Uromodulin retention in thick ascending limb of Henle's loop affects SCD1 in neighboring proximal tubule: Renal transcriptome studies in mouse models of uromodulin-associated kidney disease

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Abstract

Uromodulin-associated kidney disease (UAKD) is a hereditary progressive renal disease which can lead to renal failure and requires renal replacement therapy. UAKD belongs to the endoplasmic reticulum storage diseases due to maturation defect of mutant uromodulin and its retention in the enlarged endoplasmic reticulum in the cells of the thick ascending limb of Henle's loop (TALH). Dysfunction of TALH represents the key pathogenic mechanism of UAKD causing the clinical symptoms of this disease. However, the molecular alterations underlying UAKD are not well understood. In this study, transcriptome profiling of whole kidneys of two mouse models of UAKD, UmodA227T and UmodC93F, was performed. Genes differentially abundant in UAKD affected kidneys of both Umod mutant lines at different disease stages were identified and verified by RT-qPCR. Additionally, differential protein abundances of SCD1 and ANGPTL7 were validated by immunohistochemistry and Western blot analysis. ANGPTL7 expression was down-regulated in TALH cells of Umod mutant mice which is the site of the mutant uromodulin maturation defect. SCD1 was expressed selectively in the S3 segment of proximal tubule cells, and SCD1 abundance was increased in UAKD affected kidneys. This finding demonstrates that a cross talk between two functionally distinct tubular segments of the kidney, the TALH segment and the S3 segment of proximal tubule, exists. Copyright:

Figures

  • Table 1. List of primer sequences used for RT-qPCR.
  • Table 2. Functional classification of differentially expressed genes in kidney of UmodA227T mutant line.
  • Table 3. Functional classification of differentially expressed genes in kidney of UmodC93Fmutant line.
  • Figure 1. Verification of DEGs, identified by transcriptome profiling of whole kidneys, by RT-qPCR. Data are shown as scatter dot plot with mean (n = 5 per group). Age of mice analyzed: four months. One-way-ANOVA with Tukey’s Multiple Comparison Post hoc Test: p vs. wild-type, *, p,0.05; **, p,0.01; ***, p,0.001. doi:10.1371/journal.pone.0113125.g001
  • Figure 2. Analysis of localization and protein abundance of ANGPTL7 in healthy and UAKD-affected kidneys. (A) ANGPTL7 was predominantly detected in the cytoplasmic compartment of tubular cells, predominantly in TALH cells, of wild-type mice. Compared to the staining intensity of ANGPTL7 in TALH cells of wild-type mice, TALH cells of Umod mutant mice exhibited a lower cytoplasmic staining intensity of ANGPTL7. Age of mice analyzed: four months. Umodwt: wild-type mouse; UmodC93F: homozygous UmodC93F mutant mouse. UMOD immunohistochemistry enabled identification of TALH segments. Serial kidney sections were used for ANGPTL7 and uromodulin immunohistochemistry and corresponding kidney regions are shown. C: renal cortex; P: renal papilla. Chromogen: DAB; nuclear staining: hemalum. (B) Protein abundance of ANGPTL7 in the outer medulla of kidneys of homozygous Umod mutant mice of both lines was decreased compared to wild-type mice. Signal intensities of ANGPTL7 were corrected for GAPDH signal intensities of the same PVDF-membrane. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type) = 1]. One-way-ANOVA with Tukey’s Multiple Comparison Post hoc Test: p vs. wild-type, **, p,0.01; ***, p,0.001. Age of mice analyzed: four months. doi:10.1371/journal.pone.0113125.g002
  • Figure 3. Analysis of localization and protein abundance of SCD1 in healthy and UAKD-affected kidneys. (A) SCD1 was detected in the cytoplasmic compartment selectively of proximal tubular cells (in the straight S3 segment). In the kidney of the homozygous UmodC93F mutant mouse, SCD1 appeared to be abundant in a larger fraction of proximal tubular cells compared to the kidney of the wild-type mouse, and the average staining intensity of SCD1 positive cells appeared to be more prominent. Age of mice analyzed: four months. Umodwt: wild-type mouse; UmodC93F: homozygous UmodC93F mutant mouse. Uromodulin immunohistochemistry enabled identification of TALH segments. Proximal tubule segment are morphologically characterized by luminal microvilli. Chromogen: DAB for SCD1, Vector RED for UMOD; nuclear staining: hemalum. (B) Protein abundance of SCD1 in whole kidney lysate of homozygous Umod mutant mice of both lines was increased compared to wild-type mice. Signal intensities of SCD1 were corrected for GAPDH signal intensities of the same PVDF-membrane. Mean of protein abundance of wild-type mice was set on a value of 1 [mean (wild-type) = 1]. One-way-ANOVA with Tukey’s Multiple Comparison Post hoc Test: p vs. wild-type, **, p,0.01; ***, p,0.001. Age of mice analyzed: four months. doi:10.1371/journal.pone.0113125.g003
  • Figure 4. Evaluation of the role of TALH-dysfunction derived salt wasting state on renal Scd1 transcript abundance. Increased Scd1 transcript abundance in kidneys of homozygous Slc12a1I299T mutant mice compared to the kidneys of littermate controls were detected by RT-qPCR analysis. Data are shown as scatter dot plot with mean (n = 5 per group). Age of mice analyzed: three months. Student’s t test: p vs. wild-type, *, p,0.05. doi:10.1371/journal.pone.0113125.g004

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Horsch, M., Beckers, J., Fuchs, H., Gailus-Durner, V., De Angelis, M. H., Rathkolb, B., … Kemter, E. (2014). Uromodulin retention in thick ascending limb of Henle’s loop affects SCD1 in neighboring proximal tubule: Renal transcriptome studies in mouse models of uromodulin-associated kidney disease. PLoS ONE, 9(11). https://doi.org/10.1371/journal.pone.0113125

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