Pediat. Res. 13: 884-888 (1979) Antidiuretic hormone kidney c-AMP newborn concentrating capacity Effect of Antidiuretic Hormone Upon Urinary Concentrating Ability and Medullary c-AMP Formation in Neonatal Piglets R. JOPPICH, U. KIEMANN, G. MAYER, and D. HABERLE Universilals-h'inderklinikof the Universit.~of Munich, Lindwurmstrasse 4,8 Munich 2, Federal Republic of Germany und Ph~~siologischesInstitut of the Universily of Munich, (D. 11.). Pettenkoferstrasse 12,8 Munich 2, Federal Republic of Germany Summary uted to the structural immaturity of the renal medulla (2, 22), to the reduced GFR and to the anabolic state of the neonatal In 16 anesthetized piglets, the effects of I-deamino-8-D-argi- metabolism, which together result in low filtration-, reabsorp- nine-vasopressin (DDAVP) upon urinary flow rate, relative urinary tion-, and excretion rate of urea (5, 6). It was argued that in spite osrilolarity (U/P,,,,,,I), fractional water excretion (U/Pl,), frac- of the effects of ADH on the permeability of the collecting ducts, tional urca excretion (U/P,,,.,,/U/PI,), renal adenosine 3',5'-cyclic no significant amounts of water and urea could be reabsorbed, nlonophosphate (c-AMP) formation rate, and medullary c-AMP thus restricting the increase in urinary osmolarity. content were studied during the first 6 days of life. Glomerular More recent studies (18) focused upon the fact that the neonatal filtration rate (GFR) was 0.19 + 0.09 ml/min/g kidney wt (means concentrating system is highly insensitive to ADH. This concept + SD) on day 1 and increased to 0.51 + 0.11 ml/min/g kidney wt was strongly supported by findings on membrane preparations of on day 6. It was not affected by the application of 20 pg DDAVP/ newborn rats in vitro which demonstrated that the activity of the kg body wt. On day 1, U/P,,,,,1(1.81 + 0.29), urinary flow rate ADH dependent adenylate-cyclase is reduced in comparison to (5.36 + 1.49 pl/n~in/g kidney wt), U/PI, (31.67 + 6.10) and U/ the adult state (19, 21). Hence, it is possible that, in addition to P,,,.,,/U/PI, (0.84 + 0.12) were not affected by DDAVP applica- the immaturity of the countercurrent system and the parameters tion. Fro111 day 2 onward, the reponse of these variables to appli- mentioned above, a lack of intracellular c-AMP formation as a cation of DDAVP increased progressively. On day 6, the following consequence of the reduced enzyme activity could be responsible, responses were observed: Urinary flow rate decreased from 13.85 at least in part, for the diminished concentrating capacity of the f. 7.02 pl/nlin/g kidney wt before DDAVP to 5.07 ? 2.19 pl/min/ newborn kidney. g kidney wt after DDAVP. U/P,,,,I raised from 1.24 + 0.27 In order to examine this theory, we performed clearance studies before DDAVP to 2.64 ? 0.17 after DDAVP. U/PI, increased and measurements of the medullary c-AMP content on piglets in fro111 36.85 + 13.11 before DDAVP to 109.39 + 22.00 after the neonatal period to determine whether there exists a parallelism DDAVP. U/P,,,,,/U/P,, decreased from 0.75 ? 0.05 before between the development of renal concentrating capacity and the DDAVP to 0.55 + 0.04 after DDAVP. These data indicate that responsiveness of the ADH dependent c-AMP system to supra- the snlall solute permeability of the medullary collecting ducts maximal doses of DDAVP. becon~esincreasingly sensitive to antidiuretic hormone (ADII) during this period. The increase in responsiveness of the urinary concentrating METHODS system with age was paralleled by the effect of DDAVP on renal c-AMP fornlation rate. In the diuretic control state, no correlation GENERAL PROCEDURES between renal c-AMP fornlation rate and the number of days was Piglets (strain: Deutsche Landrasse) weighing between 600 and observed. llowever, after the application of DDAVP, the nephro- 2600 g and ranging in age between day 1 and 6 after birth were genic fraction of renal c-AMP excretion rate increased from 10.58 anesthetized by ip injection of 60-100 mg/kg of body wt of Inactin + 4.49 pnlole/min/g kidney wt on day 1 to 21.61 + 8.10 pmole/ (Byk-Gulden, Konstranz, West Germany). During the experi- n~in/gkidney wt on day 6. Similar results were observed with the ments, Nembutal (Boehringer Sohn, Ingelheim am Rhein, West nledullary c-AMP content. In diuretic control animals which had Germany) was injected iv at doeses between 5 and 10 mg/kg body riot received DDAVP, the medullary c-AMP content ranged be- wt as required. Body temperature was maintained at 37OC by a tween 636 ? 