Fig 1.
Expression of Prox1 in the adult mouse kidney.
(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.
Fig 2.
Prox1 expression during mouse kidney development.
(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.
Fig 3.
NKCC2 expression during mouse kidney development.
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.
Fig 4.
Prox1 expression in the developing mouse kidney.
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
Fig 5.
Prox1 during maturation of the AL of Henle’s loop in mouse kidney development.
Triple immunofluorescence staining of Prox1 (red), CLC-K1 (green), and THP (white) in the renal papilla of 14-day-old pups. Prox1 was observed in the transforming region from TAL to ATL (F–J) and was not expressed in the mature TAL (A–E) or mature ATL (K–O). Blue counterstain: DAPI. Scale bars: 20 μm
Fig 6.
Cells undergoing apoptosis in the Prox1- and NKCC2-positive TAL of the developing mouse kidney.
(A-O) Triple immunofluorescence staining for Prox1 (red), NKCC2 (white) and TUNEL (green) in the renal papilla from 7-day-old pups. Arrows indicate TUNEL-positive nuclei that did not co-localize with Prox1. Blue counterstain: DAPI. Scale bars: 20 μm. (P) Cells undergoing apoptosis are expressed as a percentage of the Prox1-positive TAL cells. Values are means ± SD.
Fig 7.
Co-localization of Prox1 and PCNA in the NKCC2-positive TAL of the developing mouse kidney.
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.
Fig 8.
Osmolality and Prox1 expression.
(A) Urine osmolalities were measured in mice at postnatal days 1, 4, 7, 14, 21, 28, and 56 on ad libitum 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
Fig 9.
Expression and distribution of TonEBP in response to changes in osmolality in vitro.
Immunofluorescence staining of TonEBP (green) in MDCK cells from 150 (A–D), 300 (E–H), 600 (I–L), or 1200 (M–P) mOsmol/kg H2O medium for 18 h. With an increase in osmolality, TonEBP tanslocated to the nucleus. Blue counterstain: DAPI. Scale bars: 20 μm. (Q–R) Western blot analysis of TonEBP in MDCK cells for 18 h in media of different osmolality (150–1200 mOsmol/kg H2O). TonEBP decreased in hypotonic conditions and increased in hypertonic conditions. Band intensity was normalized to β-actin. Each bar represents the mean of three independent experiments; error lines are SD.
Fig 10.
Expression and distribution of Prox1 in response to changes in osmolality in vitro.
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.
Table 1.
Water intake, body weight, and urine osmolality and volume of urine in mice maintained under different water intake conditions.
Fig 11.
Expression and distribution of Prox1 in the renal medulla of adult mice by osmolality in vivo.
Double immunofluorescence staining of Prox1 (red) and CLC-K1 (green) in control, water-restricted, and water-loaded mice for 7 days. In the control (A, D) and water-restricted mice (B, E), Prox1 immunoreactivity in ATL occurred only in the initial part of the renal medulla. However, Prox1 was also expressed in the terminal part of the renal medulla in water-loaded mice (C, F). Scale bars: 20 μm. (G–J) Western blot analysis of Prox1 in water-restricted or water-loaded mice after 7 days. Prox1 immunoreactivity significantly decreased in water-restricted mice and increased in water-loaded mice versus normal mice. Prox1 band intensity was normalized to β-actin. Values are means ± SD; n = 6 mice/group. *P < 0.05, experimental vs. control.
Fig 12.
Regulation of Prox1 in the renal medulla of developing mice with reduced osmolality.
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. *P < 0.05, experimental vs. vehicle.
Table 2.
Effect of furosemide on body weight, kidney size, and urine osmolality.
Fig 13.
Prox1 is required for transdifferentiation of the AL of Henle’s loop in the renal medulla during mouse kidney development.
Prox1 plays an important role in the maturation of the AL of Henle's loop. During the first 2 weeks after birth, some simple cuboidal epithelial cells (Sm. Cb. Ep.) of the TAL are removed by apoptosis while surviving cells are converted into the simple squamous epithelial cells (Sm. Sq. Ep.) of the ATL. Prox1 activity in TAL cells is essential for cell survival and transdifferentiation into the ATL of Henle’s loop. The location and distribution of Prox1-expressing cells is changed by increasing medullary osmolality according to the maturation of the ascending limb of Henle’s loop.