Polycystic kidney disease: the chemical signals of polycystin 1 (PC1) attract monocytes for renal cysts, stimulating its growth and contributing to the deterioration of renal function

• Grant number: 13/16376-0
• Support Opportunities: Scholarships abroad - Research
• Start date: July 01, 2014
• End date: June 30, 2015
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Marcelo Ferreira Cassini
• Grantee: Marcelo Ferreira Cassini
• Host Investigator: Lloyd Garnet Cantley
• Host Institution: Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Institution abroad: Yale University, United States

Abstract

Introduction: Polycystic kidney disease (PKD) is one of the most common genetic disorders in humans, affecting over 500,000 people in the United States alone. It is characterized by progressive growth of kidney cysts leading to end stage renal disease in ~50% of the patients. Polycystin-1 (PC1) and polycystin-2 (PC2) are the protein products of the PKD1 and PKD2 genes respectively, with mutations in PKD1 accounting for the majority of autosomal dominant PKD (ADPKD). Knock-out of Pkd1 or Pkd2 in adult mice results in slow cyst growth over weeks to months, unless there is a superimposed stimulus for tubule cell proliferation such as acute kidney injury. Thus the rate of cyst growth in ADPKD cannot be determined solely by the cell autonomous increase in proliferation that occurs in the absence of polycystins, but must also be regulated by largely unidentified external signals. It has found that tubular cell proliferation during the repair phase of kidney injury is stimulated by alternatively activated macrophages that transiently surround the tubules, and then leave or apoptose once repair is complete. When the macrophages were depleted in the whole animal, the cyst-lining cell proliferation and cyst growth were substantially decreased. This finding has been replicated in a separate model of murine PKD and extended to show that kidneys from patients with ADPKD exhibit large numbers of M2 activated macrophages that can promote proliferation of cyst derived cells in vitro. We therefore believe that macrophages are providing an external signals that accelerates cyst growth in ADPKD, and that defining the pathways that stimulate macrophage homing to cystic kidneys and the macrophage-derived factors that promote cyst progression represents an new area of research focus that will identify novel therapeutic targets to slow the loss of kidney function in patients with ADPKD without impacting normal tubular cell or macrophage function. Objectives: To identify the mechanism that promotes macrophage homing to cystic kidneys and then generating knock-out mouse models to interrupt those signals in order to define the most effective approach to prevent macrophage accumulation and slow cyst growth. Material and Methods: Animal Models: We will use Pkd1fl/fl mice that will be crossed with Pkhd1-Cre mice for mediating kidney-specific deletion of the Pkd1 allele. Deletion will be confirmed by standard genotypic PCR analysis. Pkd1fl/fl; Pkhd1-Cre neonates will be used for the experiments. All experiments involving mice will be conducted as per Yale University Institutional Animal Care and Use Committee Guidelines and Procedures. Macrophage Depletion: 400¼ls/20 g body weight of liposomal suspension containing either clodronate or saline will be administered into 10-d old mice by intraperitoneal injection on alternate days. Macrophage Migration Assay: Naïve macrophages will be plated onto the top chambers of 6-well Transwell plates and allowed to attach overnight. The next day, the medium was replaced with DMEM-F12. The bottoms of the Transwell filters will be then wiped with a Q-tip to remove any macrophages that may have migrated through. The bottom wells will be then filled with serum-free DMEM/F12, DMEM/F12 +10% FBS, Pkd1fl/- -conditioned medium, or Pkd1-/--conditioned medium. The plates will be incubated at 37 °C for 12 h, and the top of the insert will be then wiped clean with a Q-tip to remove macrophages that had not migrated through the membrane. The wells will be then fixed in 4% formaldehyde at room temperature for 15 min, followed by eosin/hematoxylin staining and counting using a light microscope. Twenty random fields will be counted at 20x magnification. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CASSINI, MARCELO F.; KAKADE, VIJAYAKUMAR R.; KURTZ, ELIZABETH; SULKOWSKI, PARKER; GLAZER, PETER; TORRES, RICHARD; SOMLO, STEFAN; CANTLEY, LLOYD G.. Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease. JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, v. 29, n. 10, p. 2471-2481, OCT 2018. (13/16376-0)