Activated Protein C Inhibits Apoptosis In Podocytes

Objective1. To study the effect of aPC on PAN induced apoptosis in podocytes.2. To explore the antiapototic mechamism of aPC in podocytes.3. To define the possible role of the downstream signaling pathway of aPC and PAR-3 in inhibiting apoptosis in podoytes.Methods1. Treated PAN stressed podocytes with different concentration of aPC, then detect the in situ cell apoptosis with TUNEL detection kit, in order to find out whether the anticoagulant function of aPC plays a role in apoptosis in podocytes, the cells were analyzed after pre-incubation aPC with specific antibody and aptamer.2. The related gene and protein(Thbd,PAR-1,PAR-2,PAR-3,PAR-4,EPCR) expressing profile of podocytes were analyzed with RT-PCR, Western Blot and Immunofluorescence.3. The key receptors which may participate in regulating apoptosis in podoytes were screened with the help of specific agonists and blocking antibodies and the results were further proved by shRNAmir gene Knock Down technology.4. Measure the intracellular Ca2+ level in podocytes under different stimuli(aPC,TRAP-3,Thrombin) with the help of a cacium sensitive fluorescent dye(Fura-2 AM) using a digital image fluorescence microscopy.5. Co-immunoprecipitation were used to study the time dependent manner of the interaction of the related receptors.6. V5 tagged wild type PAR-3 were cloned and the PAR-3 mutants were constructed with the site directed mutagenesis kit. All the constructs were transfected into mesangial cells then stable transfected cell clones were selected respectively. Related cell clones were used to study the interaction between aPC and PAR-3 and the subsequent effect on apoptosis in podocytes.7. The possible signaling pathway, such as Caveolae, RhoA and ERK1/2, which associate with the regulation of aPC in apoptosis were detected with the help of immunoprecipitation and Western Blot.Results1. Concomitant treatment with aPC reduced PAN induced podocytes apoptosis, even at concentrations as low as 0.2 nM. The anti-apoptotic effect was not impaired following pre-incubation of aPC with the antibody HAAPC, which inhibits specifically its anticoagulant function, or with the aptamer HSO2-88, which reduces the anticoagulant function of aPC without impairing the cytoprotective effect.2. Immortalized human podocytes do not express EPCR (mRNA and protein) and express very low levels of PAR-1 and PAR-4 (mRNA and protein), while PAR-2 and PAR-3 were readily detectable. Consistently, PAR3 and PAR2, but not EPCR, were readily detectable in podocytes in healthy human kidney biopsy cryo-sections.3. With the help of specific agonist peptides and blocking antibodies for PAR-1,PAR-2,PAR-3,PAR-4 we found that PAR-2 and PAR-3 were the key receptors which played vital role in regulating apoptosis in podoytes by aPC, and this was further confirmed by experiments with PAR-2 and PAR-3 shRNAmir gene Knock Down stable transfected cell lines. 4. aPC and PAR-3 agonist peptide failed to induce intracelluar Ca2+ spikes in human podoytes,while a Ca2+ peek was provoked at about 80s after stimuli of control agonist Thrombin.5. Human podocytes were treated with aPC and PAR-2/PAR-3 complex formation was determined by co-immunoprecipitation at various time points. Activated PC induced complex formation of PAR-2 and PAR-3, which was first detectable after 10 minutes and further increased at later time points. Pre-treatment of podocytes with an antibody blocking PAR-3 prevented dimerization of PAR-3 and PAR-2.6. RT-PCR results showed the cell lines were successful transfected. Transfection with either PAR-3 expression construct increased binding of aPC to the cells. Despite binding equally to PAR-3-wt-V5, mutant PAR-3-m-V5 and mutant PAR-3-TEM1-V5 only the wild type PAR-3 was cleaved by aPC, resulting in easily detectable free V5 epitope in the cellular supernatant by Western Blot. Following exposure to staurosporine aPC failed to inhibit apoptosis in non-transfected, PAR-3-m-V5 expressing mesangial cells. Conversely, aPC significantly reduced apoptosis in staurosporine treated mesangial cells expressing PAR-3-wt-V5. Similar results were obtained in glucose stressed mesangial cells.7. Co-immunoprecipitation experiments found out that in resting podocytes an interaction of PAR-2 and PAR-3 with caveolin-1 is readily detectable. Following treatment with aPC, PAR-3 dimerized with PAR-2, as shown above, but the association of these receptors with caveolin-1 decreased. The cholesterol depleting molecular MβCD impaired the aPC induced dissociation of PAR-2 and PAR-3 from caveolin-1 and the dimerization of PAR-3 with PAR-2. In addition to modulating the PAR-2/PAR-3 dimerization and interaction with caveolin-1, aPC time-dependently reduces phosphorylation of caveolin-1. Furthermore shRNAmir gene Knock down of caveolin-1 abolished the antiapoptotic effect of aPC in PAN-stressed podocytes. To identify intracellular pathways mediating the aPC-PAR-2-PAR-3 anti-apoptotic effect in podocytes we inhibited various signaling pathways, including those known to be relevant for aPC dependent cytoprotection in other cell types. Inhibition of G a i, EGF-receptor, or sphingosine receptor-1 did not alter the anti-apoptotic effect of aPC in PAN-stressed podocytes. Conversely, inhibition of the Rho-associated protein kinase p160ROCK or of MEK1/MEK2 abolished the anti-apoptotic effect of aPC. Interestingly, phosphorylation of ERK1/2 and activation of RhoA was first detectable after 10 min, coinciding with the dephosphorylation of caveolin-1.Conclusions1. These data establish that aPC cell autonomously inhibits apoptosis in podocytes independent of its anticoagulant properties, implying a receptor dependent mechanism.2. aPC can bind and cleave PAR-3,PAR-2 and PAR-3 are expressed in human podocytes and their dimerization is required for aPC dependent inhibition of podocyte apoptosis.3. aPC induces dephosphorylation of and dissociation of PAR-2 and PAR-3 from caveolin-1, and these dynamic, caveolin-1 dependent changes are required for aPC dependent apoptosis inhibition in podocytes. These data also indicate that aPC mediated apoptosis inhibition depends on Gα12/13 and RhoA/ERK1/2 dependent signalling.Significances of the study1. These observations establish for the first time that aPC can mediate cytoprotection independent of both PAR-1 and EPCR, which were previously considered the two bona fide receptors of aPC and provide a new reference for the further study of podocytes related diseases.2. Our works provided the first evidence that aPC can bind and cleave PAR-3, it lays a foundation for further study of the interaction between aPC and related homogeneous receptors.3. Our study found that aPC can effectively prevent the apoptosis in a PAN stressed podocyte model, suggesting that aPC can also be applied to treat acute kidney injury diseases independent of its classical application in severe sepsis.