Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment

RESEARCH ARTICLE Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment Munkyung Kim1, Alessandro Piaia2, Neeta Shenoy3, David Kagan3, Berangere Gapp1, Benjamin Kueng1, Delphine Weber1, William Dietrich4, Iwona Ksiazek1* 1 Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Basel, Switzerland, 2 Preclinical Safety, Novartis Institute for Biomedical Research, Basel, Switzerland, 3 Preclinical Safety, Novartis Institute for Biomedical Research, Cambridge, Massachusetts, Unites States of America, 4 Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Cambridge, Massachusetts, Unites States of America * OPEN ACCESS Citation: Kim M, Piaia A, Shenoy N, Kagan D, Gapp B, Kueng B, et al. (2015) Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment. PLoS ONE 10(11): e0141231. doi:10.1371/journal.pone.0141231 Editor: David Long, UCL Institute of Child Health, UNITED KINGDOM Received: May 7, 2015 Accepted: October 5, 2015 Published: November 10, 2015 Copyright: © 2015 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome. Data Availability Statement: All relevant data are within the paper. Funding: Funding for this work was provided by the Novartis Institute of Biomedical Research (NIBR). The funder provided support in the form of salaries and research materials for authors (MK, AP, NS, DK, BG, BK, DW, WD and IK), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Introduction Alport syndrome is an inherited genetic disease which affects approximately 1 in 5000 people and is caused by mutations in the type IV collagen genes [1]. In particular, mutations in the type IV collagen α5 chain gene (COL4A5) are responsible for the X-linked form of the disease, which accounts for ~85% of the patients and mutations in the type IV collagen α3 or α4 chain gene (COL4A3 or COL4A4) lead to the autosomal form of the Alport syndrome [2]. Type IV collagen assembles primarily as α3α4α5 heterotrimers in the adult glomerular basement PLOS ONE | DOI:10.1371/journal.pone.0141231 November 10, 2015 1 / 17 Macrophage Contribution to Progression of Kidney Alport Disease Competing Interests: All authors were employees of the Novartis Institute of Biomedical Research at the time of the study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. membrane (GBM) and is one of the main structural components essential for GBM integrity and function. Mutations in any of the three collagen chains can result in defective assembly of the GBM leading to the renal pathology of Alport syndrome manifested by irregular thickening and splitting of the GBM, podocyte effacement, glomerulosclerosis with extracellular matrix deposition, kidney fibrosis, and ultimately, end stage renal disease (ESRD) early in life [3,4]. Col4a3-deficient (Col4a3KO) mice, one of murine models of Alport syndrome [5–8], are developed by gene targeting at the Col4A3 locus and raised on a 129/SvJ genetic background [6]. In the absence of type IV collagen α3, α4, and α5 chains, mice develop progressive glomerulonephritis as well as ESRD and die at an age of approximately 10 weeks [6,9]. The structural and functional manifestation of renal pathology of Col4a3KO mice closely resembles that of human Alport syndrome, making Col4a3KO mice an ideal model to understand Alport pathology. The translatability of Col4a3KO model for the autosomal recessive form of Alport syndrome is demonstrated by animal studies with Col4a3KO mice that have successfully assisted in identifying effective therapies for Alport patients. Well-established evidence comes from RAAS blockage with ACE inhibitors which delays progression to renal replacement therapies in humans with Alport syndrome [10,11] and is effective in delaying renal failure in Col4a3KO mice [9]. While i) human autosomal form of Alport syndrome is shown to affect males and females equally [12], and ii) mice bearing Col4a4 splice site mutation, another model of Alport syndrome, show similar progression of albuminuria in males and females [5], relatively little is known about sex-specific susceptibility to disease progression in Col4a3KO mice. Especially, sex of the mice was not specified in the previously published studies which Col4a3KO mice [6,7,13]. One of the goals of this study was to determine whether sex has a significant impact on the onset and progression of kidney disease in Col4a3KO mice. It is well established that interstitial inflammation is a prominent feature of progressive renal diseases including, Alport syndrome. As early as 1961, Whalen and colleagues reported the presence of CD68-positive foam cells in human Alport syndrome [14]. Foam cells belong to the monocyte-macrophage lineage and acquire their ‘foamy’ appearance owing to accumulation of fat. Extensive macrophage infiltration is also reported for the Col4a3KO kidney with a strong correlation to the severity of kidney injury and fibrosis [15,16]. In spite of the association of macrophages with Alport syndrome, the contribution o (...truncated)