Neonatal nephron loss during active nephrogenesis - detrimental impact with long-term renal consequences

Abstract

Neonatal nephron loss may follow hypoxic-ischemic events or nephrotoxic medications. Its long-term effects on the kidney are still unclear. Unlike term infants, preterm neonates less than 36 weeks gestational age show ongoing nephrogenesis. We hypothesized that nephron loss during nephrogenesis leads to more severe renal sequelae than nephron loss shortly after the completion of nephrogenesis. Rats show nephrogenesis until day 10 of life resembling the situation of preterm infants. Animals were uninephrectomized at day 1 (UNX d1) resulting in nephron reduction during nephrogenesis and at day 14 of life (UNX d14) inducing nephron loss after the completion of nephrogenesis. 28 days after uninephrectomy the compensatory renal growth was higher in UNX d1 compared to UNX d14. Nephrin was reduced and collagen deposition increased in UNX d1. At 1 year of age, glomerulosclerosis and markers of tubulointerstitial damage were most prevalent in UNX d1. Moreover, the number of desmin-positive podocytes was higher and nephrin was reduced in UNX d1 indicating podocyte damage. Infiltration of inflammatory cells was heightened after UNX d1. Uninephrectomized animals showed no arterial hypertension. We conclude that neonatal nephron loss during active nephrogenesis leads to more severe glomerular and tubulointerstitial damage, which is not a consequence of compensatory arterial hypertension.

Figure 1

Glomeruli 28 days after uninephrectomy (UNX). (A) Perimeter of glomerular cross sections. (B) Cell number of glomerular cross sections. (C) Representative photomicrographs of PAS-stained renal tissue. UNX d1, uninephrectomy at day 1 of life with respective controls (Co d1). UNX d14, uninephrectomy at day 14 of life with respective controls (Co d14). n = 9 for UNX d1 and n = 9 for respective controls, n = 10 for UNX d14 and n = 6 for respective controls. Data are mean ± SEM. **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 2

Glomerular collagen IV deposition 28 days after uninephrectomy (UNX). (A) Computer-assisted quantification after immunohistochemical detection of collagen IV. (B) Representative photomicrographs of collagen IV-stained renal tissue. UNX d1, uninephrectomy at day 1 of life with respective controls (Co d1). UNX d14, uninephrectomy at day 14 of life with respective controls (Co d14). n = 8 for UNX d1 and n = 8 for respective controls, n = 10 for UNX d14 and n = 6 for respective controls. Data are mean ± SEM. ***p < 0.001 (Student’s t-test)

Figure 3

Detection of nephrin in glomeruli 28 days after uninephrectomy (UNX). (A), Computer-assisted quantification after immunohistochemical detection of nephrin. (B) Representative photomicrographs of nephrin-stained renal tissue. UNX d1, uninephrectomy at day 1 of life with respective controls (Co d1). UNX d14, uninephrectomy at day 14 of life with respective controls (Co d14). n = 9 for UNX d1 and n = 9 for respective controls, n = 9 for UNX d14 and n = 6 for respective controls. Data are mean ± SEM. ** p < 0.01 (Student’s t-test).

Figure 4

Renal fibrosis at 52 weeks of age. (A) Glomerulosclerosis score with representative photomicrographs of PAS-stained glomeruli. (B) mRNA expression analysis of collagen I in renal cortical tissue. UNX d1, uninephrectomy at day 1 of life. UNX d14, uninephrectomy at day 14 of life. Co, control group with two kidneys. n = 8 for UNX d1, n = 7 for UNX d14, n = 8 for controls. Data are mean ± SEM. *p < 0.05 (one-way ANOVA, followed by Bonferroni post hoc test).

Figure 5

Glomerular tuft-to-capsule adhesions and tubular casts at 52 weeks of age. (A) Quantification of the number of tubular casts in PAS-stained renal sections. Arrows point to tubular casts. (B) Evaluation of the percentage of glomeruli with tuft-to-capsule adhesions. Arrows point to tuft-to-capsule adhesions. (C) Exemplary photomicrographs of glomeruli of control group, UNX d1 and UNX d14 group. UNX d1, uninephrectomy at day 1 of life. UNX d14, uninephrectomy at day 14 of life. Co, control group with two kidneys. n = 8 for UNX d1, n = 7 for UNX d14, n = 8 for controls. Data are mean ± SEM. * p < 0.05, *** p < 0.001 (one-way ANOVA, followed by Bonferroni post hoc test).

Figure 6

Markers of podocyte damage at 52 weeks of age. (A) Glomerular desmin score assessed in PAS-stained renal sections. (B) Nephrin mRNA expression in renal cortical tissue. UNX d1, uninephrectomy at day 1 of life. UNX d14, uninephrectomy at day 14 of life. Co, control group with two kidneys. n = 8 for UNX d1, n = 7 for UNX d14, n = 8 for controls. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA, followed by Bonferroni post hoc test).

Figure 7

Renal immune cell infiltration at 52 weeks of age. (A) Quantification of glomerular and interstitial macrophage infiltration with exemplary photomicrographs of renal tissue stained for ED-1. (B) Quantification of tubulointerstitial T-cell infiltration. (C) Quantification of tubulointerstitial cytotoxic T-cell infiltration. UNX d1, uninephrectomy at day 1 of life. UNX d14, uninephrectomy at day 14 of life. Co, control group with two kidneys. n = 8 for UNX d1, n = 7 for UNX d14, n = 8 for controls. Data are mean ± SEM. * p < 0.05, ** p < 0.01 (one-way ANOVA, followed by Bonferroni post hoc test).