Aging increases UT-B urea transporter protein abundance in brains of male mice

Abstract

Facilitative UT-B urea transporters in the brain play an important role in regulating levels of urea in various cell types, including astrocytes. Numerous studies have reported increased UT-B RNA expression with aging and in neurological disorders, such as Alzheimer's Disease. However, much less is known about the effects of these conditions on UT-B transporter protein abundance. This current study compared the levels of UT-B RNA and protein in young and aged male C57BL/6 mice. Endpoint RT-PCR experiments showed UT-B RNA expression increased in both aged cortex and aged hippocampus. Importantly, these changes were coupled with an increase in protein abundance, as western blotting revealed that 30-35 kDa UT-B1 protein was significantly increased in aged mouse brain tissues compared with tissue from young animals. An increased UT-B1 protein abundance was observed in the hippocampus, cerebellum, frontal cortex, and occipital cortex. In contrast, no such changes were observed in the abundance of MCT1 short-chain fatty acid transporters in these aged tissues. These data therefore confirmed that specific increases in UT-B1 protein abundance occur in multiple regions of the aged male mouse brain. Further studies are now needed to determine cell-specific changes and the functional consequence of increased UT-B1 protein in aged brain tissues.

Keywords: UT‐B transporter; aging; brain; protein; urea.

FIGURE 1

Schematic diagram of the mouse UT‐B gene structure. The UT‐B gene contains 10 exons, which are differentially transcribed to produce either UT‐B1 or UT‐B2 transcripts. The positions of various forward (F1 and F4) and reverse (R5 and R7) primers used for detecting UT‐B isoforms in PCR experiments are also shown.

FIGURE 2

UT‐B1 and UT‐B2 RNA expression in young and aged mouse brain. (a) RT‐PCR experiments using F1/R5 UT‐B primer sets to detect both UT‐B1 and UT‐B2 RNA expression in young (3–5 months) and aged (18–24 months) mouse brain samples. +RT, +ve Reverse Transcriptase reaction; −RT, −ve Reverse Transcriptase reaction. (b) Bar graphs summarizing the densitometry data for mUT‐B1, mUT‐B2 and GAPDH expression in all cortex and hippocampus. * = p < 0.05, ** = p < 0.01.

FIGURE 3

Peptide inhibition of UT‐Bc19 antibody signals detected in the mouse brain. Western blots of mouse brain protein samples (~10 μg per lane) probed with 1:1000 UT‐Bc19 antibody, pre‐incubated with either non‐specific (NSP) immunizing peptide (UT‐A6) or specific (SP) immunizing peptide (c19). Strong 30–35 kDa and 100 kDa signals were detected with the UT‐Bc19 antibody after NSP incubation. In contrast, these signals were almost completely absent after SP incubation. NSP, Non‐specific peptide; SP, Specific peptide.

FIGURE 4

UT‐B RNA expression and protein abundance in C8D1A mouse astrocyte cells, and hippocampal tissue from young and aged mice. (a) RT‐PCR experiments using C8D1A astrocyte cell and aged mouse hippocampus cDNA samples. Using F4/R7 primers, no UT‐B expression was detected in C8D1A cells, whereas a strong PGK1 signal was detected. (b) Western blots of urea‐treated C8D1A, young (3–5 months) hippocampus and aged (18–24 months) hippocampus protein samples (~10 μg per lane) probed with 1:1000 UT‐Bc19 antibodies. The strong 30–35 kDa UT‐B signals detected in all hippocampus samples were absent in the C8D1A protein samples. In contrast, the non‐UT‐B 100 kDa signal is present in C8D1A protein and was decreased with urea treatment. +, +ve Reverse Transcriptase reaction; −, −ve Reverse Transcriptase reaction; AMH, Aged Mouse Hippocampus; Co, Control; 5, 5 mM urea (24 h); 10, 10 mM urea (24 h); 20, 20 mM urea (24 h); ag, aged; yo, young.

FIGURE 5

UT‐B protein abundance in cortex and hippocampus from young and aged mice. (a) Western blot of young (3–5 months) and aged (18–24 months) mouse cortex protein samples (~10 μg per lane) probed with 1:1000 UT‐Bc19 antibodies. Strong 30–35 kDa UT‐B signals were similar between young and aged samples. In contrast, non‐UT‐B 100 kDa signals were much reduced in aged samples. (b) Western blot of young (3–5 months) and aged (18–24 months) mouse hippocampus protein samples (~10 μg per lane) probed with 1:1000 UT‐Bc19 antibodies. Strong 30–35 kDa UT‐B signals were increased in aged hippocampus samples, whereas non‐UT‐B 100 kDa signals were not increased. (c) Bar graphs summarizing densitometry data (normalization using UT‐B signal/protein) values for Non‐UT‐B and UT‐B protein abundance. ag, aged; Yo, young. * = p < 0.05, ** = p < 0.01.

FIGURE 6

UT‐B and MCT1 protein abundance in specific brain regions from young and aged mice. Western blots of cerebellum, frontal cortex, occipital cortex, and parietal/temporal cortex protein samples (~10 μg per lane) probed with (a) 1:1000 UT‐Bc19 and (b) 1:2000 MCT1 antibodies. The abundance of the 30–35 kDa UT‐B protein was increased in all aged samples, whereas no such changes were observed for MCT1 protein. (c) Bar graphs summarizing densitometry values for UT‐B and MCT1 abundance across young and aged mouse cerebellum, hippocampus, frontal cortex, occipital cortex, and parietal/temporal cortex protein samples. CB, Cerebellum; FC, Frontal Cortex; OC, Occipital Cortex; PTC, Parietal/Temporal Cortex. * = p < 0.05.