Human tissue and mouse models

Formalin-fixed, paraffin-embedded human kidney (n = 4) and liver (n = 3) tissue from normal margins of cancer resections were obtained from the Cleveland Clinic Lerner Research Institute Biorepository. Three transgenic mouse lines expressing a 47 kb human genomic fragment in a bacterial artificial chromosome (BAC) encompassing the promoter and coding regions of the human APOL1 gene for each G0, G1, or G2 alleles have been previously described [17, 18]. Each of the BAC-APOL1 transgenic lines were ≥10 generations backcrossed to FVB/Nj, a genetic background susceptible to HIVAN. The mouse HIVAN model used to induce proteinuria was the Tg26 HIVAN4 congenic [19] that develops proteinuria and progressive focal segmental glomerulosclerosis as the parental Tg26 model (Jackson Laboratory #22354) but disease progression is slower. For all studies, kidney disease was monitored weekly after weaning by measuring proteinuria (i.e. amount of protein in spontaneously voided urine) by urinalysis using diagnostic dipsticks (Uristix, Siemens Healthcare). The IACUC-approved humane endpoint for kidney disease in this model was proteinuria reaching 4+ on dipstick. No animal reached this humane endpoint before the predetermined study endpoint, as this study required an early stage of renal dysfunction (proteinuria on dipstick of 2+ to 3+) as functioning kidneys still capable of filtration and reabsorption were needed. All animals maintained normal weights and exhibited typical grooming and activity levels, and were not in pain or distress during the study. All animals were monitored twice a week by veterinary technicians not associated with this study for overall health and well-being, and no animals required analgesics or other supportive care. Each BAC-APOL1 transgenic mouse line was intercrossed with the HIVAN4 mouse model and were sacrificed at 8–10 weeks of age when their kidney disease progressed to proteinuria levels of ≥2+ by urine dipstick (G0 n = 15, G1 n = 11, G2 n = 9). Terminal urine, blood, and tissue collections were performed under deep isoflurane anesthesia followed immediately (while still under anesthesia) by euthanasia using cervical dislocation. Albuminuria was assessed by polyacrylamide gel electrophoresis of 1μl urine, followed by Coomassie staining. Use of human tissue was reviewed and approved by the Cleveland Clinic IRB (IRB-06-050). Informed consent was waived because tissue was considered discard and no identifiable data were collected. All animal studies were reviewed and approved by Institutional Animal Care and Use Committees at the Cleveland Clinic and Case Western Reserve University (IACUC-2430).