title: Quantification and Comparison of Anti-Fibrotic Therapies by Polarized SRM and SHG-Based Morphometry in Rat UUO Model.
abstract: Renal interstitial fibrosis (IF) is an important pathologic manifestation of disease progression in a variety of chronic kidney diseases (CKD). However, the quantitative and reproducible analysis of IF remains a challenge, especially in experimental animal models of progressive IF. In this study, we compare traditional polarized Sirius Red morphometry (SRM) to novel Second Harmonic Generation (SHG)-based morphometry of unstained tissues for quantitative analysis of IF in the rat 5 day unilateral ureteral obstruction (UUO) model. To validate the specificity of SHG for detecting fibrillar collagen components in IF, co-localization studies for collagens type I, III, and IV were performed using IHC. In addition, we examined the correlation, dynamic range, sensitivity, and ability of polarized SRM and SHG-based morphometry to detect an anti-fibrotic effect of three different treatment regimens. Comparisons were made across three separate studies in which animals were treated with three mechanistically distinct pharmacologic agents: enalapril (ENA, 15, 30, 60 mg/kg), mycophenolate mofetil (MMF, 2, 20 mg/kg) or the connective tissue growth factor (CTGF) neutralizing antibody, EX75606 (1, 3, 10 mg/kg). Our results demonstrate a strong co-localization of the SHG signal with fibrillar collagens I and III but not non-fibrillar collagen IV. Quantitative IF, calculated as percent cortical area of fibrosis, demonstrated similar response profile for both polarized SRM and SHG-based morphometry. The two methodologies exhibited a strong correlation across all three pharmacology studies (r2 = 0.89-0.96). However, compared with polarized SRM, SHG-based morphometry delivered a greater dynamic range and absolute magnitude of reduction of IF after treatment. In summary, we demonstrate that SHG-based morphometry in unstained kidney tissues is comparable to polarized SRM for quantitation of fibrillar collagens, but with an enhanced sensitivity to detect treatment-induced reductions in IF. Thus, performing SHG-based morphometry on unstained kidney tissue is a reliable alternative to traditional polarized SRM for quantitative analysis of IF.
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: Hu Sheng
authors.identifier: http://orcid.org/0000-0001-5296-1573
authors.initials: HS
authors.lastname: Qian
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: Steve M
authors.initials: SM
authors.lastname: Weldon
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: Damian
authors.initials: D
authors.lastname: Matera
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: ChungWein
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Haichun
authors.initials: H
authors.lastname: Yang
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Fryer
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Agnes B
authors.initials: AB
authors.lastname: Fogo
authors.affiliation: CardioMetabolic Disease Research, Boehringer Ingelheim Pharmaceutics, Inc, Ridgefield, Connecticut, United States of America.
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Reinhart
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D001391:Azo Compounds / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004656:Enalapril / Q000627:therapeutic use; D005355:Fibrosis; D007674:Kidney Diseases / Q000188:drug therapy* / Q000473:pathology*; D008297:Male; D009173:Mycophenolic Acid / Q000627:therapeutic use; D024041:Non-Fibrillar Collagens / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014517:Ureteral Obstruction / Q000188:drug therapy* / Q000473:pathology*
chemical_list: D000911:Antibodies, Monoclonal; D001391:Azo Compounds; D024041:Non-Fibrillar Collagens; D004656:Enalapril; C062246:Direct Red 81; D003094:Collagen; D009173:Mycophenolic Acid