Cyclophilin D promotes tubular cell damage and the development of interstitial fibrosis in the obstructed kidney

Abstract

Cyclophilin D (CypD) is an important component in mitochondrial-dependent tubular cell death in acute kidney injury. However, it is not known whether CypD contributes to tubular cell damage in chronic interstitial fibrosis. We investigated this question in the unilateral ureter obstruction (UUO) model of renal interstitial fibrosis. Groups of CypD and wild type (WT) mice were killed 7 or 12 days after UUO surgery. The significant tubular cell apoptosis seen in WT UUO was significantly reduced in CypD UUO based on TUNEL and cleaved caspase 3 staining. Other markers of tubular cell damage; loss of E-cadherin and AQP1 expression, were also reduced in the CypD UUO kidney. This reduced tubular damage was associated with less inflammation and a partial protection against loss of peritubular capillaries. The prominent accumulation of α-SMA+ myofibroblasts and interstitial collagen deposition seen in WT UUO was significantly reduced in CypD UUO on day 12, but not day 7. Activation of several pro-fibrotic signalling pathways (p38 MAPK, JNK and Smad3) was unaltered in CypD UUO, arguing that CypD acts independently to promote renal fibrosis. CypD deletion in cultured tubular cells attenuated oxidative stress-induced pro-inflammatory, pro-fibrotic and apoptotic responses; however, responses to angiotensin II and LPS were unaffected. In contrast, CypD deletion in cultured renal fibroblasts did not affect PDGF-induced proliferation or TGF-β1-induced collagen I expression, suggesting no direct role of CypD in the fibroblast response. In conclusion, we have identified a role for CypD in chronic tubular cell damage and in the development of renal interstitial fibrosis.

A Pan-Cyclophilin Inhibitor, CRV431, Decreases Fibrosis and Tumor Development in Chronic Liver Disease Models

“…The physicochemical properties of MM284 dictate that it targets extracellular cyclophilins, which suggests that fibrosis is lowered at least partly by attenuating Cyp A-CD147–mediated inflammation (Iordanskaia et al, 2015). Similarly, in the unilateral ureter obstruction model, preservation of cell viability and lowering of inflammation by Cyp D knockout partly prevents renal fibrosis (Hou et al, 2018). Direct, antifibrotic effects on hepatic stellate cells, the primary cell type implicated in liver fibrosis, is another proposed mode of action.…”

Protease‐activated receptor 2 does not contribute to renal inflammation or fibrosis in the obstructed kidney

“…Reverse transcription was performed using the SuperScript III First‐Strand Synthesis System with random primers (Thermo Fisher Scientific, Scoresby, Australia). Real‐time polymerase chain reaction (PCR) using Taqman probes was performed on a StepOne Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described . The primer/probes for α‐SMA, NOS2 and CD206 have been published previously, and the other primer/probes were purchased from Applied Biosystems.…”

“…Immunostaining for Na/K‐ATPase (1 in 400 dilution) and p‐ERK1/2 (1 in 200 dilution) was performed on formalin‐fixed 4 μm paraffin sections with antigen retrieval (sodium citrate pH6) using the ABC peroxidase method as previously described . Immunostaining for collagen IV (1 in 400 dilution) and α‐SMA (1 in 2000 dilution) was performed on methylcarn fixed 4 μm paraffin sections using the ABC peroxidase method.…”

“…Frozen kidney samples were homogenized in 0.4 mL of a 6 M urea‐based lysis buffer as previously described . Samples were separated on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with antibodies to Na/K‐ATPase, p‐ERK1/2 or p‐Akt (all at 1 in 1000 dilution) in Odyssey Blocking Buffer with 0.05% Tween 20 overnight, and then incubated with goat anti‐rabbit Alexa fluor 680 or donkey anti‐mouse IRDye 800 (Molecular Probes) and detected using the Odyssey Infrared Image Detecting System (LICOR).…”

“…Real-time polymerase chain reaction (PCR) using Taqman probes was performed on a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described. 19 The primer/probes for α-SMA, NOS2 and CD206 have been published previously, 17,18 and the other primer/probes were purchased from Applied Biosystems. The relative amount of mRNA was determined using the comparative Ct (ΔCt) method.…”