The Effect And Mechanism Of Increased Levels Of Serum Pigment Epithelium-derived Factor In Proteinuria In Diabetic Kidney Disease

BackgroundDiabetic kidney disease(DKD)is a severe microvascular complication of diabetes mellitus,which has become a leading cause of end-stage renal disease(ESRD)worldwide.As a multifactorial disease,the mechanism of DKD remains unclear.Podocytes form the final layer of the glomerular filtration barrier,which stabilize the glomerular capillaries and maintain glomerular permselectivity.In recent years,more and more studies have shown that podocyte damage plays a critical role in the pathogenesis of DKD.The role of podocytes is highly dependent on their actin-based cytoskeletal architecture.Loss of these actin-driven membrane extensions is tightly connected to foot process effacement(FPE),podocyte loss,and proteinuria.Thus,further studies to examine factors that interfere with the podocyte actin cytoskeleton might provide us with new insight regarding DKD pathogenesis and suggest potential therapeutic treatments.Pigment epithelium-derived factor(PEDF)is a 50-kDa endogenous secreted glycoprotein,which has been found elevated in patients with diabetes.However,the role of serum PEDF in DKD is much less known.There is evidence that serum PEDF can increase vascular endothelial permeability by regulating F-actin arrangement in sepsis patients.Based on the cytoskeleton-related filtration function of podocytes and the permeability-related activity of PEDF,it is reasonable to hypothesize that PEDF may be involved with the development of proteinuria.Whereas,few studies have been reported regarding the role of PEDF in the cytoskeletal changes of podocytes.In the current study,we aimed to elucidate the effect of PEDF on proteinuria by investigating the role of PEDF in regulating the F-actin arrangement of podocytes,and exploring the underlying molecular mechanisms.Material and Methods1.Type 1 and Type 2 diabetic mice model were induced by intraperitoneal injection with different dosages of streptozotocin,and were divided into five week or ten week groups according to the length of diabetic course.2.Exogenous PEDF protein or recombinant adeno-associated virus vector(AAV)expressing PEDF was delivered to normal or diabetic mice by intravenous injection to induce higher levels of serum PEDF in mice body.3.Mice urine was collected to assess the urinary albumin/creatinine ratio(ACR).Blood samples were obtained to measure the serum PEDF and serum creatinine levels.Kidneys were removed for hematoxylin-eosin(HE)staining and transmission electron microscopy(TEM)observation.4.In vitro the conditionally immortalized mouse podocyte cell line was cultured to detect the permeability by measuring the leakage of fluorescein isothiocyanate(FITC)-dextran tracer after the stimulation of different concentrations of PEDF in different time.5.The conditionally immortalized mouse podocyte cell line was used to measure the leakage of FITC-dextran tracer,F-actin rearrangement in phalloidin staining,podocyte apoptosis in flow cytometry,the expressing levels of Zonula occludens-1(ZO-1),nephrin and podocin protein in western blotting(WB)assays after the stimulation with high glucose and high dose PEDF.6.The podocyte was used to measure the RhoA activity and Rho-associated coiled-coil protein kinase1(ROCK1)protein expressing levels in WB assays after further stimulation with RhoA inhibitor(C3 transferase).And the leakage of FITC-dextran tracer,F-actin rearrangement,podocyte apoptosis,the expressing levels of ZO-1,nephrin and podocin were also assessed.Results1.Diabetic mice exhibited elevated serum PEDF and ACR levels at the fifth week after diabetes induction compared with normal mice.HE staining showed no significant change in the capsular space volume.FPE was observed by TEM.2.Mice at the tenth week after diabetes induction exhibited higher serum PEDF,serum creatinine and ACR levels compared with the mice of five weeks.The capsular space volume was increased and FPE worsened.3.After injection of high dosage of exogenous PEDF protein or recombinant AAV expressing PEDF,the serum PEDF levels of both normal and diabetic mice were increased compared with non-injected mice,accompanied by increased serum creatinine,ACR levels,and capsular space volume.FPE was observed in normal mice,and worsened in diabetic mice.4.PEDF induced an increase in FITC-dextran tracer leakage across cultured podocyte monolayers in a dose-and time-dependent manner.5.High glucose stimulation increased the FITC-dextran leakage,decreased the ZO-1 expression,rearranged podocyte F-actin,increased podocyte apoptosis and decreased nephrin and podocin expression.PEDF stimulation aggravated the above effects under both normal and high glucose conditions.6.High glucose or PEDF or co-stimulation of high glucose and PEDF promoted RhoA activity and increased the ROCK1 expression.7.C3 transferase alleviated PEDF and(or)high glucose induced FITC-dextran leakage,improved the F-actin rearrangement,decreased podocyte apoptosis,restored ZO-1,nephrin and podocin expression and blocked RhoA and ROCK1 activation.ConclusionIn summary,the present study demonstrated that elevated serum PEDF aggravated the development of proteinuria and renal dysfunction in diabetic mice through promoting actin arrangement and apoptosis of podocytes via activating the RhoA/ROCK1 signaling pathway.The RhoA inhibitor blocked the PEDF-induced effects in podocytes,which suggests that inhibition or antagonism of serum PEDF may provide a new potential therapeutic strategy for proteinuria in DKD patients.