NDT Advance Access originally published online on December 10, 2008
Nephrology Dialysis Transplantation 2009 24(5):1422-1428; doi:10.1093/ndt/gfn676
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Splice variants of neuronal nitric oxide synthase are present in the rat kidney
1 Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL 32610 2 Core Proteomics Laboratory, University of Louisville, Louisville, KY 40208, USA 3 1st Department of Pediatrics, Semmelweis University, Budapest, Hungary 4 Chang Gung Memorial Hospital-Kaohsiung Medical Center, College of Medicine, Chang Gung University, Kaohsiung, Taiwan 5 Veterans Administration Medical Center, Louisville, KY 40206 6 Department of Medicine, University of Florida, Gainesville, FL 36210, USA
Correspondence and offprint requests to: Chris Baylis, Department of Physiology and Functional Genomics and Department of Medicine, 1600 SW Archer Road, Room M554, University of Florida, POB 100274, Gainesville, FL 36210, USA. Tel: +1-352-392-7869; Fax: +1-352-392-7935; E-mail: baylisc{at}ufl.edu
 |
Abstract |
|---|
Background. Decreased renal cortical neuronal NO synthase (nNOS) abundance/activity correlates with progression of chronic kidney disease (CKD) in a number of animal models.
Methods. Western blotting with both N-terminal and C-terminal antibodies, immunoprecipitation, proteomics, RT-PCR and in situ hybridization were used to identify nNOS splice variants in the rat kidney.
Results. We have identified two nNOS proteins and transcripts in the rat kidney; nNOS
(
160 kDa) and nNOSβ (140 kDa), a catalytically active exon-2 deletion variant, lacking both the PDZ and protein inhibitor of nNOS (PIN) domains. We also report that nNOSβ protein abundance is increased in the kidney at 11 weeks following 5/6th nephrectomy (5/6NX)-induced CKD while nNOS protein abundance is diminished. The transcript data parallel the protein data in 5/6NX. By in situ hybridization, there is abundant nNOS mRNA widely distributed throughout the normal kidney cortex, with very sparse nNOSβ mRNA confined to a few proximal tubules. In a second injury model (6 weeks after 5/6 renal mass reduction by combined right kidney ablation and infarction of 2/3 of the left kidney; 5/6 A/I), nNOS mRNA almost disappears from the kidney cortex while nNOSβ mRNA abundance increases in tubules and tubulo-interstitium.
Conclusion. The renal cortical nNOSβ protein is present in low abundance in the normal kidney and increases with injury, in an inverse pattern of change with the nNOS.
Keywords: 5/6 nephrectomy; in situ hybridization; nNOS; nNOSβ; proteomics
Received for publication: 31. 3.08
Accepted in revised form: 13.11.08