Inhibition of fibronectin polymerization alleviates kidney injury due to ischemia-reperfusion

Abstract

Fibrosis is a common feature of chronic kidney disease; however, no clinical therapies effectively target the progression of fibrosis. Inhibition of fibronectin polymerization with the small peptide pUR4 attenuates fibrosis in the liver and heart. Here, we show that pUR4 decreases renal fibrosis and tissue remodeling using a clinically relevant model of kidney injury, unilateral ischemia-reperfusion. This work highlights the benefits of inhibiting matrix polymerization, alone or in conjunction with cell-based therapies, as a novel approach to diminish the maladaptive responses to ischemic kidney injury that lead to chronic renal failure.

Keywords: acute kidney injury; chronic kidney disease; fibronectin; fibrosis; ischemia-reperfusion.

Fig. 1.

Inhibition of fibronectin polymerization abrogates kidney damage after unilateral ischemia-reperfusion (UIR). A: whole organ imaging and tissue-level analysis of fluorescence-labeled peptides demonstrates their delivery throughout the kidney. Scale bar = 50 μm. The epifluorescence scale represents radiant efficiency {×10⁹ [(photons/s/cm²/steradian)/(μW/cm²)]}. B: experimental timeline for UIR injury and pUR4 peptide treatment. C: quantitative PCR analysis of kidney lysates (graphs; left) showing the increase in both lipocalin-2 (Lcn2) and hepatitis A virus cellular receptor 1 (Havcr1) transcripts in injured (Inj) versus uninjured contralateral (Con) kidneys 14 days after UIR when mice were treated with PBS or III-11C (control peptide, 25 mg/kg). These expressions were diminished with pUR4 treatment (25 mg/kg). n = 6 for peptide-treated groups and n = 5 for others. *P < 0.04. Changes between injured kidneys were not significant and are not indicated on the graph. Representative images (right) of immunofluorescent staining (n = 5/group) likewise revealed an abrogation of injury-induced protein expression of both neutrophil gelatinase-associated lipocalin (NGAL)/Lcn2 and T cell immunoglobulin and mucin domain 1 (Tim-1)/ kidney injury molecule 1 (Kim-1) after pUR4 treatment. Scale bar = 50 μm. Arrows denote specific staining in tubular cells.

Fig. 2.

pUR4 attenuates postinjury fibrosis and immune cell infiltrate. A: picrosirius red staining was increased significantly after unilateral ischemia-reperfusion (UIR) but was decreased with pUR4 treatment. Imaging of second harmonic generation (SHG) of fibrillar collagen also demonstrated alleviation of fibrotic collagen deposition in animals treated with pUR4. Furthermore, hematoxylin and eosin (H&E) staining showed an expected deterioration in tubule morphology and increase in cell infiltration after injury, with a corresponding increase in CD45 immunostaining. pUR4 treatment reduced all of these injury-induced phenomena. n = 5/group. Scale bar = 100 μm. B: quantitation of picrosirius red staining showing the alleviation of fibrosis with pUR4 treatment, in addition to the partial recovery of the kidney weight-to-body weight ratio. n = 8 for sham or n = 11 for peptide treatment. *P < 0.001 vs. sham; +P < 0.001, III-11C vs. pUR4 treatment.

Fig. 3.

Injury-induced extracellular matrix organization and cellular activation are altered after pUR4 treatment. A: immunostaining illustrating the increase and reorganization of the fibronectin and collagen type I network as well as increase in α-smooth muscle actin (α-SMA)-positive cells in kidneys 14 days after unilateral ischemia-reperfusion (UIR). Treatment with pUR4 abrogated these changes in injured tissue. Scale bar = 50 μm. B: higher-magnification images showing the changes in fibronectin deposition and α-SMA-positive cells immediately surrounding glomeruli after injury, which were both attenuated with the inhibition of fibronectin polymerization. Scale bar = 100 μm.

Fig. 4.

Periglomerular cell activation and the big picture. A, left: vimentin was normally expressed in podocytes within glomeruli but was more highly detected in the interstitial space after injury (×20 images; scale bar = 50 μm). This change in the expression pattern was reduced with pUR4 treatment. Higher magnification (×80 images; scale bar = 100 μm) revealed that the majority of glomeruli were “encapsulated” by vimentin in injured kidneys, but this was significantly decreased with inhibition of fibronectin polymerization with pUR4. Masson’s trichrome was included for orientation and assessment of the tissue surrounding glomeruli. Right, the top graph shows the percentage of glomeruli surrounded by vimentin in each condition; no changes in the average total number of glomeruli/image were observed (bottom graph). Data were obtained from three ×10 images/kidney. n = 4–5/group. *P < 0.002 vs. sham, +P < 0.002, III-11C vs. pUR4 treatment. B: schematic of the timeline and major findings of the study. UIR, unilateral ischemia-reperfusion; CKD, chronic kidney disease.