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Assessment of Kidney Function in Mouse Models of Glomerular Disease
Published on: June 30, 2018
Proteomic identification and immunolocalization of increased renal calbindin-D28k expression in OVE26 diabetic mice
Visith Thongboonkerd1, Shirong Zheng, Kenneth R McLeish
1Core Proteomics Laboratory, Kidney Disease Program, Department of Medicine, University of Louisville, Louisville, KY, USA. thongboonkerd@dr.com
Abstract:
Diabetic nephropathy is a common diabetic complication that is associated with alterations in the expression of several renal proteins and abnormal calcium homeostasis. We performed proteomic analysis to screen for global changes of renal protein expression in diabetic kidney. Proteins extracted from the whole kidney of 120-day-old OVE26 (a transgenic model of Type 1 diabetes) and FVB (non-diabetic background strain) mice were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by SYPRO Ruby staining (n = 5 in each group). Quantitative intensity analysis revealed 41 differentially expressed proteins, of which 30 were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. One of the altered proteins with the greatest magnitude of change was the calcium-binding protein, calbindin-D28k, whose expression was increased 6.7-fold in diabetic kidney. We confirmed the increase in calbindin-D28k expression in diabetic kidney by Western blot analysis. Immunohistochemical study demonstrated that calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules (DCT), collecting ducts (CD), and proximal convoluted tubules (PCT) in diabetic kidney. Calbindin-D28k plays a critical role in maintaining calcium homeostasis. The elevation in renal calbindin-D28k expression in our model may indicate a compensatory mechanism to overcome hypercalciuria in diabetes.
Insights
Diabetic nephropathy alters kidney proteins and calcium balance. This study found increased calbindin-D28k in diabetic mouse kidneys, suggesting a compensatory response to abnormal calcium levels.
Area of Science:
- Nephrology
- Molecular Biology
- Biochemistry
Background:
- Diabetic nephropathy is a significant complication of diabetes mellitus.
- It involves changes in renal protein expression and calcium homeostasis.
- Understanding these molecular changes is crucial for therapeutic development.
Purpose of the Study:
- To investigate global changes in renal protein expression in a mouse model of Type 1 diabetes.
- To identify specific proteins involved in diabetic kidney alterations.
- To elucidate the role of calcium-binding proteins in diabetic nephropathy.
Main Methods:
- Proteomic analysis using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and SYPRO Ruby staining.
- Protein identification via matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting.
- Validation of protein expression changes using Western blot and immunohistochemistry.
Main Results:
- Identified 41 differentially expressed proteins in the diabetic kidney, with 30 successfully identified.
- Observed a significant 6.7-fold increase in the calcium-binding protein calbindin-D28k expression.
- Confirmed elevated calbindin-D28k in tubular epithelial cells of distal convoluted tubules, collecting ducts, and proximal convoluted tubules.
Conclusions:
- Increased renal calbindin-D28k expression in diabetic nephropathy may represent a compensatory mechanism.
- This elevation could be a response to manage hypercalciuria in diabetes.
- Further research is warranted to explore the therapeutic implications of calbindin-D28k modulation.
