Immunoexpression Pattern of Autophagy-Related Proteins in Human Congenital Anomalies of the Kidney and Urinary Tract

Abstract

The purpose of this study was to evaluate the spatiotemporal immunoexpression pattern of microtubule-associated protein 1 light chain 3 beta (LC3B), glucose-regulated protein 78 (GRP78), heat shock protein 70 (HSP70), and lysosomal-associated membrane protein 2A (LAMP2A) in normal human fetal kidney development (CTRL) and kidneys affected with congenital anomalies of the kidney and urinary tract (CAKUT). Human fetal kidneys (control, horseshoe, dysplastic, duplex, and hypoplastic) from the 18th to the 38th developmental week underwent epifluorescence microscopy analysis after being stained with antibodies. Immunoreactivity was quantified in various kidney structures, and expression dynamics were examined using linear and nonlinear regression modeling. The punctate expression of LC3B was observed mainly in tubules and glomerular cells, with dysplastic kidneys displaying distinct staining patterns. In the control group's glomeruli, LAMP2A showed a sporadic, punctate signal; in contrast to other phenotypes, duplex kidneys showed significantly stronger expression in convoluted tubules. GRP78 had a weaker expression in CAKUT kidneys, especially hypoplastic ones, while normal kidneys exhibited punctate staining of convoluted tubules and glomeruli. HSP70 staining varied among phenotypes, with dysplastic and hypoplastic kidneys exhibiting stronger staining compared to controls. Expression dynamics varied among observed autophagy markers and phenotypes, indicating their potential roles in normal and dysfunctional kidney development.

Keywords: CAKUT; GRP78; HSP70; LAMP2A; LC3B; autophagy; congenital anomalies of the kidney and urinary tract; nephrogenesis.

Figure 1

Human fetal kidney immunofluorescence staining using the microtubule-associated protein 1 light chain 3 beta (LC3B) antibody (a–d). Indicated on the 4′,6-diamidino-2-phenylindole-stained image (DAPI), the expression pattern of LC3B (green signal) is represented by arrows in the glomeruli (g), convoluted tubules (ct), proximal convoluted tubules (pct), distal convoluted tubules (dct), and dysplastic tubules (dt). Immunoexpression of LC3B, DAPI staining, and merged LC3B and DAPI in control (CTRL) at the 30th dw (a), horseshoe kidney (HK) at the 27th dw (b), dysplastic kidney (DYS) at the 35th dw (c), duplex kidney (DU) at the 30th dw (d), and hypoplastic kidney (HYP) at the 37th dw (e). Inserts corresponding to the dashed boxes represent the most prominent region of protein expression. At a ×40 magnification, images were captured. The scale bar is 100 μm, which refers to all images. The LC3B (f) area percentages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidneys are displayed as the mean ± SD (vertical line) and underwent a Tukey’s multiple comparison test after a standard one-way ANOVA test. Significant differences were indicated by **** p < 0.0001. Ten sample images were evaluated at each time interval. By using linear and nonlinear regression modeling of area percentages over developmental stages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidney tissues at the 18th, 22nd, 24th, 27th, 30th, 35th, 37th, and 38th dw, the expression dynamics of LC3B (g) were demonstrated. The mean is used to present the data.

