Data from: Quantification and comparison of anti-fibrotic therapies by polarized SRM and SHG-based morphometry in rat UUO model

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.肾间质纤维化（Renal interstitial fibrosis, IF）是多种慢性肾脏病（CKD）疾病进展过程中的重要病理表现。然而，对IF进行定量且可重复的分析仍是一项挑战，尤其是在进行性IF的实验动物模型中。本研究将传统偏振天狼星红形态计量法（polarized Sirius Red morphometry, SRM）与基于新型二次谐波生成（Second Harmonic Generation, SHG）的未染色组织形态计量法进行对比，用于定量分析大鼠5天单侧输尿管梗阻（unilateral ureteral obstruction, UUO）模型中的IF。为验证SHG检测IF中纤维状胶原成分的特异性，本研究采用免疫组化（Immunohistochemistry, IHC）对I、III、IV型胶原开展共定位分析。此外，本研究考察了偏振SRM与基于SHG的形态计量法在检测三种不同治疗方案的抗纤维化效果时的相关性、动态范围、灵敏度及检测能力。本研究共开展三项独立实验，分别使用三种作用机制不同的药物对动物进行处理：依那普利（enalapril, ENA, 15、30、60 mg/kg）、霉酚酸酯（mycophenolate mofetil, MMF, 2、20 mg/kg）以及结缔组织生长因子（connective tissue growth factor, CTGF）中和抗体EX75606（1、3、10 mg/kg）。研究结果显示，SHG信号与I、III型纤维状胶原存在显著共定位，但与非纤维状IV型胶原无共定位。以纤维化皮质面积百分比计算的定量IF结果显示，偏振SRM与基于SHG的形态计量法具有相似的响应特征。两种方法在三项药理学实验中均呈现极强的相关性（决定系数r²=0.89~0.96）。然而，与偏振SRM相比，基于SHG的形态计量法在治疗后展现出更大的动态范围及更高的IF降幅绝对值。综上，本研究证实，未染色肾组织的基于SHG的形态计量法在定量纤维状胶原方面与传统偏振SRM具有可比性，且在检测治疗诱导的IF降低方面灵敏度更高。因此，对未染色肾组织开展基于SHG的形态计量法，是传统偏振SRM用于IF定量分析的可靠替代方法。