We used Rapp DSS rats (Ontology: SS/JrHsdMcwi, RS:0002576) that have been inbred for more than 50 generations at the Medical College of Wisconsin. This strain is a widely used and physiological model for the study of salt-sensitive hypertension and kidney injury (3133). Male rats were obtained at weaning from colonies under controlled environmental conditions with parents and offspring. Rats were fed a purified AIN-76A rodent food (#D113755, Dyets Inc., Bethlehem, PA) containing 0.4% NaCl with water provided ad libitum. At 7 to 8 weeks of age, the salt content of the chow was either maintained at 0.4% (in the group fed a normal diet) or increased to 4.0% NaCl (Dyets Inc., #D113756) for either 7 or 21 days. Urine samples were collected for 24 hours using metabolic cages (40615; Laboratory Products) to measure albuminuria. Albuminuria was quantified using an Albumin Blue 580 (Molecular Probes) fluorescence assay. At the completion of the study, rats were surgically prepared by cardial perfusion, blood was flushed out using phosphate-buffered saline (PBS), and rats were humanely euthanized. The kidneys were removed, and part of the left kidneys was formalin-fixed and paraffin-embedded. Tissue sections were deparaffinized and stained with hematoxylin and eosin for analysis of kidney injury and glomerular morphology. The right and the other half of the left kidneys were removed and used for glomeruli and cortex fraction isolation, respectively. C3 immunohistochemistry was performed as described previously (34). Animal use and welfare procedures adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.

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