Transgenic overexpression of p23 induces spontaneous hydronephrosis in mice

Abstract

p23 is a cochaperone of heat shock protein 90 and also interacts functionally with numerous steroid receptors and kinases. However, the in vivo roles of p23 remain unclear. To explore its in vivo function, we generated the transgenic (TG) mice ubiquitously overexpressing p23. The p23 TG mice spontaneously developed kidney abnormalities closely resembling human hydronephrosis. Consistently, kidney functions deteriorate significantly in the p23 TG mice compared to their wild-type (WT) littermates. Furthermore, the expression of target genes for aryl hydrocarbon receptor (AhR), such as cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1A1) and cytochrome P450, family 1, subfamily B, polypeptide 1 (Cyp1B1), were induced in the kidneys of the p23 TG mice. These results indicate that the overexpression of p23 contributes to the development of hydronephrosis through the upregulation of the AhR pathway in vivo.

Figure 1

Generation of mice overexpressing p23. (a) Schematic representation of the p23 transgenic (TG) construct and a result of PCR genotyping are shown. The positions of restriction sites for cloning and linearization are indicated. Primers used for genotyping are represented by black arrows. Cytomegalovirus (CMV); CMV-IE enhancer; Ckβ, chicken β-actin promoter; pA, poly A signal. (b) Western blot analysis with p23 antibody shows a representative expression of endogenous and transgenic p23 in mouse tissues (p23 TG line G). Proteins were isolated from the lung (Lu), spleen (Sp), brain (Br), heart (He), liver (Li), muscle (Mu), thymus (Th), kidney (Ki), intestine (In), stomach (St) and testis (Te) of WT and p23 transgenic (TG) mice. GAPDH is used as a loading control. The representative data shown were obtained from the line G mice. (c) The p23 transgene expression patterns of four TG lines (lines B, C, D and G) were compared in various organs as indicated. The relative expression of transgene hemagglutinin (HA)-p23 was normalized to endogenous p23.

Figure 2

Transgenic (TG) mice overexpressing p23 exhibit hydronephrosis. (a) Kidney of p23 TG mice showed distension and dilatation of renal pelvis (white arrowheads) and calyces (white arrows). (b) p23 TG mice exhibited swollen kidneys with perinephric pseudocysts (blue arrowhead). yellow arrowhead, blood vessel; red arrowhead, ureter.

Figure 3

Histological analysis of hydronephrotic kidney of p23 transgenic (TG) mice. (a) Normal and hydronephrotic kidney sections from WT and p23 TG mice. The renal pelvis was grossly dilated in p23 TG mice. *The dilatation regions of the renal pelvis in hydronephrotic kidney of p23 TG mice. (b) Kidney sections from 10-month-old WT and p23 TG mice were stained with H&E. The cortex regions from normal and hydronephrotic kidneys of WT and p23 TG mice, respectively, are shown. Detailed images of sections from WT and hydronephrotic p23 TG kidney do not show detectable structural changes in the glomeruli (yellow arrowheads). (c) Immunohistochemical staining using an anti-HA antibody showing representative expression of the p23 protein in the kidney (Scale bar = 200 μm). yellow arrows, glomeruli; red arrows, distal convoluted tubules; blue arrows, proximal tubules; c, cortex; md, medulla; rp, renal pelvis; p, papilla.

Figure 4

Histological analysis of kidney and dynamic contrast computed tomography (CT) in WT (a–c) and p23 transgenic (TG) (d–f) mice. (a) Representative images after ink injection into the pyelocaliceal space of kidney. Higher magnification images of the ureter are shown as insets. (b) Precontrast image (a, d). Coronal CT image 30 s after contrast injection via tail vein showed collection of the contrast dye in the renal pelvis without significant dilatation (b). At 180 s after contrast injection, most of the contrast dye was washed out in the renal pelvis and filled in the bladder (c). Coronal CT images 30 s after contrast injection showed dye accumulation along the marked dilated renal pelvis (red arrowheads) (e). CT image 600 sec after contrast injection showed that the contrast dye still remained in the renal pelvis (f). Contrast emptying in the bladder was incomplete even though it occurred over a prolonged time. White arrowhead, kidney.

Figure 5

Hydronephrotic kidneys of p23 transgenic (TG) mice show renal failure. Blood urea nitrogen (BUN) and creatinine levels in serum from WT and p23 TG mice are shown. (a) BUN level in male p23 TG mice (43 ± 21 mg/dl, n = 18) was significantly higher than that of controls (30 ± 5 mg/dl, n = 17). This difference was not observed in female mice. (b) The creatinine level in male p23 TG mice (0.32 ± 0.31 mg/dl, n = 18) was markedly higher than that of controls (0.13 ± 0.03 mg/dl, n = 17). This difference was not observed in female mice. *P < 0.015; **P < 0.016.

Figure 6

Altered expression of hydronephrosis-related genes in kidneys of WT and p23 transgenic (TG) mice at 9 months of age. (a) mRNA transcripts of Cyp1B1, Aqp2, Avpr2, Id-2 and p23 in kidneys from WT (lanes 1–4) and p23 TG (lanes 5–8) mice were quantified by RT-PCR. Each lane represents transcripts from an individual mouse. The transcript level was normalized to Gapdh values. A representative graph is shown (WT, n = 4; TG, n = 4). (b) The protein expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (Cyp1A1), cytochrome P450, family 1, subfamily B, polypeptide 1 (Cyp1B1) and cyclooxygenase-2 (Cox-2) in kidneys from WT (lanes 1–4) and p23 TG (lanes 5–8) mice was analysed by Western blotting using the indicated antibodies. The expression level was normalized to β-actin values. A representative graph is shown (WT, n = 4; TG, n = 4). *P < 0.01; **P = 0.0117; ***P = 0.0131.