-related inflammation has not been documented in existing literature.Chapter I Relationship between PCSK9 expression and renal function and renal tubule injury in T2 DM patients and DN mouse modelsObjective: The primary emphasis of research on PCSK9 and diabetes pertains to dysglycolipid metabolism associated with diabetes and macrovascular complications,particularly cardiovascular diseases.However,there is a scarcity of literature addressing the relationship between PCSK9 and common microvascular complications,such as DN.This chapter endeavors to ascertain the presence of PCSK9 in the serum of patients with Type 2 diabetes mellitus(T2DM)and in both serum and renal tissue of DN mice.Additionally,it aims to explore the correlation between PCSK9 and renal function as well as tubular injury.Methods:(1)From June 2019 to January 2020,serum samples were collected from 145 T2 DM patients diagnosed by Central South University’s Third Xiangya Hospital.During the same period,serum samples were collected from 37 healthy control groups from Central South University’s Health Management Center(gender,age,and other basic information were matched with T2DM).The concentration of PCSK9 in the serum of human subjects was quantified using a human enzyme-linked immunosorbent assay(ELISA)kit.Concurrently,various biochemical markers such as the urinary microalbumin to urinary creatinine ratio(UACR),serum creatinine(Scr),blood urea nitrogen(BUN),among others,were gathered,and the estimated glomerular filtration rate(e GFR)was computed.(2)The DN mouse model was created through the administration of a high fat and high glucose(HFD)diet,along with intraperitoneal injection of STZ.Following the successful establishment of the model,the mice were subjected to a 12-week period of HFD feeding.Urine,serum,and renal cortex samples were then collected from both Control mice fed a normal diet and HFD mice that had been induced with STZ(HFD/STZ).The ELISA kit was utilized to detect the content of PCSK9 in mouse serum.The levels of Scr,BUN,and UACR were assessed in both Control mice and HFD/STZ mice.The renal cortex was subjected to detection of PCSK9 levels through RT-PCR,Western blot,and immunohistochemistry techniques.Additionally,HE staining was employed to identify renal tubule injury in mice.(3)HK-2cells were subjected to treatment with low glucose(LG,5mmol/l)and high glucose and high lipid(HGPA,HG 30mmol/l+PA 100 mmol/l)for a duration of 24 hours.Subsequently,the expression of PCSK9 at both m RNA and protein levels,as well as the m RNA levels of KIM-1 and NGAL,which serve as markers for renal tubule injury,were assessed.Results:(1)In comparison to the healthy control group,the serum PCSK9 content in patients with type 2 diabetes mellitus(T2DM)exhibited a significant increase(P<0.05).Furthermore,there were positive correlations observed between the serum PCSK9 level and Scr(r=0.189,P=0.024),BUN(r=0.213,P=0.010),and UACR(r=0.365,P<0.001),while a negative correlation was found with e GFR(r=-0.173,P=0.037).(2)The levels of serum PCSK9 content,PCSK9 m RNA,and protein in the renal tubules of HFD/STZ mice were found to be significantly elevated compared to the control group(P<0.05).Additionally,a significant positive correlation was observed between the serum PCSK9 content in HFD/STZ mice and the levels of Scr(r=0.789,P=0.012),BUN(r=0.838,P=0.005),and UACR(r=0.748,P=0.020).The immunohistochemical analysis revealed a higher intensity of PCSK9 staining in the renal tubules of mice subjected to a high-fat diet and streptozotocin treatment(HFD/STZ),while the hematoxylin and eosin staining demonstrated a more significant tubular damage in the HFD/STZ mice compared to the control group.Furthermore,the correlation analysis demonstrated a positive association between the expression of PCSK9 in the tubules and the severity of tubular injury(r= 0.604,P= 0.014).