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Reliable Detection of SGLT2 Protein by Knockout-Based Antibody Characterization
Takuo Hirose1,2, Hiroki Ito1, Akari Endo1
1Division of Nephrology and Hypertension (T.H., H.I., A.E., T.K., K.Y., R.I., A.K., I.O.-Y., W.Y., T.M.), Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan.
Background:
SGLT2 (sodium-glucose cotransporter 2) mediates renal glucose reabsorption, and its pharmacological inhibition exerts cardio- and renoprotective benefits. Despite widespread clinical interest, reliable detection of SGLT2 protein remains challenging due to concerns regarding antibody specificity.
Methods:
Eight commercially available anti-SGLT2 antibodies were evaluated by immunohistochemistry and Western blotting using kidneys and hearts from genetically engineered Sglt2-deficient mice and rats. Human kidney tissues, including renal cell carcinoma samples, were also examined.
Results:
Among the antibodies tested, ab306558 and HPA041603 showed specific immunostaining in rodent kidneys, with minimal background in wild-type tissues and complete absence of staining in Sglt2-deficient samples. However, ab306558 was unsuitable for human samples because of nonspecific staining. In renal cell carcinoma, HPA041603 detected SGLT2 immunostaining in proximal tubules of nontumor regions but not in tumor areas. Subcellular analyses revealed that SGLT2 was enriched within proximal tubular microvilli, partially overlapping with PDZK1IP1 (MAP17), but not with LRP2 (megalin) or NHE3. Western blotting identified an SGLT2-specific band at ≈55 kDa in rodent kidney lysates using ab306558, 20802, 24654-1-AP, and HPA041603 under optimized conditions, whereas no SGLT2-specific signals were detected in heart lysates. In contrast, ab85626 detected a weak ≈55 kDa band even in Sglt2-deficient kidneys. N-linked glycan removal shifted the SGLT2-specific band from ≈55 kDa to ≈45 kDa.
Conclusions:
HPA041603 consistently detected SGLT2 in rodent and human kidney tissues, whereas other antibodies showed limited specificity. Knockout-based antibody validation and optimized experimental conditions are essential for accurate interpretation of SGLT2 protein expression in experimental and translational studies.
