Effect of Urodilatin on Cgmp Accumulation in the Kidney1
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Effect of Urodilatin on cGMP Accumulation in the Kidney1 Jun Koike, Hiroshi Nonoguchi, Yoshio Terada, Kimio Tomita,2 and Fumiaki Marumo myocytes (1 -4). The brain and liver have also been J, Koike, H, Nonoguchi, Y. Terada, K. Tomita, F. Mar- shown to produce ANP (6). The main target sites of umo, Second Department of Internal Medicine, Tokyo ANP in the kidney are the gbomeruli, the inner med- Medical & Dental University, Tokyo, Japan ubbary collecting ducts (IMCD), and the vascular sys- tem (1 -5.7- 1 2). Functional effects of ANP were also (J. Am. Soc. Nephrol. 1993; 3:1705-1709) reported in proximal convoluted tubules (PCT), prox- imal straight tubules, medullary thick ascending limbs (MAL). and cortical collecting ducts (1-4.13). Recently. a new native peptide. urodilatin, was ABSTRACT identified in human urine (14-18). Urodibatin is re- Urodibatin is found in the urine and is thought to be sistant to protease. which completely destroys un- produced in the kidney. The effect of urodilatin on nary ANP (1 9). This protease is rich in the brush cGMP accumulation in the kidney was investigated. border membrane of proximal tubules. Therefore, cGMP accumulation by urodilatin and atrial natri- urinary excretion of ANP is quite small, whereas uretic peptide (ANP) were compared with tubule urodibatin can be found In the urine. Urodibatin is a suspensions from renal cortex, outer medulla, inner 32-amino-acid peptide and a member of the ANP medulla, and microdissected nephron segments. family. Physiologic effects of urodilatin have been Urodibatin-stimulated cGMP accumulation was higher recently reported (15,20-22). Urodilatin had almost in tubule suspensions from the inner medulla than in equal ability to ANP in causing diuresis and natri- those from the cortex and outer medulla. The highest uresis. The concentration of urodibatin in plasma is too bow to detect, whereas that of ANP can be meas- accumulation stimulated by I 0 M urodilatin among ured, suggesting that urodibatin is a local, probably nephron segments was observed in glomerubi and of renal origin. hormone rather than a circulating inner medullary collecting ducts (1MCD). Small ac- one (14,15,17,18). cumulations were seen in proximal convoluted tu- Previous studies reported that ANP-like protein is bules and medullary thick ascending limbs. Urodila- produced in distal cortical nephrons, probably in dis- tin-stimulated cGMP accumulation was almost equal tab convoluted and connecting tubules ( 1 5.23). It Is to that stimulated by the same concentrations of ANP not known if this protein is urodilatin. However. it in these nephron segments. Urodilatin (10_8 M) did seems likely that the kidney can produce natniuretic not stimulate cGMP accumulation in gbomeruli, but it peptide. stimulated cGMP accumulation in IMCD by threefold. Membrane-bound guanybate cyclase itself is a This pattern was quite similar to that with ANP. It was receptor for ANP (24). and the second messenger of concluded that urodilatin has a similar ability to ANP ANP is cGMP in endothelial cells and epithelial cells. cGMP mimicked some functional effects of ANP in stimulating cGMP synthesis and that the main tar- ( 1 0, 1 2). Urodibatin is also known to increase cGMP get sites are gbomeruli and IMCD. accumulation in several tissues (25), including cell Key Words: Urodiatin, atrialnatriuretic peptide, cGMP, neph- suspensions of IMCD (22). Therefore. we investigated ron segments. inner medullary collecting ducts. glomerull the effects of urodilatin on cGMP accumulation to learn the target sites of urodilatin in the kidney. A trial natriuretic peptide (ANP) is known to in- duce natriuresis, diuresis, decrease in blood METHODS pressure. and smooth muscle relaxation ( 1 -4). ANP Pathogen-free male Sprague-Dawley rats weighing stimulates particulate guanylate cyclase and in- 100 to 250 g were used. Urodibatin and collagenase creases intracellular cGMP content (1-4). However. were purchased from Sigma Chemical Co. (St. Louis, the mechanisms of natriuresis by ANP are still poorly MO). ANP was obtained from Peptide Institute understood (5). ANP is synthesized mainly in atriab (Osaka, Japan). Other chemicals were of first grade. I Received september 1 1, 1992. Accepted October 22, 1992. Preparation of Tubule Suspensions 2 Correspondenceto K. Tomito, second Departmentoflntemal Medicine. Tokyo Mdical & Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo I 13, Japan. Tubule suspensions were prepared as described 1046-6673/03 10-1 705$03.OO/O previously (9). In brief, both kidneys were perfused Journal of the American society of Nephrology Copyright C 1993 by the American Society of Nephrology with ice-cold dissection solution containing 0. 1 % col- Journal of the American Society of Nephrology I 705 Effect of Urodilatin on Renal cGMP Accumulation lagenase and 0. 