257 pnlole/g wet wt on day 1 and 622 + 75 pmole/g heated operating table. The trachea was cannulated and the right wet wt on day 6. IIowever, in animals which had been infused with external jugular vein was catheterized for infusion of inulin and DDAVP, niedullary c-AMP content increased from 684 + 274 isotonic glucose solutions. The right femoral artery was cathe- pnrole/g wet wt on day 1 to 1536 ? 316 pmole/g wet wt on day 6. terized in order to sample blood and measure arterial blood pressure. The urinary bladder was exposed by a median supra- Speculation pubical incision and each ureter cannulated for sampling of urine specimens. The unresponsiveness of the medullary adenylatecyclase to AD11 in the newborn kidney might restrict, in addition to other EXPERIMENTAL PROCEDURE parameters, the renal concentrating ability during the neonatal period. Control Measurements. After completion of surgery, the piglets received an infusion of isotonic glucose solution at a rate of 30 ml/hr/kg body wt and an infusion of inulin at a rate of 18.75 It is generally acknowledged that the concentrating ability of mg/min/kg body wt over a period of about 3 hr. This procedure the newborn kidney is reduced in comparison to that of the mature induced a diuresis. After this period, both urinary flow rate kidney. In the past, this phenomenon was predominately attrib- and inulin concentration in plasma reached steady state. Inulin-, EFFECT OF ADH ON CONCENTRATING ABILITY AND C-AMP FORMATION 885 urea-, and c-AMP-clearance as well as U/P,,,,,,I were then deter- above. The age of the animals ranged between day I and day 6 mined for control. Three ml of arterial blood were sampled at the after birth. One group which served for control, received only midpoint of each clearance period. glucose infusions for about 3 hr. The kidneys were then removed Clearance measurements during and after the infusion of DDA VP. and frozen in liquid nitrogen. The second group, which was used After the control measurements were finished, glucose infusion to study the effi:cts of DDAVP on medullary c-AMP content, was was stopped in order to avoid fluid imbalance, but the inulin handled according to a protocol identical to that mentioned infusion was continued at the same rate. Thirty min later, when previously. urinary flow rate had decreased markedly, 20 &kg body wt DDAVP (Ferring Sveden) was infused iv over a period of 20 min. ANALYSES Even at such supramaximal doses, the drug has no significant Inulin was analyzed in urine and in serum by the anthrone vasoconstrictor effects (26). Clearance measurements as described method (7). Plasma was deproteinized with 0.33 N perchloric acid. above, were then performed for at least 3 hr. The response to Urea in plasma and urine was analyzed using the Berthelot DDAVP was assumed to be complete when urinary osmolarity reaction (Asid Diagnostics, Lohhof-Munich, West Germany). Os- didn't increase further during three consecutive clearance periods. molarity in plasma and urine was determined by freezing point When measurements were finished, both kidneys were immedi- depression. Using the test kit for the radio isotope dilution test ately removed and frozen in liquid nitrogen. with A-3:5-MP-binding protein of Boehringer (Mannheim, West Measurement of medullary c-AMP content. In order to measure Germany), c-AMP was determined. This method was first de- medullary c-AMP content, experiments on two groups of 16 scribed by Gilman (9). Determinations of c-AMP in: I) urine: animals were performed according to the protocol described depending upon its osmolarity, urine was diluted by double Table 1. Development of renal response in urinary concentration to DDA VP during neonatal period' Days after birth 1 2 3 4 5 6 Number of animals 3 3 3 3 2 2 Kidney wt (g) 8.9 + 2.7 8.8 + 0.4 9.3 + 1.8 12.7 + 3.3 17.2 + 6.7 18.6 + 1.9 Number of samples Before DDAVP After DDAVP C,,, (ml/min g kidney wt) Before DDAVP 0.19 + 0.09 0.23 + 0.06 0.34 + 0.12 0.33 + 0.07 0.44 + 0.17 0.51 + 0.1 I After DDAVP 0.18 + 0.04 0.20 + 0.10 0.38 + 0.07 0.27 + 0.03' 0.45 + 0.18 0.55 + 0.05 Ctr (ml/min g kidney wt) Before DDAVP 0.16 + 0.04 0.17 + 0.03 0.26 + 0.10 0.23 + 0.08 0.3 1 + 0.09 0.38 + 0.12 After DDAVP 0.15 + 0.05 0.14 + 0.09 0.26 + 0.06 0.17 + 0.03" 0.25 + 0.04' 0.30 + 0.15' v (pl/min g kidney wt) Before DDAVP 5.36 + 1.49 7.82 + 2.66 9.25 + 5.38 7.35 + 4.85 14.79 + 8.