(3)In comparison to the LG group,the expression of PCSK9 in HGPAstimulated HK-2 cells exhibited a statistically significant increase(P<0.05).This finding aligns with the expression pattern observed for KIM-1 and NGAL,widely recognized markers of kidney injury in clinical settings.Conclusion: The serum levels of PCSK9 in patients with T2 DM are elevated and demonstrate a correlation with renal function.Similarly,in DN rats,there is an increase in PCSK9 content in the serum,which is also correlated with renal function and UACR.Furthermore,the upregulation of PCSK9 expression in renal tissue is associated with renal tubule injury.Additionally,the expression of PCSK9 in HK-2 cells,when stimulated by HGPA,is elevated,aligning with the expression pattern of KIM-1 and NGAL,both recognized markers of renal injury.Chapter II PCSK9 mAb improves renal function and reduces tubular damage and inflammation in HFD/STZ diabetic miceObjective: PCSK9 is known to have significant implications in nephrotic syndrome,acute kidney injury,and chronic kidney disease.Furthermore,PCSK9’s involvement in lipid metabolism is wellestablished,and it also plays a role in inflammatory reactions associated with cardiovascular diseases and sepsis.However,the precise role of PCSK9 in DN and its potential involvement in the inflammatory response of DN remain uncertain.PCSK9 mAb,a specific inhibitor of PCSK9,is the focus of this chapter,which aims to explore its impact on renal function,tubular injury,and the inflammatory response in HFD/STZ diabetic mice.Methods:(1)The DN mouse model was established through the combination of HFD feeding and intraperitoneal injection of streptozotocin(STZ).Following successful modeling,the mice were subjected to treatment with PCSK9 mAb while being fed with HFD for a duration of 12 weeks.The control group consisted of mice on a normal diet and receiving vehicle treatment(Control+vehicle),whereas the experimental group involved mice on a normal diet receiving PCSK9 mAb treatment(Control+PCSK9 mAb).The urine,serum,and renal cortex samples were collected from rats in the Control+PCSK9 mAb group,the HFD/STZ+vehicle group,and the HFD/STZ+PCSK9 mAb group,which served as the diabetes control group and the diabetes mellitus PCSK9 mAb treatment group,respectively.(2)The levels of Scr,BUN,UACR,LDL-C,TC,and TG in mice were quantified using biochemical laboratory techniques.Mouse serum PCSK9 concentration was determined using an ELISA kit.The presence of PCSK9 and F4/80 in renal tissues was assessed through immunohistochemical analysis.Lipid deposition in renal tissue was identified using oil red O staining.The measurement of the ratio of PAS-positive area and mesangial area to glomerular area was conducted through PAS staining.Renal tubule injury was detected using HE staining.The thickness of the mouse glomerular basement membrane(GBM)was measured using electron microscopy.The m RNA expression levels of inflammatory cytokines(IL-1β,MCP-1,and TNF-α),PCSK9,KIM-1,and NGAL were determined through RTPCR.The expression of PCSK9 was assessed using Western blot analysis.Results:(1)UACR in mice treated with a HFD/STZ and vehicle was found to be significantly higher compared to mice in the control group treated with vehicle alone(P<0.05).Following treatment with PCSK9 monoclonal antibody(mAb),the UACR in DN mice was significantly reduced(P<0.05).Additionally,the levels of Scr and BUN in HFD/STZ+vehicle mice were significantly elevated compared to Control+vehicle mice(P<0.05).However,after intervention with PCSK9 mAb,the levels of Scr and BUN in DN rats showed a certain degree of reduction(P<0.05).(2)In comparison to the HFD/STZ+vehicle group,the HFD/STZ+PCSK9 mAb group exhibited a significant decrease in serum PCSK9 levels(P<0.01).Western Blot and immunohistochemical analyses further confirmed that the PCSK9 level in the HFD/STZ+PCSK9 mAb group was significantly lower than that in the HFD/STZ+vehicle group(P<0.01).Additionally,oil red O staining revealed that PCSK9 mAb treatment partially mitigated diabetes-induced renal lipid accumulation(P<0.01).The turbidity of the serum in HFD/STZ+vehicle mice was found to be greater than that of the control group.However,the administration of PCSK9 mAb partially mitigated the observed turbidity in the serum of HFD/STZ+vehicle mice.Furthermore,the use of PCSK9 mAb resulted in a significant reduction in LDL-C,TC,and TG levels in DN mice(P<0.01).Additionally,PAS staining revealed that PCSK9 mAb treatment effectively reduced glomerular area enlargement induced by HFD/STZ and increased the ratio of PAS-positive area to glomerular area in the mesangial region(P<0.05).Furthermore,the