Effect of Urodilatin on Cgmp Accumulation in the Kidney1

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 system (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), proximal 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 significant. Preparation of Microdissected Nephron Seg- ments RESULTS Microdissection of nephron segments was performed 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 urodilatin 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). Small cGMP accumulations by urodibatin were Log [ANP or urodllatin] Ml observed also in PCT (basal. 0.02 ± 0.02; urodibatin, Figure 3. Dose-dependent stimulation of cGMP accumuba- 0.07 ± 0.0 1 ; ANP, 0.07 ± 0.03 fmob/mm per 3 mm; tion by urodilatin and ANP in gbomeruli (glm). Significant N = 4) and MAL (basal. 0.01 ± 0.01; urodilatin. 0.09 increases of cGMP accumulations by urodilatin and ANP ± 0.03; ANP, 0.13 ± 0.05 fmol/mm per 3 mm; N = were observed at doses of higher than 10e M (N = 6). P < 4). The effect of urodibatin on cGMP accumulation 0.05 versus control. was almost equal to that of ANP in the nephron segments tested. 1.’ The dose-dependent increase of cGMP accumula- I control EJ1oM ANP tion in gbomenuli by urodibatin was compared with _ 108M urodilatin that by ANP (Figure 3). The threshold concentration for both urodilatin and ANP to stimulate cGMP ac- * cumulation was between 10_8 and i0’ M. In con- E I trast, 108 M urodibatin and ANP significantly stim- 05 ubated cGMP accumulation in IMCD (Figure 4). The 0 E threshold concentration of urodilatin and ANP to E stimulate cGMP in IMCD was lower than that in 0 E glomerubi. 0

= E DISCUSSION =

Urodibatin was originally found in the urine. and it 0.

CD is thought to be of renal origin. Our results show that U urodibatin-stimulated cGMP accumulation was higher in tubule suspensions from inner medulla

15 control GIm IMCD r EJ10#{176}MANP 10 6M Figure 4. Effects of low doses of urodilatin (10 M) and ANP E (10 M) on cGMP accumulation in gbomeruli (gbm) and be IMCD. Low doses of urodilatin and ANP were used in this study (N= 4). ‘P< 0.05 versus control.

I than in those from cortex and outer medulla. Micro-

0 dissected tubule experiments revealed that urodibatin 1 stimulated cGMP accumulation markedly in glomer- E uli and IMCD among nephron segments. Urodilatin- i0.5 - stimulated cGMP accumulation in gbomeruli and IMCD were almost equal to that stimulated by the 1_LI -r same concentrations of ANP. These data show that GIm PCT MAL IMCD urodilatin has an ability almost equal to that of ANP In stimulating cGMP accumulation and the same dis- Figure 2. Effects of urodibatin (10_6 M) and ANP (10 M) on cGMP accumulation along the nephron. Nephron segments tnibution of target sites along the nephron as ANP. were dissected after collagenase treatment of the kidney, ANP is known to be produced by atnial myocytes. and they were incubated with urodilatin, ANP, or vehicle ANP synthesis has also been identified in the brain for 3 mm at 37#{176}C(N = 4 to 8). gbm, gbomerubi. and lung (6). It has been thought that the kidney is

Journal of the American Society of Nephrology I707 Effect of Urodilatin on Renal cGMP Accumulation ......

the target site of ANP and is not a site of ANP pro- tubular actions of ANP rather than effects on gb- duction. However, recently. urodilatin was found in meruli (33). Our findings that the threshold concen- the human urine, suggesting the production of natni- tration of both urodilatin and ANP was lower in IMCD uretic peptide in the kidney. Greenwald et al. have than in gbomeruli may be compatible with these data. reported that ANP-bike protein was produced in distal IMCD has only guanylate cyclase-coupled ANP recep- cortical nephrons. especially in the nephrotic kidney tons, whereas two thirds of gbomerular ANP receptors (23). It is not known if this protein is urodilatin. If are clearance receptors (34). This could be one of the this protein is urodibatin, the action site must be reasons for the differences between gbomerubi and beyond the connecting tubules, namely, collecting IMCD. These results may also suggest greater physi- ducts. Our data showing that the main target site is obogic importance of IMCD rather than glomeruli for IMCD are quite compatible with this idea. The natni- the action of both urodilatin and ANP. uretic peptide produced in distal convoluted and con- In summary. urodilatin showed almost equal ability necting tubules descends in the urine to IMCD and to ANP in stimulating cGMP accumulation. The main shows some functional effects. The same idea has target sites of urodilatin were glomerubi and IMCD. been proposed for bradykmnmn (26). The role of urodilatin in renal ion transport must be The urinary excretion of ANP is small because of investigated. degradation in the kidney. Degradation of ANP by nephron segments was observed in almost all neph- ACKNOWLEDGMENTS ron segments (27). The degradation of ANP was high- This study was supported by a grant-in-aid for Scientific Research est in proximal convoluted and straight tubules and of Japan (02670273. 02770326. 04454234. and 04670383). lowest in gbomerubi and IMCD (27). indicating that the main target sites of ANP have low degrading REFERENCES ability. The brush border membrane is rich in endopeptidase. and the luminal membrane fractions of 1 . Brenner BM, Ballermann BJ, Gunning ME, Zei- del ML: Diverse biological actions of atniab natri- cortical homogenate were most active in metabolizing uretic peptide. Physiol Rev 1 990;70:665-699. ANP among subcelluban fractions (27). Therefore, fib- 2. Cogan MG: Renal effects of atnial natniuretic tered ANP is thought to be destroyed by the brush factor. Annu Rev Physiol 1990;52:699-708. border of proximal tubules. However, urodibatin is 3. 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However, it is quite interesting that for atrial natriuretic factor. Proc Natl Acad Sci immunoreactive staining of ANP was found only in USA 1 986;83:6697-670 1. the luminal cytoplasm in intercalated cells in the 7, Berl T, Mansour J, Teltelbaum I: ANP stimu- connecting tubules and collecting ducts (30). Sonnen- bates phosphobipase C in cultured RIMCT cells: Roles of protein kinases and G protein. Am J berg et al. demonstrated that luminal ANP reduced Physiol 1 99 1 ;260:F590-F595. Na transport (3 1). Judging from the finding that uro- 8. GunnIng M, Ballermann B..!, Silva P, Brenner dilatin-stimulated cGMP accumulation was highest BM, Zeidel ML: Brain natriuretic peptide: Inter- in IMCD, the luminal and basolateral effect of urodi- action with renal ANP system. Am J Physiol latin on water and urea transport must be examined 1 990;258:F467-F472. 9. Nonoguchi H, Knepper MA, Manganiello VC: in IMCD. Gunning et al. reported that urodilatin stim- Effects of atrial natriuretic factor on cyclic guan- ulated cGMP accumulation and inhibited Na trans- osine monophosphate and cyclic adenosine port in suspensions of IMCD cells (22). Although a monophosphate accumulation in microdissected preliminary report suggested that the luminal admin- nephron segments from rats. J Clin Invest 1987; 79:500-507. istration of urodilatin had no effect on osmotic water I 0. Nonoguchl H. Sands JM, Knepper MA: Atrial permeability in IMCD (32). the interaction with va- natniuretic factor inhibits vasopressin-stimu- sopressin. which is a key hormone in the regulation bated osmotic water permeability in nat inner of water and Ion transport in various portions of the medullary collecting duct. J Clin Invest 1988; 82:1383-1390. kidney, must be examined further. 1 1 . Ujile K, Nonoguchi H, Tomita K. 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Journal of the American Society of Nephrology 1709