FC 015LACK OF PLASMINOGEN RELATES TO A HYPERCOAGULABLE STATE IN MICE WITH EXPERIMENTAL NEPHROTIC SYNDROME
Authors: Xiao Mengyun , Hammer Stefanie , Khalel Wissam A , Pelzl Lisann , Bohnert Bernhard N , Althaus Karina , Bakchoul Tamam , Birkenfeld Andreas L , Artunc Ferruh 
Published: May 2021

Background and Aims

Urinary excretion of the fibrinolytic enzyme plasminogen has been identified as a characteristic feature of nephrotic syndrome (NS) in both human and experimental mouse models. Lack of plasminogen may lead to a hypercoagulable state and thrombosis, and mice with plasminogen deficiency have been shown to suffer from developing spontaneous thrombosis. However, the role of plasminogen in hypercoagulable state and thrombosis in an experimental nephrotic syndrome has not been investigated before.

Method

We investigated the relationship between plasminogen and a hypercoagulable state in an inducible nephrotic mouse model with conditional podocyte-specific podocin deletion (Nphs2Δipod * Plg+/+, n=12). The Nphs2Δipod mice with constitutive plasminogen knockout were used as negative plasminogen control (Nphs2Δipod * Plg-/-, n=15). All mice received a daily oral doxycycline administration for 2 weeks for NS induction. The last day of doxycycline treatment was set as day 0. Spot urine was collected daily for proteinuria and urinary plasmin activity measurement. Citrate blood was collected from each mouse before induction of NS, 7 days and 21 days after induction, respectively (Nphs2Δipod * Plg+/+ mice, n=4/timepoint; Nphs2Δipod * Plg-/- mice, n=5/timepoint). A global assessment of coagulation (extrinsic coagulation test, EX test) was examined by ClotPro® system. Besides, fibrinolysis was tested by adding tissue plasminogen activator (TPA test).

Results

According to the EX test, uninduced mice with plasminogen deficiency showed a significantly reduced clotting time (CT, Plg-/- vs. Plg+/+, 42 ± 1s vs. 54 ± 4s, p=0.0213), and decreased clot formation time (CFT, Plg-/- vs. Plg+/+, 82 ± 5s vs. 206 ± 28s p&lt;0.0001) with a larger alpha-angle (Plg-/- vs. Plg+/+, 75 ± 1° vs. 66 ± 2°, p=0.0041). The maximum clot firmness (MCF) was significantly increased in uninduced plasminogen knockout mice (Plg-/- vs. Plg+/+, 45 ± 0.5mm vs. 32 ± 2.5mm p&lt;0.0001). According to the TPA test, uninduced Nphs2Δipod *Plg-/-mice had a faster velocity of clot formation (α-angle, 75.6 ± 0.2° vs. 66.5 ± 1.6°, p=0.0254) and did not show any clot lysis in contrast to uninduced nphs2Δipod * plg+/+mice. After induction of NS, both Nphs2Δipod * Plg-/-mice and Nphs2Δipod * Plg+/+ mice developed massive proteinuria to a comparable extent (Plg-/- vs. Plg+/+on day 21, 218 ± 46mg/mg crea vs. 203 ± 28mg/mg crea), and plasminuria was detectable in nephrotic nphs2Δipod * plg+/+ mice. With the ongoing loss of plasminogen in the urine, CT and CFT was significantly reduced in nephrotic Nphs2Δipod * Plg+/+ mice. MCF was significantly increased with a faster velocity of clot formation measured by both the EX and TPA test. Moreover, clot lysis was significantly reduced. In nephrotic nphs2Δipod *plg-/-mice at day 21, there was also a tendency towards a decrease in CT, CFT and an increased velocity of clot formation. According to both EX and TPA test, there were no significant differences between the genotypes in nephrotic mice any more.

Conclusion

The results highlight that loss of plasminogen in the nephrotic state contributes to a hypercoagulable state with shortened clotting time, clot formation time, increased clot firmness, and most strikingly, loss of clot lysis. Changes in nephrotic wild-type mice were similar to mice with constitutive plasminogen deficiency, indicating that loss of plasminogen plays a role in the hypercoagulable state of nephrotic syndrome.