findings from electron microscopy analysis demonstrated a significant reduction in the thickness of the glomerular basement membrane(GBM)in DN mice following treatment with PCSK9 mAb(P<0.05).The expression of F4/80 in mice with high-fat diet/streptozotocin-induced diabetes(HFD/STZ+vehicle)was notably elevated(P<0.05).However,after administration of PCSK9 mAb,the expression of F4/80 was observed to be lower compared to untreated HFD/STZ mice(P<0.05).Furthermore,the m RNA levels of IL-1β,MCP-1,and TNF-α were significantly increased in HFD/STZ+vehicle mice compared to Control+vehicle mice(P<0.05).Nevertheless,treatment with PCSK9 mAb resulted in a decrease in the m RNA levels of IL-1β,MCP-1,and TNF-α(P<0.05).Conclusion: The administration of PCSK9 mAb effectively decreases the levels of PCSK9 in both the serum and kidney tissue of mice subjected to a HFD/STZ-induced diabetic condition.This intervention leads to notable enhancements in renal function,as well as reductions in tubular damage and inflammation in the HFD/STZ diabetic mouse model.Chapter III Study on the mechanism of PCSK9 on inflammation of DNObjective: PCSK9 is closely associated with inflammation.The objective of this chapter is to investigate the potential involvement of PCSK9 in the inflammatory response of DN via the TLR4 /mtDNA /cGAS /STING pathway.Methods:(1)The DN mouse model was created through a combination of HFD feeding and intraperitoneal injection of STZ.Once the model was successfully established,the mice were subjected to treatment with PCSK9 mAb while being fed with HFD for a duration of 12 weeks.Samples were collected from the renal cortex,specifically from the groups designated as Control+vehicle,Control+PCSK9 mAb,HFD/STZ+vehicle,and HFD/STZ+PCSK9 mAb.(2)The HK-2 cell line was subjected to treatment with LG+si Ctrl,LG+siPCSK9,HGPA+ si Ctrl,and HGPA+ siPCSK9.(3)The HK-2 cells were subjected to treatment conditions including LG+si Ctrl,LG+si TLR4,HGPA+ si Ctrl,and HGPA+ si TLR4.(4)The treatment of HK-2 cells involved the application of LG+DMSO,LG+C-176,HGPA+DMSO,and HGPA+C-176,as reported by the study.(5)The structural changes of renal mitochondria were observed through the utilization of electron microscopy.The presence of STING in mouse kidney tissue was identified via immunohistochemistry.The m RNA expression levels of KIM-1,NAGL,and inflammatory factors(IL-1β,MCP-1,and TNF-α),as well as mtDNA damage,were determined using RT-PCR.Western blot analysis was conducted to ascertain the expression of TLR4,cGAS,STING,and downstream target proteins(p-TBK1,p-NF-κB p65,and IL-1β).The immunofluorescence technique was employed to detect the expression of STING in HK-2 cells.Results:(1)In comparison to LG,the m RNA levels of KIM-1,NAGL,IL-1β,MCP-1,and TNF-α in HGPA-stimulated HK-2 cells exhibited a significant increase(P<0.05).Following interference with PCSK9,HGPA prompted the induction of KIM-1,NAGL,IL-1β,and MCP-1 in HK-2 cells,while the m RNA levels of TNF-α were significantly reduced(P<0.05).(2)The electron microscope findings revealed a notable enhancement in the renal mitochondrial structural impairment of HFD/STZ mice upon treatment with PCSK9 mAb.In comparison to the untreated group,the renal mtDNA damage in HFD/STZ mice treated with PCSK9 mAb exhibited a significant reduction(P<0.05).Moreover,the levels of TLR4,cGAS,STING,and their downstream target proteins(PTBK1,P-NF-κB p65,and IL-1β)in the kidney exhibited a significant decrease(P<0.05).(3)In cellular investigations,the interference of PCSK9 resulted in a notable reduction in the mtDNA level of HK-2 cells(P<0.05),along with significant decreases in TLR4,cGAS,STING,and downstream target proteins(P<0.05).Conversely,the interference of TLR4 led to a significant decrease in the mtDNA level of HK-2 cells by HGPA(P<0.05),accompanied by decreased m RNA levels of inflammatory factors(IL-1β,MCP-1,and TNF-α)induced by HGPA(P<0.05).(4)C-176 demonstrated inhibitory effects on the upregulation of HGPA-stimulated inflammatory cytokines(P<0.05).Furthermore,it significantly suppressed the elevation of P-TBK1,P-NF-κB p65,and IL-1β protein levels in HGPA-treated HK-2 cells(P<0.05).Conclusion: PCSK9 is involved in the inflammatory response of DN via the TLR4/mtDNA/cGAS/STING pathway.