Role Of Scaffolding Protein JLP In Kidney Interstitial Fibrosis

Background and AimRenal interstitial fibrosis is the common feature and final outcome of chronic kidney disease(CKD)of various causes,which is characterized by the interstitial extracellular matrix deposition(ECM),tubule atrophy and inflammatory infiltration.It is well documented that tubular epithelial cells(TECs)play a vital role in kidney fibrosis.Recent,attention has been payed to the potential role of autophagy of TECs in kidney fibrosis,and although it is confirmed that during kidney fibrosis,the autophagic activity of TECs is upregulated,the precise role is quite confused and under controversy.In addition,the underlying mechanism of autophagy in kidney fibrosis still needs to be explored.JNK-interacting proteins(JIPs)are kinds of scaffolding proteins in JNK or p38 MAPK signaling pathway,which consist of JIP1,JIP2,JIP3,JIP4 and JNK-associated leucine-zipper protein(JLP).It is recently reported that JLP may play a negative role in regulating activity of autophagy of mouse cerebellar purkinje cells.Our preliminary study has shown that JLP is highly expressed in TECs,and the fibrotic responses in jlp deficient mice after unilateral ureteral obstruction(UUO)are more severe compared with jlp wildtype mice,suggested that JLP may participate in progression of kidney fibrosis.Combined with current data and our previous findings,we assume that JLP may regulate the progression of kidney fibrosis through mediating the activation of autophagy.In present study,we established the UUO model in jlp deficient mice and explored the potential role of JLP in progression of kidney fibrosis and the activity of autophagy during fibrosis.Moreover,in vitro human TECs cell lines HK-2 cells were carried to investigate the molecular mechanism of JLP in regulating autophagic activation.The objection of this work is to explore the potential role and underlying mechanism of JLP during renal fibrogenesis based on our previous results,and prove new theoretical basis and therapeutic approaches for clinical treatment of kidney fibrosis.MethodsPart I:Experimental animal groups:(1)jlp +/+ plus sham;(2)jlp +/+ plus UUO;(3)jlp-/-plus sham;(4)jlp-/-plus UUO.Immunostaining was performed to detect the expression and distribution of JLP,and the co-localization with renal resident cells in mouse kidney tissue.Immunostaining,Western blotting,immunohistochemistry(IHC)and real-time PCR was performed to examine the alteration of JLP expression in patients with different stage of CKD,and different time after UUO treatment.HE stain and MTS stain were carried to examine the histopathology and the deposition of collagen fibrils in each group.Immunostaining,Western blotting,immunohistoch-emistry(IHC)and real-time PCR was used to observe the expression of a-SMA,Fibronectin(FN)and collagen-I in each group.In addition,we performed Immunostaining and IHC to explore the expression of F4/80+ cells and caspase-3 in different groups.At the end,we detected the alteration of transforming growth factor beta(TGF-?)and the activation of Smad signaling pathway in jlp +/+ and jlp-/-mice during UUO duration.Experimental cell groups:(1)scramble siRNA plus medium;(2)scramble siRNA plus TGF-?;(3)jlp siRNA plus medium;(4)jlp siRNA plus TGF-?.Immunostaining and Western blotting was used to assess the expression of a-SMA,FN and collagen-I in each group.Flow cytometry was performed to detected the cell cycle and cell apoptosis in each group.Part ?:Experimental animal groups were same as part ?.Western blotting was performed to examine the expression of LC3 and beclin-1 in each group;Immunostaining was used to observe the LC3 puncta in each group;In addition,we used electron microscopy to directly observe the autophagosome in given group.Experimental cell groups:(1)Starvation treatment plus scramble siRNA or jlp siRNA transfected HK-2 cells;(2)Bafilomycin A1(Baf A1)treatment plus scramble siRNA or jlp siRNA transfected HK-2 cells;(3)TGF-? stimulation plus scramble siRNA or jlp siRNA transfected HK-2 cells.Western blotting was performed to detect the expression of LC3,beclin-1 and SQSTM1 in each given group.(4)Cells were co-transfected with mRFP-GFP-LC3 plasmid and scramble RNA or jlp siRNA.Con-focal was performed to observe the mRFP-LC3 and GFP-LC3 in each group.Part ?:Experimental animal groups:(1)jlp +/+ plus sham plus medium;(2)jlp+/+ plus UUO plus medium;(3)jlp-/-plus sham plus medium;(4)jlp-/-plus UUO plus medium;(5)jlp +/+ plus sham plus chloroquine;(6)jlp +/+ plus UUO plus chloroquine;(7)jlp-/-plus sham plus chloroquine;(8)jlp-/-plus UUO plus chloroquine.MTS stain was performed to examine the deposition of collagen fibrils in given group,Western blotting was carried to detect the expression of LC3,SQSTM1 and a-SMA in each group.Experimental cell groups:(1)scramble siRNA plus medium;(2)scramble siRNA plus medium plus Baf A1;(3)scramble siRNA plus TGF-? plus medium;(4)scramble siRNA plus TGF-? plus Baf Al;(5)jlp siRNA plus medium;(6)jlp siRNA plus medium plus Baf A1;(7)jlp siRNA plus TGF-? plus medium;(8)jlp siRNA plus TGF-? plus Baf A1.Western blotting was carried to detect the expression of LC3,SQSTMI and a-SMA in each group.ResultsPart ?:JLP was dominantly expressed in proximal tubule,slightly expressed in distal tubule but not expressed in glomeruli and renal resident fibroblasts.Expression of JLP was gradually downregulated either after UUO or in different stage of CKD,which was negatively correspondent with the severity of fibrosis.Tubule atrophy and ECM deposition was more worsen in jlp-/-mice after UUO,as well as the expression of?-SMA,FN and collagen-I.In addition,cell apoptosis and G2/M cell cycle arrest was significantly increased in jlp-/-mice compared with wildtype mice.Moreover,the expression of TGF-P and the activation of Smad singling pathway was dramatically upregulated in jlp-/-mice after UUO engagement.In vitro cell experiments also confirmed the observation.Part ?:Western blotting and immunostaining revealed that activation of autophagy was increased in jlp-/-mice after UUO treatment,compared with jlp +/+mice.Electron microscopy confirmed that more autophagosome was observed in jlp-/-mice.In vitro experiments showed that JLP deficiency led to increased autophagic activity either in starvation or in TGF-? stimulated HK-2 cells.In addition,mRFP+GFP-and mRFP+GFP+ LC3 puncta were significantly increased in jlp knockdown cells after transfected with mRFP-GFP-LC3 plasmid.Part ?:Pre-treatment with chloroquine showed significant inhibition of autophagy activation in kidney tissue after UUO engagement,moreover,pre-treatment with chloroquine significantly reversed UUO-induced fibrotic responses either in jlp+/+ or in jlp-/-mice.In vitro,pre-treatment HK-2 cells with Baf Al also showed a significant attenuation of fibrotic responses after TGF-? stimulation in scramble siRNA and jlp siRNA-transfected cells.ConclusionJLP is dominantly expressed in proximal TECs and JLP deficiency leads to deteriorated fibrotic responses and over-activation of autophagy after UUO engagement.In vitro cell culture experiments indicate that JLP regulates autophagy in early autophagosome formation stage.Pharmaceutical inhibition of autophagy could suppress such fibrotic responses during UUO duration either in jlp +/+ and jlp-/-mice.These data suggest that JLP mediated activation of autophagy may participate in the progression of kidney fibrosis.For the first time,our investigation has demonstrated a novel role of JLP in regulating kidney interstitial fibrosis through mediating the autophagic activation,which may be a potential therapeutic strategy on kidney fibrosis.