Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background

Abstract

Chronic kidney disease (CKD) begins with renal injury; the progression thereafter depends upon a number of factors, including genetic background. Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and as such is considered a model of CKD. We used an improved reversible unilateral ureteral obstruction (rUUO) model in mice to study the strain dependence of development of CKD after obstruction-mediated injury. C57BL/6 mice developed CKD after reversal of three or more days of ureteral obstruction as assessed by blood urea nitrogen (BUN) measurements (>40 mg/dl). In contrast, BALB/c mice were resistant to CKD with up to 10 days ureteral obstruction. During rUUO, C57BL/6 mice exhibited pronounced inflammatory and intrinsic proliferative cellular responses, disruption of renal architecture, and ultimately fibrosis. By comparison, BALB/c mice had more controlled and measured extrinsic and intrinsic responses to injury with a return to normal within several weeks after release of ureteral obstruction. Our findings provide a model that allows investigation of the genetic basis of events during recovery from injury that contribute to the development of CKD.

Fig. 1.

Development of chronic kidney disease (CKD) after varying times of ureteral obstruction followed by its reversal. C57BL/6 mice had their right ureter obstructed (RUO) for the indicated number of days followed by the release of this obstruction (RUOR), 7 days of recovery, and then removal of contralateral kidney function (left ureteral obstruction; LUO). Blood urea nitrogen (BUN) levels were measured post-LUO to assess function of the previously obstructed right kidney. C57BL/6 mice developed CKD with times of obstruction (t_O) ≥3 days (3d) as evidenced by persistently elevated BUN levels. Development of CKD exhibited a dose-response relationship with t_O. Error bars indicate SE. *P < 0.03 vs. RUOR. †P < 0.03 vs. 3d RUO. ‡P < 0.05 vs. 5d RUO.

Fig. 2.

Susceptibility to development of CKD after reversible unilateral ureteral obstruction (rUUO) in murine strains. C57BL/6 (blue lines) and BALB/c (red lines) mice underwent 6 days of RUO followed by RUOR (obstructed, solid lines) or surgical manipulation but no ureteral obstruction (sham, dashed lines). Seven days later, the contralateral left kidney function was removed (LUO), and BUN values were measured over time, as reflective of the function of the previously obstructed right kidney alone. The C57BL/6 strain demonstrated significant chronic loss of renal function after 6 days of rUUO, while the BALB/c strain was resistant to loss of function after rUUO. Error bars indicate SE. *P < 0.03 vs. RUOR. †P < 0.02 vs. sham. ‡P < 0.05 vs. BALB/c.

Fig. 3.

Hematoxylin and eosin (H&E) staining at various times following release of ureteral obstruction. A: representative H&E-stained sections from C57BL/6 and BALB/c at various times following release of 6 days of RUO (post-RUOR; ×20 magnification). In C57BL/6 mice, inflammatory cells persisted through 7 days post-RUOR and were accompanied by significant and lasting disruption of tubular architecture. In BALB/c mice, inflammation developed rapidly and then resolved. Tubular architecture was better preserved throughout with a return to near-normal structure by day 28 post-RUOR. B: average inflammation score at 0, 2.5, 7, and 28 days post-RUOR. H&E-stained sections from C57BL/6 and BALB/c mice were scored (0–3) for severity of global interstitial inflammation by a renal pathologist blinded to their origin (strain and rUUO time point). A score of 0 was used for absence of interstitial inflammation. Scores of 1, 2, or 3 reflected the presence of inflammation involving 1–25, 26–50, or >50% of the interstitium, respectively. *P < 0.05 vs. BALB/c (Kruskal-Wallis test).

Fig. 4.

F4/80⁺ cellular infiltration in rUUO. C57BL/6 and BALB/c mice underwent a 6 day obstruction followed by release and were studied using flow cytometry and immunohistochemistry at the indicated times post-RUOR. A, top: representative flow cytometric data showing side scatter (SSC-A) vs. F4/80 staining for whole kidney cell preparations at 0, 2, and 7 days post-RUOR. Representative control data with omission of anti-F4/80 antibody (unstained) is shown at the top. Bottom: summary of percentage of F4/80⁺ cells in whole kidney cell preparations at 0, 2, and 7 days after release of a 6-day obstruction in C57BL/6 (blue) and BALB/c (red) mice. Error bars indicate SE. Data from controls are shown for each strain (dashed lines). Controls for each strain included data from both contralateral kidneys as well as sham-operated kidneys as no difference was seen between either types of control. *P < 0.01 vs. control. **P < 0.05 vs. control. †P < 0.005 vs. BALB/c. B: kidney tissue sections stained at 7 days post-RUOR with F4/80 antibody.

Fig. 5.

Renal fibrosis after rUUO. A: interstitial fibrosis scores at 28 days post-RUOR in C57BL/6 (blue circles) and BALB/c (red circles) mice (see text for details). Bars indicate the mean score for each group. *P < 0.001 vs. BALB/c. Representative Sirius red-stained sections from each strain are shown (bottom). B: relative renal amounts of collagen III (α1) mRNA post-RUOR in C57BL/6 (blue line) and BALB/c mice (red line) compared with sham controls. Error bars indicate SE. *P < 0.05 vs. BALB/c.

Fig. 6.

Strain-dependent differences in S100A4 during recovery from ureteral obstruction. C57BL/6 and BALB/c mice underwent a 6-day obstruction followed by release and were studied using quantitative PCR and immunohistochemistry at the indicated times post-RUOR. A: S100A4 mRNA levels post-RUOR in C57BL/6 (blue line) and BALB/c mice (red line) relative to sham controls for each strain. Error bars indicate SE. C57BL/6 vs. BALB/c, P ≤ 0.01 at all time points. B: anti-S100A4 staining of paraffin-embedded tissue sections. S100A4 staining is observed in the interstitial space in C57BL/6 kidneys (white arrows) while remaining in the tubules and tubular lumina (white asterisks) in BALB/c kidneys. C: dual staining of frozen kidney sections with fluorescent-labeled anti-S100A4 (blue) and anti-laminin (red). S100A4 staining is observed traversing the tubular basement membrane (as outlined by anti-laminin staining) and in the interstitial space in C57BL/6 kidneys (white arrows).