011 - Prolonged nephrogenesis in offspring exposed to maternal low protein during gestation

Publication Number: 11.6446

Sage Timberline, University of Virginia, Charlottesville, VA, United States; Jennifer R Charlton, University of Virginia School of Medicine, charlottesville, VA, United States; Meredith P. Schuh, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States; Sunitha Yarlagadda, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States; Shalini Indugula, University of Cincinnati, Mason, OH, United States; Wyatt Johnson, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, United States; Kimberly deRonde, university of Virginia, Charlottesville, VA, United States

Background: Low birthweight is a risk factor for chronic kidney disease (CKD). Infants born growth restricted have a decreased nephron number, which has been modeled in the offspring of pregnant mice fed a low protein (LP) diet. However, the mechanism for this reduction in nephron number is unknown. Alterations in the duration of or signaling during nephrogenesis could provide a mechanism for the lower nephron number and resultant CKD.

Objective: To determine if nephrogenesis duration is affected by gestational protein restriction in offspring.

Design/Methods: Pregnant CD-1 dams were fed a normal protein (NP, 18%) or low protein (LP, 8%) diet from conception through lactation. The LP and NP offspring were euthanized on postnatal days (PN) 2-7 (n=4/group) as nephrogenesis is normally completed by PN4. Kidneys were prepared for histologic evaluation and stained with Periodic acid Schiff and Lotus tetragonolobus lectin to visualize the nephrogenic zone. Immunofluorescent staining on P4 kidneys (n=3/diet) was used to assess for the presence of nephron progenitor cells (NPC, Six2 with or without ZO-1), nascent nephron patterning (notch ligand Jag1) and canonical Wnt target/effector of Wnt signaling (transcription factor Lef1).

Results: The histologic appearance of the cap mesenchyme was absent in the NP group on PN4. However, it was not absent in the LP group until PN6. The NP group completed nephrogenesis, as evidenced by lack of Six2+ NPC and Jag1+ nascent nephrons, with only one sample having faint LEF1+ staining in the final differentiating structures (Figure). Conversely, Six2+/ZO-1+ NPC were seen in one LP animal, and all three LP animals demonstrated high levels of Jag1+ nascent nephrons with strong LEF1+ protein expression. This demonstrates that although the NPC pool was committed to differentiation, the completion of nephron formation was ongoing.

Conclusion(s): Despite their decreased nephron number, offspring exposed to low protein during gestation demonstrated a prolonged window of nephrogenesis, characterized by histologic evidence of immature structures and persistent expression of Six2, Jag1 and Lef1 in the differentiating NPC and nascent nephrons. These data suggest that there is a delay in the final wave of nephron differentiation. Unlike models of hyperglycemia and maternal diabetes, the NPC do not appear to be arrested in a progenitor state where they lack the ability to differentiate. This delayed formation of nephrons has several potential implications for susceptibility to nephron injury and loss in neonates born with growth restriction.

Immunofluorescent staining of LP and NP kidneys
fig_PAS.pdfSix2+ nephron progenitor cells, Jag1+ nascent nephrons, and Lef1+ expression indicate delayed but ongoing nephron formation in the LP kidneys. In comparison, the NP kidneys do not have expression of Six2 and Jag1 with minimal expression of Lef1.