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Glomerular expression of transforming growth factor-beta (TGF-beta) isoforms in mice lacking CD2-associated protein
Robert P Woroniecki1, Mario Schiffer, Andrey S Shaw
1Section of Pediatric Nephrology, Children's Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, NY 10467, USA.
Abstract:
Mice lacking CD2-associated protein (CD2AP-/-) develop glomerular lesions resembling human focal segmental glomerulosclerosis (FSGS) between 3-4 weeks of age and die approximately 2 weeks later from massive proteinuria and renal failure. The mechanisms involved in the glomerular injury in this model are unclear. In this study, we used laser capture microdissection (LCM) and real-time PCR, and examined expression of TGF-ss isoforms in CD2AP-/- mice at the level of isolated glomeruli. Total RNA yield from cryosections of 30 glomeruli was 10.71 ng (SD, 5.45) in CD2AP+/+ group (n =7), and 4.20 ng (SD, 2.04) in CD2AP-/- group (n =8), p =0.008. Expression of TGF-ss1 mRNA was increased 1.5-fold in the whole kidney (p =0.030), and twofold in isolated CD2AP-/- glomeruli (p =0.026). Whole kidney mRNA of TGF-ss receptor I (RI) and II (RII) was not different in CD2AP-/- and CD2AP+/+ animals, but it was increased in CD2AP-/- glomerular samples by 4.38-fold (p =0.001) and 11.37-fold (p =0.0163), respectively. By using LCM we confirmed increased glomerular expression levels of TGF-ss isoforms previously described by our group in glomeruli isolated by sieving in CD2AP KO mice and underscored the importance of local factors in the development of glomerulosclerosis.
Insights
Mice lacking CD2-associated protein develop kidney disease. Glomerular TGF-β1 and its receptors are upregulated, indicating local factors drive glomerulosclerosis in CD2AP-/- mice.
Area of Science:
- Nephrology
- Molecular Biology
- Genetics
Background:
- Mice lacking CD2-associated protein (CD2AP-/-) exhibit glomerular lesions similar to human focal segmental glomerulosclerosis (FSGS).
- The precise mechanisms underlying glomerular injury in this model remain largely unknown.
- Understanding these mechanisms is crucial for developing targeted therapies for FSGS.
Purpose of the Study:
- To investigate the expression of transforming growth factor-beta (TGF-β) isoforms and their receptors in the glomeruli of CD2AP-/- mice.
- To elucidate the role of local glomerular factors in the pathogenesis of FSGS in this model.
- To confirm previous findings using a more precise method for isolating glomeruli.
Main Methods:
- Laser capture microdissection (LCM) was employed to isolate glomeruli from CD2AP-/- and wild-type (CD2AP+/+) mice.
- Real-time PCR was utilized to quantify the mRNA expression levels of TGF-β isoforms and their receptors (RI and RII).
- RNA yield was assessed to ensure the quality and quantity of genetic material for analysis.
Main Results:
- A significant reduction in total RNA yield was observed in glomeruli from CD2AP-/- mice compared to controls.
- Glomerular expression of TGF-β1 mRNA was significantly increased (twofold) in CD2AP-/- mice.
- Expression of TGF-β receptor I and II mRNA was markedly upregulated in CD2AP-/- glomeruli (4.38-fold and 11.37-fold, respectively).
Conclusions:
- The study confirms increased glomerular expression of TGF-β isoforms and their receptors in CD2AP-/- mice.
- These findings highlight the critical role of local glomerular factors, particularly the TGF-β signaling pathway, in the development of glomerulosclerosis.
- LCM provides a robust method for analyzing gene expression in isolated glomeruli, advancing the study of kidney disease mechanisms.
