Wnt4 is a novel biomarker for the early detection of kidney tubular injury after ischemia/reperfusion injury

Abstract

Earlier intervention after acute kidney injury would promote better outcomes. Our previous study found that Wnt proteins are promptly upregulated after ischemic kidney injury. Thus, we assessed whether Wnt4 could be an early and sensitive biomarker of tubular injury. We subjected mice to bilateral ischemia/reperfusion injury (IRI). Kidney and urinary Wnt4 expression showed an early increase at 3 hours and increased further at 24 hours post-IRI and was closely correlated with histopathological alterations. Serum creatinine slightly increased at 6 hours, indicating that it was less sensitive than Wnt4 expression. These data were further confirmed by clinical study. Both kidney and urinary Wnt4 expression were significantly increased in patients diagnosed with biopsy-proven minimal change disease (MCD) with tubular injury, all of whom nevertheless had normal estimated glomerular filtration rate (eGFR) and serum creatinine. The increased Wnt4 expression also strongly correlated with histopathological alterations in these MCD patients. In conclusion, this is the first demonstration that increases in both kidney and urinary Wnt4 expression can be detected more sensitively and earlier than serum creatinine after kidney injury. In particular, urinary Wnt4 could be a potential noninvasive biomarker for the early detection of tubular injury.

Figure 1. Characterization of mice after IRI.

(a) Serum creatinine at different time points in sham-operated and IRI mice. (b) Survival (%) of IRI mice. (c) Serum creatinine and (d) BUN level at different time points after 20 minutes of bilateral renal ischemia or sham operation. (e) Representative images of HE-stained sections at different time points after 20 minutes of bilateral renal ischemia (magnification, 200x). Scale bar, 100 μm. (f) Renal tubular injury scores from HE staining. The data are expressed as the mean ± SD. *P < 0.05, ***P < 0.001 versus the sham group (n = 8 in each group).

Figure 2. Kidney Wnt4 expression markedly increases in IRI mice, in correlation with tubular injury and kidney KIM-1 expression.

(a) Representative immunofluorescence images of kidney Wnt4 and KIM-1 in the IRI mice at different time points and in sham-operated mice (magnification, 200x). Scale bar, 100 μm. (b) Quantification of the immunofluorescence staining of Wnt4 in each group. (c) Quantification of the immunofluorescence staining of KIM-1 in each group. (d) Correlation between kidney Wnt4 expression and tubular injury. (e) Correlation between kidney Wnt4 and KIM-1 by immunofluorescence staining. (f) Western blot analysis and quantification of kidney Wnt4 expression in each group. (g) Western blot analysis and quantification of kidney KIM-1 expression in each group. (h) Correlation between kidney Wnt4 expression and tubular injury. (i) Correlation between kidney Wnt4 and KIM-1 expression by western blot. The gels were run under the same experimental conditions. *P < 0.05, **P < 0.01, ***P < 0.001 versus the sham group (n = 8 in each group).

Figure 3. Enhanced expressed Wnt4 was localized in both the injured proximal and distal tubules following IRI, and almost all TUNEL positive cells and majority of Ki67 positive cells co-labeled with Wnt4.

(a) Representative images of TUNEL staining (brown) in each group (magnification, 400x). (b) Quantification of TUNEL-positive cells in each group. (c) Quantification of Ki67-positive cells in each group. (d) Representative Ki67 immunofluorescence images in each group (magnification, 200x). (e) Co-staining for TUNEL and Wnt4 (magnification, 200x). (f) Co-staining for Ki67 and Wnt4 (magnification, 200x). (g) Co-staining for Wnt4 and segment-specific tubular markers, AQP1 (h) and NCC, in injured kidneys 12 hours after IRI (magnification, 200x). All scale bars, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 versus the sham group (n = 8 in each group).

Figure 4. Urinary Wnt4 excretion is significantly elevated early in IRI mice, in correlation with tubular injury and urinary KIM-1.

(a) Western blot analysis and quantification of urinary Wnt4 in each group. (b) Western blot analysis and quantification of urinary KIM-1 in each group. (c) ELISA analysis of urinary Wnt4 normalized to uCr in each group. (d) ELISA analysis of urinary KIM-1 normalized to uCr in each group. (e) Correlation between urinary Wnt4 levels and tubular injury. (f) Correlation between urinary Wnt4 excretion and kidney Wnt4 expression. (g) Correlation between urinary Wnt4 and KIM-1 excretion in mice. Cropped blots are shown (full-sized blots are presented in Supplementary Fig. S5). *P < 0.05, **P < 0.01, ***P < 0.001 versus the sham group (n = 8).

Figure 5. Kidney Wnt4 is upregulated in MCD patients with early AKI, similar to the expression of KIM-1.

(a) Representative light microscopy images of HE, PAS and Masson-stained sections of the renal tissue in MCD patients with or without tubular injury (magnification, 200x). Scale bar, 100 μm. (b) Renal tubular injury score from HE staining. (c) Quantification of the fibrotic area from Masson staining. (d) Immunohistochemistry of kidney Wnt4 and KIM-1 (magnification, 200x). Scale bar, 100 μm. (e) Quantification of immunohistochemistry for Wnt4. (f) Quantification of immunohistochemistry for KIM-1. (g) Correlation between kidney Wnt4 expression and tubular injury in MCD patients. (h) Correlation between kidney Wnt4 and KIM-1 expression by immunohistochemistry. ***P < 0.001 versus MCD only.

Figure 6. Urinary Wnt4 is elevated in MCD patients with early AKI but normal serum creatinine, similar to KIM-1.

(a) Western blot analysis and quantification of urinary Wnt4 excretion in MCD patients with or without tubular injury. (b) Western blot analysis and quantification of urinary KIM-1 excretion in MCD patients with or without tubular injury. (c) ELISA analysis of urinary Wnt4 normalized to uCr in each group. (d) ELISA analysis of urinary KIM-1 normalized to uCr in each group. (e) Correlation between urinary Wnt4 level and tubular injury. (f) Correlation between urinary Wnt4 excretion and kidney Wnt4 expression (by immunochemical staining). (g) Correlation between urinary Wnt4 and KIM-1 excretion. Cropped blots are shown (full-sized blots are presented in Supplementary Fig. S5). *P < 0.05, **P < 0.01, versus MCD only.