Role of Prox1 in the Transforming Ascending Thin Limb of Henle's Loop during Mouse Kidney Development

<div><p>The homeobox transcription factor Prox1 is critical to the development of many embryonic organs and tissues, although current understanding of its expression in the developing renal medulla is limited. We examined the functional role of Prox1 during mouse kidney development with particular emphasis on the developing loop of Henle. Our data show that Prox1 is expressed in the transdifferentiating region from the NKCC2-positive thick ascending limb, into the CLC-K1-positive ascending thin limb of Henle’s loop beginning at embryonic day 18. From 1 to 14 days of age, Prox1-positive cells gradually disappeared from the papillary tip, and remained in the initial part of inner medulla after 21 days. In this transforming area, no Prox1 was observed in cells undergoing apoptosis but was expressed strongly in the remaining cells, which differentiated into ascending thin limb epithelial cells. <i>In vitro</i> and <i>in vivo</i> approaches showed that Prox1 expression increases where the osmolality is near optimal range, but decreases at below- or above-optimal ranges. Renal hypoosmolality induced by furosemide (NKCC2 inhibitor) inhibited Prox1 expression and delayed maturation of the ascending limb of Henle’s loop. Together, these studies suggest that Prox1 appears to be a critical stage specific regulator of specifying ascending thin limb cell fate and that its expression is regulated by osmolality.</p></div

Osmolality and Prox1 expression.

<p>(A) Urine osmolalities were measured in mice at postnatal days 1, 4, 7, 14, 21, 28, and 56 on <i>ad libitum</i> fluid intake. Within 1 week after birth, urine osmolality was no more than 500 mOsmol/kg H₂O; it began to increase from day 14, reaching peak levels at days 28 and 56. Each bar represents the mean of three independent experiments; error lines are SD. (B) At all developmental stages, Prox1 expression correlated closely with urine osmolality. R² = 0.8870; P = 0.0015</p

Water intake, body weight, and urine osmolality and volume of urine in mice maintained under different water intake conditions.

<p>Weight before treatment initiation was 22.44 ± 0.79. Values are means ± SD; n, no. of animals.</p><p>*<i>P</i> < 0.05 vs. control.</p><p>Water intake, body weight, and urine osmolality and volume of urine in mice maintained under different water intake conditions.</p

NKCC2 expression during mouse kidney development.

<p>Immunostaining of NKCC2 in the kidneys from 18-day-old fetuses (A) and from 1- (B), 4- (C), 7- (D), 14- (E) and 21-day-old pups (F) and adults (G). Scale bars: 200 μm. (A’) Inset: higer magnification view of rectangle in A, demonstrated that the descending thin limb (arrow) continues directly into the NKCC2-positive thick ascending limb. Scale bars: 10 μm.</p

Prox1 expression in the developing mouse kidney.

<p>Double immunofluorescence staining for CLC-K1 (A, green) and Prox1 (B, red), NKCC2 (D, green) and Prox1 (E, red), AQP1 (G, green) and Prox1 (H, red), AQP2 (J, green) and Prox1 (K, red), and CD31 (M, green) and Prox1 (N, red) in renal papilla of 4-day-old pups. Prox1 was co-expressed in CLC-K1-positive ATL (C) or NKCC2-positive TAL (F), but not in AQP1-positive DTL (I), AQP2-positive CD (L), and CD31-positive VR (O). Scale bars: 20 μm</p

Expression of Prox1 in the adult mouse kidney.

<p>(A-C) Single immunostaining for Prox1. Scale bars: 200 μm. Co, cortex; G, glomerulus; AA, arcuate artery; LV, lymphatic vessel. (A’) Inset: higer magnification view of rectangle in A. Scale bars: 10 μm. (D–O) Double immunofluorescence staining for Prox1 (red) and LYVE-1 (green), Prox1 (red) and NKCC2 (green), Prox1 (red) and AR (green), and Prox1 (red) and AQP2 (green). Blue counterstain: DAPI. Scale bars: 20 μm. OMCD, outer medullary collecting duct; IMCDi, initial inner medullary collecting duct; IMCDt, terminal inner medullary collecting duct. (P–Q) Western blot analysis of Prox1 in the outer medulla (OM), and initial (IMi), middle (IMm), and terminal (IMt) part of the inner medulla. Band intensity was normalized to β-actin. Each bar is the mean of three independent experiments; error lines are SD.</p

Regulation of Prox1 in the renal medulla of developing mice with reduced osmolality.

<p>Double immunofluorescence staining of Prox1 (red) and NKCC2 (green) in 7-day-old pups treated with vehicle or furosemide since birth. Prox1 was strongly expressed in the transforming NKCC2-positive TAL cells (A) and in the fully transformed NKCC2-negative ATL (C) in the vehicle group. In the furosemide-treated group, Prox1 was not expressed in the NKCC2-positive TAL cells in the base of the renal papilla due to delayed transformation of Henle’s loop (B); Prox1 was expressed in the transforming NKCC2-positive TAL cells in the tip of renal papilla (D). Scale bars: 20 μm. (J–K) Western blot analysis of Prox1 in 7-day-old pups treated with vehicle or furosemide since birth. The intensity of Prox1 immunoreactivity was significantly decreased in the furosemide-treated group vs. the vehicle group. Band intensity was normalized to β-actin. Values are means ± SD; n = 6 mice/group. *<i>P</i> < 0.05, experimental vs. vehicle.</p

Prox1 expression during mouse kidney development.

<p>(A-N) Immunofluorescence staining of Prox1 (red) in the kidneys from 18-day-old fetuses (A, E, I) and from 4- (B, F, J), 7- (C, G, K, M) and 21-day-old pups (D, H, L, N). Blue counterstain: DAPI. Scale bars: 20 μm. (O-P) Western blot analysis of Prox1 in the mouse renal medulla at postnatal days 1, 4, 7, 14, 21, 28, and 56. Band intensity was normalized to β-actin. Each bar is the mean of three independent experiments; error lines are SD.</p

Expression and distribution of Prox1 in response to changes in osmolality <i>in vitro</i>.

<p>Immunofluorescence staining of Prox1 (red) in MDCK cells from 150 (A–D), 300 (E–H), 600 (I–L), or 1200 (M–P) mOsmol/kg H₂O medium for 18 h. Prox1 was expressed in the nucleus and cytoplasm; nuclear intensity was strong in isotonic conditions but not in hypotonic or hypertonic conditions. Blue counterstain: DAPI. Scale bars: 20 μm. (Q–R) Western blot analysis of Prox1 in MDCK cells after 18 h in media of different osmolality (150–1200 mOsmol/kg H₂O). Prox1 was most strongly expressed in isotonic conditions and weakened with increasing or decreasing osmolality. Band intensity was normalized to β-actin. Each bar represents the mean of three independent experiments; error lines are SD.</p

Co-localization of Prox1 and PCNA in the NKCC2-positive TAL of the developing mouse kidney.

<p>Triple immunofluorescence staining for Prox1 (red), NKCC2 (white) and PCNA (green) in the renal papilla from 7-day-old pups. PCNA co-localized with Prox1 signals in the nuclei of TAL cells. Blue counterstain: DAPI. Scale bars: 20 μm.</p