Chronic administration of bumetanide upregulates calbindin D28k mRNA and protein abundance in rat distal convoluted tubules.
OBJECTIVE
Calbindin D28k has been reported to be involved in transcellular calcium transport along the rat distal convoluted tubule (DCT). It has also been shown that administration of bumetanide is associated with hypercalciuria. The experiments reported here were designed to verify whether chronic infusion of this diuretic affects the gene expression and protein abundance of calbindin D28k along rat kidney DCT.


METHODS
Bumetanide was subcutaneously infused by an osmotic minipump for 7 days at a rate of 1.5 mg x h(-1) x kg(-1). cDNA was synthesized from total RNA extracted from DCT microdissected from collagenase-treated kidneys.


RESULTS
Calbindin D28k mRNA abundance, quantified by competitive PCR, was found to be 13.7 +/- 1.9 amol x ng(-1) total RNA in DCT of control rats (n = 4) as compared to 24.2 +/- 2.4 amol x ng(-1) total RNA in DCT of bumetanide- treated rats (n = 5) (p < 0.01). This effect was associated with a 52% increase (p < 0.005) in calbindin D28k protein abundance, as detected by Western blot performed on tissue slices from renal cortex (n = 4).


CONCLUSIONS
These data not only demonstrate that bumetanide upregulates the mRNA and protein abundance of calbindin D28k in rat DCT, but also suggest that DCT calcium reabsorption is increased following the administration of this loop diuretic.
Rizzo
Maria
M
Chair of Nephrology and Research Center for Cardiovascular Diseases, School of Medicine, Second University of Naples, Naples, Italy.
Metafora
Salvatore
S
Morelli
Francesco
F
Russo
Ferdinando
F
Ciani
Francesca
F
Capasso
Giovambattista
G
D000818:Animals; D002034:Bumetanide / Q000008:administration & dosage / Q000494:pharmacology*; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000652:urine; D004232:Diuretics / Q000008:administration & dosage / Q000494:pharmacology*; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D015854:Up-Regulation; D014556:Urine
C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D004232:Diuretics; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D002034:Bumetanide; D002118:Calcium