1 % BSA. Kidneys were divided into sodium acetate buffer were added to blank samples. three parts: cortex, outer medulla, and inner medulla. After the samples were acetybated. the content of Each pant was then minced with a razor blade and cGMP was assayed by use of an RIA kit (Dupont. NEN Incubated In the collagenase solution at 37#{176}Cfor 30 Research Products, Boston, MA). mm for tubule suspensions from cortex. for 45 mm for tubule suspensions from outer medulla, and for Measurement of Protein Content 60 mm for tubule suspensions from inner medulla. Suspensions were passed through three layers of Protein contents of tubule suspensions were meas- gauze. Then, the tubule suspensions were washed ured by use of the Bio-Rad assay kit (Bio-Rad Labo- with dissection solution by centnifugation four to five ratories, Richmond, CA). Albumin was used as a times. Tubule suspensions were kept on ice until the standard. experiment. The composition of dissection solution was as follows (in millimolar amounts): 130. NaC1: 5, KC1: Statistics 1 , NaH2PO4; 2, CH3COONa: 1 , MgSO4: 1 . calcium Results were expressed as mean ± SE. Statistical lactate: 5.5. glucose: 5. L-alanine; 2. L-leucine: 10. evaluation was performed by analysis of variance N-2-hydroxyethylpiperazine-N’ -2-ethanesulfonic followed by multiple comparison of Dunnett’s test. A acid (HEPES); pH was adjusted to 7.4 with NaOH. P value <0.05 was considered statistically signifi- cant. Preparation of Microdissected Nephron Seg- ments RESULTS Microdissection of nephron segments was per- formed as described previously (9. 1 1 ). Briefly. the left cGMP Accumulation in Tubule Suspensions kidney was perfused with 1 0 mL of ice-cold collagen- At first. cGMP accumulation in tubule suspensions ase solution. Coronal slices were incubated in colla- from each pant of the kidney was compared. The genase solution for 30 mm at 37#{176}C.Microdissection results are summarized in Figure 1 . Urodilatin-stim- was performed under a microscope with needles. ulated cGMP accumulation was highest in tubule Microdissected nephron segments were as follows: suspensions from the inner medulla. Small accumu- glomeruli. PCT. MAL. and IMCD. Gbomeruli and PCT bations were also observed in tubule suspensions were dissected from the cortex. MAL was dissected from the cortex and outer medulla. ANP showed quite from the inner stripe of the outer medulla. IMCD was similar results. The effects of urodibatin on cGMP dissected from the inner medulla. Because ANP-stim- accumulation were not statistically different from ulated cGMP accumulation was not different between those of ANP in cortical. outer medulbany. and inner initial and terminal IMCD (1 1), we did not separate medullany tubule suspensions. initial and terminal IMCD. The length of tubules was measured with an ocular micrometer. 15 cGMP Accumulation Study cGMP accumulation stimulated by ANP and urodi- latin was examined as described previously (9. 1 1). ANP .-. urodilatin Inner 0 50 Twenty microliters of solution containing tubule sus- Medulla pensions or microdissected nephron segments was E preincubated for 10 mm at 37#{176}C.Then, 20 L of ANP or urodilatin with 3-isobutyl- 1 -methylxanthine (IBMX) (final, 0.5 mM) was added. After 3 mm of 25 incubation, the reaction was stopped by the addition Outer of 50 ML of ice-cold 1 0% tnichlonoacetic acid (TCA). Medulla CD Cortex Seventy-five microliters of supernatant was stored at -20#{176}Cuntil cGMP assay. Blank samples with TCA 0-9-8-1-6 0-9-8-7-6 0-9-8-1-6 and medium without tubule suspensions or micro- Log [ANP or urodilatin] (M) dissected tubules were stored for use as standards. Figure 1 . Effects of urodilatin and ANP on cGMP accumula- On the day of assay. each sample was thawed at tion in tubule suspensions from the cortex, outer medulla, room temperature. TCA was extracted by water-sat- and inner medulla. Tubule suspensions were incubated with urated ether. The aqueous phase was dried, and 100 various concentrations (10 to 10 M) of urodilatin, ANP, zL of 50 mM sodium acetate buffer was added to each or vehicle for 3 mm at 37#{176}C(N = 4). cGMP contents were sample. Appropriate concentrations of standards in determined by RIA. prot, protein. 1706 Volume 3 . Number 10 . 1993 c:o ANP Koike et al cGMP Accumulation in Microdissected Neph- 15 ron Segments Next, cGMP accumulation by microdissected neph- E ron segments was investigated. Figure 2 shows 10-6 - 10 M urodibatin- and 1 0’6 M ANP-induced cGMP accu- 0 E mubation in nephron segments. Urodibatin (10’6 M) stimulated cGMP accumulation markedly in gbomer- E uli (basal. 0.21 ± 0.14; 10’6 M unodibatin, 9.8 ± 1.2: 10_6 M ANP, 13.0 ± 2.0 fmob/gbomerulus per 3 mm; 0. N = 5). In IMCD. urodibatin stimulated an 1 1 -fold CD cGMP accumulation (basal, 0.06 ± 0.02; urodilatin, 0.62 ± 0.17; ANP. 0.83 ± 0.23 fmob/mm per 3 mm; (‘ ,- I - 0 -9 -8 -7 -6 N = 6).