(853.16) Serial Intravital 2-photon Imaging Deciphers in Vivo Renal Myofibroblast Dynamics at the Fork of Acute Kidney Injury-induced Tubular Repair and Nephron Loss
Tuesday, April 5, 2022
10:15 AM – 12:15 PM
Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Myofibroblasts were suggested to play a pivotal role in the transition of acute kidney injury (AKI) to chronic kidney disease, by engaging in progressive and self-perpetuating tubulointerstitial fibrosis. Wound healing from AKI gives rise to myofibroblast-activation from renal interstitial PDGFRβ-cells. In this study, we exploited a novel model of partial ischemia reperfusion injury (IRI), to track dynamic PDGFRbeta-cell interactions with injured and non-injured tissue over time and addressed the question if and how myofibroblast-derived renal interstitial fibrosis may progress and deteriorate non-injured renal tissue after AKI.
PDGFRβ-CreERT2-Salsa6F reporter mice identified PDGFRβ-cells by tdTomato expression. IRI was performed by unilateral occlusion of only one branch of the left renal artery for 21 min, resulting in half of the left kidney being ischemic, while the other half remained non-ischemic. This was followed by abdominal imaging window implantation for serial imaging of the same kidney cells in border areas for 2 to 3 weeks (N=5). To test for myofibroblast differentiation, we performed ex vivo staining for αSMA on the same tissue regions as previously imaged in vivo.
IRI-induced necrotic tubular cell death, as detected in vivo by Propidium Iodide (PI)-staining, was only observed on day 0 (D0) post IRI. Proximal tubules (PTs) with intermediate necrotic injury (mean ± SD) (21±15% dead cells of total cell number per segment) underwent successful self-repair within 7 days through dedifferentiation as indicated by shedding of the apical membrane (peaking at D2), followed by the temporal flattening of the epithelium. In highly injured PT segments (39±15% cell death), dedifferentiation was followed by epithelial vacuolization (starting at D4/D7), resulting in failed repair in 35% of the cases as indicated by collapsed and karyolitic tubules evident between D7 and D14.
Tubule injury initiated pronounced PDGFRβ-cell recruitment, which resulted in remarked enclosure of the remodeling epithelium. Thus, PDGFRβ-cell recruitment peaked at D4, and D7 with a maximum coverage of PT cell surface of 29±6% and 44±14% for surviving tubules of initially low and high necrotic injury, respectively. In non-surviving tubules, PDGFRβ-cell recruitment peaked D7 with 63±18% of the remodeling PT cells covered. Ex vivo stainings for αSMA confirmed PDGFRβ-cell differentiation to myofibroblasts. Importantly, regardless of respective tubule faith, PDGFRβ-cell accumulation resolved over the period from D7-D14/21 and did not progress towards non-injured tubules. Further, ex vivo stainings at D14/21 indicated αSMA-negativity of persistent and previously injury-interacting PDGFRβ-cells, indicating the reversibility of myofibroblast differentiation.
Our data indicate locally restricted, injury-dependent and reversible PDGFRβ-cell accumulation and myofibroblast-differentiation in ischemic tissue regions. There is no indication for the existence of self-perpetuating interstitial fibrosis and deterioration of healthy nephrons. Furthermore, our data indicates the reversibility of myofibroblast-differentiation upon completed repair programs.
