Age-related changes in expression in renal AQPs in response to congenital, partial, unilateral ureteral obstruction in rats

Abstract

Previously we demonstrated that neonatally induced partial unilateral ureteral obstruction (PUUO) in rats is associated with changes in the abundance of renal acid-base transporters that were paralleled by reduction in renal functions dependent on the severity and duration of obstruction. The aim of the present study was to identify whether changes in renal aquaporin abundance are age-dependent. Semiquantitative immunoblotting and immunohistochemistry were used to examine the changes in abundance of AQP1, AQP2, p-S256AQP2 (AQP2 phosphorylated at consensus site Ser(256)) and AQP3 in the kidneys of rats with neonatally induced PUUO within the first 48 h of life, and then monitored for 7 or 14 weeks. Protein abundance of AQP2 and AQP3 increased in both obstructed and non-obstructed kidneys 7 weeks after induction of neonatal PUUO (PUUO-7W). In contrast, AQP1 and AQP2 protein abundance in the obstructed kidney were reduced after 14 weeks of PUUO (PUUO-14W). Importantly, pS256-AQP2 protein abundance was reduced in obstructed kidneys of both PUUO-7W and PUUO-14W. Immunohistochemistry confirmed the persistent pS256-AQP2 downregulation in both PUUO-7W and PUUO-14W rats. The study shows that the protein abundance of AQP1, AQP2, and AQP3 in the obstructed kidney is increased in PUUO-7W, which may be a compensatory phenomenon and reduced in PUUO-14W rats suggesting a time-/age-dependent dysregulation in response to PUUO. pS256-AQP2 protein abundance is reduced consistent with obstruction-induced direct effects in the apical part of the collecting duct principal cells in response to PUUO.

Fig. 1

Semiquantitative immunoblotting of membrane fractions of obstructed whole kidneys (OBS) and contralateral non-obstructed kidneys (CON) from (partial unilateral ureteral obstruction) PUUO-7W and PUUO-14W rats, and kidneys from matched sham-operated control rats (SHAM). a and c Immunoblots reacted with affinity-purified AQP2 antibody revealed 29- and 35- to 50-kDa bands. b Densitometry analysis in PUUO-7W revealed a marked increase in the abundance of AQP2 to 143 ± 12% in OBS and 125 ± 7% in CON (P < 0.05 separately, compared with SHAM). d Densitometry analysis in PUUO-14W revealed a marked decrease in the abundance of AQP2 to 80 ± 8% in OBS (P < 0.05 compared with SHAM). In CON, AQP2 expression did not change compared with SHAM. Immunohistochemistry of AQP2 in the collecting ducts of OBS, CON, and SHAM from 7- and 14-week-old PUUO and SHAM rats. In PUUO-7W the immunolabeling was intense in e OBS and f CON compared with g SHAM, whereas in PUUO-14W the immunolabeling was much weaker in h OBS and comparable in i CON compared with j SHAM. Magnification: ×250

Fig. 2

Semiquantitative immunoblotting of membrane fractions of obstructed whole kidneys (OBS) and contralateral non-obstructed kidneys (CON) from PUUO-7W and PUUO-14W rats, and kidneys from matched sham-operated control rats (SHAM). a and c Immunoblots reacted with affinity-purified phosphorylated AQP2 (pS256-AQP2) antibody revealed 29- and 35- to 50-kDa bands. b Densitometry analysis in PUUO-7W revealed a marked decrease in abundance of p-AQP2 to 78 ± 6% in OBS. d Densitometry analysis in PUUO-14W revealed a marked decrease in expression levels of pS256-AQP2 to 71 ± 6% in OBS (*P < 0.05 compared with SHAM, ^# P < 0.05 compared with CON). In CON, p-AQP2 expression did not change in either PUUO-7W or -14W. Immunohistochemistry of pS256-AQP2 in the principal cells of collecting duct (CD) of OBS, CON, and SHAM from 7- and 14-week-old PUUO and Sham rats. In the apical parts of the collecting duct principal cells of CD, pS256-AQP2 labeling was much weaker in OBS in both e PUUO-7W and h -14W rats compared with g, j SHAM. The immunolabeling in f, i CON was comparable to that seen in SHAM. Magnification: ×250

Fig. 3

Semiquantitative immunoblotting of membrane fractions of obstructed whole kidneys (OBS) and contralateral non-obstructed kidneys (CON) from PUUO-7W and PUUO-14W rats, and kidneys from matched sham-operated rats (SHAM). a, c Immunoblots reacted with affinity-purified AQP3 antibody and revealed 27- and 33- to 40-kDa bands. b Densitometry analysis in PUUO-7W revealed a marked increase in abundance of AQP3 to156 ± 21% in OBS and 152 ± 5% in CON (P < 0.05 separately, compared with SHAM). d Densitometry analysis in PUUO-14W revealed an unchanged abundance of AQP3. In PUUO-7W rats, the immunolabeling of AQP3 in the collecting duct was intense in e OBS and f CON compared with g SHAM, whereas in PUUO-14W the immunolabeling was comparable in h OBS and i CON compared with j SHAM. Magnification: ×250

Fig. 4

Semiquantitative immunoblotting of membrane fractions of obstructed whole kidneys (OBS) and contralateral non-obstructed kidneys (CON) from PUUO-7W and PUUO-14W rats, and kidneys from matched sham-operated control rats (SHAM). a, c Immunoblots reacted with affinity-purified AQP1 antibody revealed 29- and 35- to 50-kDa bands. b Densitometry analysis in PUUO-7W revealed increased abundance of AQP1 to 118 ± 2% in OBS (P < 0.05). In CON, AQP1 expression did not change. d Densitometry analysis in PUUO-14W revealed a markedly decreased abundance of AQP1 to 76 ± 8% in OBS and 83 ± 6% in CON (P < 0.05 separately, compared with SHAM). Immunohistochemistry of AQP1 in the proximal tubule of OBS, CON, and SHAM kidneys from 7- and 14-week-old PUUO and Sham rats. In PUUO-7W rats the immunolabeling was intense in e OBS, and comparable in f CON compared with g SHAM. In PUUO-14W the immunolabeling was much weaker in h OBS and i CON compared with j SHAM. Magnification: ×250