Advanced age portends a poorer prognosis in FSGS. To understand the impact of age on glomerular podocytes and parietal epithelial cells (PECs), experimental FSGS was induced in 3m-old mice (20-year old human age) and 27m-old mice (78-year old human age) by abruptly depleting podocytes with a cytopathic anti-podocyte antibody. Despite similar binding of the disease-inducing antibody, podocyte density was lower in aged FSGS mice compared to young FSGS mice. Activated PEC density was higher in aged versus young FSGS mice, as was the percentage of total activated PECs. Additionally, the percentage of glomeruli containing PECs with evidence of phosphorylated ERK and EMT was higher in aged FSGS mice. Extracellular matrix, measured by collagen IV and silver staining, was higher in aged FSGS mice along Bowman's capsule. However, collagen IV accumulation in the glomerular tufts alone and in glomeruli with both tuft and Bowman's capsule accumulation were similar in young FSGS and aged FSGS mice. Thus, the major difference in collagen IV staining in FSGS was along Bowman's capsule in aged mice. The significant differences in podocytes, PECs and extracellular matrixaccumulation between young mice and old mice with FSGS might explain the differences in outcomes in FSGS based on age.

The current studies used genetic fate mapping to prove that adult podocytes can be partially replenished following depletion. Inducible NPHS2-rtTA/tetO-Cre/RS-ZsGreen-R reporter mice were generated to permanently label podocytes with the ZsGreen reporter. Experimental focal segmental glomerulosclerosis (FSGS) was induced with a cytotoxic podocyte antibody. On FSGS day 7, immunostaining for the podocyte markers p57, synaptopodin, and podocin were markedly decreased by 44%, and this was accompanied by a decrease in ZsGreen fluorescence. The nuclear stain DAPI was absent in segments of reduced ZsGreen and podocyte marker staining, which is consistent with podocyte depletion. Staining for p57, synaptopodin, podocin, and DAPI increased at FSGS day 28 and was augmented by the ACE inhibitor enalapril, which is consistent with a partial replenishment of podocytes. In contrast, ZsGreen fluorescence did not return and remained significantly low at day 28, indicating replenishment was from a nonpodocyte origin. Despite administration of bromodeoxyuridine (BrdU) thrice weekly throughout the course of disease, BrdU staining was not detected in podocytes, which is consistent with an absence of proliferation. Although ZsGreen reporting was reduced in the tuft at FSGS day 28, labeled podocytes were detected along the Bowman's capsule in a subset of glomeruli, which is consistent with migration from the tuft. Moreover, more than half of the migrated podocytes coexpressed the parietal epithelial cell (PEC) proteins claudin-1, SSeCKS, and PAX8. These results show that although podocytes can be partially replenished following abrupt depletion, a process augmented by ACE inhibition, the source or sources are nonpodocyte in origin and are independent of proliferation. Furthermore, a subset of podocytes migrate to the Bowman's capsule and begin to coexpress PEC markers.

Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow-colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.