Figure 2

Human fetal kidney immunofluorescence staining using the lysosomal-associated membrane protein 2A (LAMP2A) antibody (a–d). Indicated on the 4′,6-diamidino-2-phenylindole-stained image (DAPI), the expression pattern of LAMP2A (green signal) is represented by arrows in the glomeruli (g), convoluted tubules (ct), proximal convoluted tubules (pct), distal convoluted tubules (dct), and dysplastic tubules (dt). Immunoexpression of LAMP2A, DAPI staining, and merged LAMP2A and DAPI in control (CTRL) at the 35th dw (a), horseshoe kidney (HK) at the 27th dw (b), dysplastic kidney (DYS) at the 35th dw (c), duplex kidney (DU) at the 30th dw (d), and hypoplastic kidney (HYP) at the 37th dw (e). Inserts corresponding to the dashed boxes represent the most prominent region of protein expression. At a ×40 magnification, images were captured. The scale bar is 100 μm, which refers to all images. The LAMP2A (f) area percentages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidneys are displayed as the mean ± SD (vertical line) and underwent a Tukey’s multiple comparison test after a standard one-way ANOVA test. Significant differences were indicated by **** p < 0.0001. Ten sample images were evaluated at each time interval. By using linear and nonlinear regression modeling of area percentages over developmental stages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidney tissues at the 18th, 22nd, 24th, 27th, 30th, 35th, 37th, and 38th dw, the expression dynamics of LAMP2A (g) were demonstrated. The mean is used to present the data.

Figure 3

Human fetal kidney immunofluorescence staining using the glucose-regulated protein 78 (GRP78) antibody (a–d). Indicated on the 4′,6-diamidino-2-phenylindole-stained image (DAPI), the expression pattern of GRP78 (green signal) is represented by arrows in the glomeruli (g), convoluted tubules (ct), proximal convoluted tubules (pct), distal convoluted tubules (dct), and dysplastic tubules (dt). Immunoexpression of GRP78, DAPI staining, and merged GRP78 and DAPI in control (CTRL) at the 22nd dw (a), horseshoe kidney (HK) at the 30th dw (b), dysplastic kidney (DYS) at the 35th dw (c), duplex kidney (DU) at the 24th dw (d), and hypoplastic kidney (HYP) at the 38th dw (e). Inserts corresponding to the dashed boxes represent the most prominent region of protein expression. At a ×40 magnification, images were captured. The scale bar is 100 μm, which refers to all images. The GRP78 (f) area percentages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidneys are displayed as the mean ± SD (vertical line) and underwent a Tukey’s multiple comparison test after a standard one-way ANOVA test. Significant differences were indicated by *** p < 0.001, **** p < 0.0001. Ten sample images were evaluated at each time interval. By using linear and nonlinear regression modeling of area percentages over developmental stages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidney tissues at the 18th, 22nd, 24th, 27th, 30th, 35th, 37th, and 38th dw, the expression dynamics of GRP78 (g) were demonstrated. The mean is used to present the data.

Figure 4

Human fetal kidney immunofluorescence staining using the heat shock protein 70 (HSP70) antibody (a–d). Indicated on the 4′,6-diamidino-2-phenylindole-stained image (DAPI), the expression pattern of HSP70 (green signal) is represented by arrows in the glomeruli (g), convoluted tubules (ct), proximal convoluted tubules (pct), distal convoluted tubules (dct), and dysplastic tubules (dt). Immunoexpression of HSP70, DAPI staining, and merged HSP70 and DAPI in control (CTRL) at the 22nd dw (a), horseshoe kidney (HK) at the 30th dw (b), dysplastic kidney (DYS) at the 35th dw (c), duplex kidney (DU) at the 24th dw (d), and hypoplastic kidney (HYP) at the 38th dw (e). Inserts corresponding to the dashed boxes represent the most prominent region of protein expression. At a ×40 magnification, images were captured. The scale bar is 100 μm, which refers to all images. The HSP70 (f) area percentages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidneys are displayed as the mean ± SD (vertical line) and underwent a Tukey’s multiple comparison test after a standard one-way ANOVA test. Significant differences were indicated by ** p < 0.01, **** p < 0.0001. Ten sample images were evaluated at each time interval. By using linear and nonlinear regression modeling of area percentages over developmental stages in the cortex of CTRL, HK, DYS, DU, and HYP fetal kidney tissues at the 18th, 22nd, 24th, 27th, 30th, 35th, 37th, and 38th dw, the expression dynamics of HSP70 (g) were demonstrated. The mean is used to present the data.