j . Inher. Metab. Dis. 1 (1978) 75-77
Cyclic Adenosine Monophosphate Excretion in Urine of Patients and Carriers of Congenital Nephrogenic Diabetes Insipidus W. S. UTTLEY, B. ATKINSONand A. ADAMS
Department of Child Life a~d Health, University of Edinburgh, Royal Hospital fi~r Sick Children, Sciennes Road, Edinburgh EH9 i l l , Scotland D. SItIRLING
Department of Medicine, Western General Hospital, Edinburgh EH4 2X~J, Scotland Urinary excretion of cyclic adenosine monophosphate (cAMP) is assessed in response to pitressin stimulation in three patients with nephrogenic diabetes insipidus, four carriers and seven controls. There is no significant difference in cAMP excretion between these groups when corrected for surface area, nor is there any significant increase in excretion after pitressin stimulation. There is very close correlation between urinary cAMP and both urinary concentration and urinary creatinine excretion. Urinary cAMP afl:er pitressin stimulation does not discriminate between carriers of nephrogenic diabetes insipidus and control subjects.
There is considerable evidence to suggest that cyclic adenosine monophosphate (cAMP) and the adenylate cyclase (E.C.4.6.1.t) perform as a mediator or second messenger in the action of antidiuretic hormone (ADH) on the tubules of the renal medulla. In the isolated toad bladder, cAMP mimics the action of ADH with regard to its effect on permeability (Orloff and Handler, I962). Further, adding vasopressin to homogenized rat renal medulla increases its content of cAMP. Specificity of this phenomenon is shown by the lack of a similar effect by substances such as adrenalin, glycogen and parathormone, known to stimulate adenylate cyclase (Chase and Aurbach, 1968). The mode of action has been discussed by OrIoff and Handler (I967). Two hormone-specific receptors for ADH are postulated. These are sited on the basal surface of tubular epithelial cells and one is calcium-dependent. Cyclic AMP released within the cell is thought to be responsible for changes in the apicat surface of epithelial cells by affkcting their permeability to salt and water. Speculation has arisen that a defect of the adenylate cyclase-cAMP system or its receptors may be the biochemical abnormality resulting in X-linked congenital nephrogenic diabetes insipidus (McKusick 30480) (Bell et aI., 1972; Fichman and Brooker, 1972). This condition still has a neonatal mortality of between 5 and 10~o in affected males (Royer, 1974) with a significant incidence of brain damage due to hypernatraemic episodes. It causes considerable family distress, and clearly reliable prenatal diagnosis is desirable. At present this is not possible and, furthermore, current methods of carrier-detection are not entirely reliable (Uttley and Thistlethwaite, 1972). The present investigation examines whether urinary cAMP following stimulation with pitressin can detect carriers of X-linked nephrogenic diabetes insipidus.
SUBJECTS AND METHODS Three male patients aged three, five and six years were studied. Each had a pitressin-resistant hyposthenuria and family studies had shown concentrating defects to be present. Four female carriers, average age 31 years, range 26~1:1 years, agreed to investigation. Diagnosis in three of these was based on presently accepted methods of carrier-detection (Carter and Simpkiss, 1956) and in the tburth on genetic criteria only (Uttley and Thistlethwaite, 1972). Three of these carriers were mothers of the children studied and gave their informed consent to the investigation. Control information was obtained from seven healthy colleagues (four male, three female), average 25 years, range 21-35. The protocol was designed to be reproducible in a ward situation and to be non-invasive. A water load of 500 ml/1.73 m 2 was administered to inhibit endogenous ADH release. The bladder was emptied after 30 min and a basal collection of urine over a further period of 1 h was obtained (water load). A subcutaneous injection of aqueous pitressin 5 ml/1.73 m 2 was given and urine collected over two fhrther periods of 1 h (P1 and P2). Urinary cAMP was measured by a competitive proteinbinding isotope dilution method (Brown et aL, 1972) as modified by Porter (1974). An extract of bovine adrenals was used as the specific binding protein and separation of bound and free cAMP was achieved using charcoal. The standard error of the method is 0.08~o (n = 19) at 0. I nmol/ml. Negligible cross-reactivity occurred with other cyclic nucleotides. RESULTS All subjects demonstrated peripheral vasoconstriction, confirming the potency of the pitressin used. The effect ofpitressin on urinary concentration is shown in Figure 75
U#ley, Atkinson, Adams and Shirling
76 urine concentration mmosmol/kg
l a, which demonstrates the expected clear difference between patients and other groups but fails to distinguish between carriers and controls. Urinary concentration of cAMP is shown in Figure 1b. There is no response to pitressin stimulation in the patients. Carriers and controls both show a striking increase in concentration following pitressin, but this does not discriminate between the two groups. Urinary output of cAMP in nmol/(1.73m2h) is similar in all groups and there is no change with pitressin (Figure lc). A similar result is obtained when urinary cAMP output is related to urinary creatinine. The scattergram (Figure 2a) obtained by plotting paired values for urinary cAMP (nmol/ml) and urinary concentration (mosmol/kg) shows a very close relationship between the t w o (correlation coefficient: r = 0.84, n = 47). Derivations of these observations, e.g. cAMP excretion rate against free water clearance, continue to bear a similar relationship. The scattergram (Figure 2b) shows a close relationship between urinary cAMP (nmol/mI) and urinary creatinine (mg/ml) (correlation coefficient: r = 0.87, n = 44). Comparison of the regression lines obtained for the individual groups of subjects from the data expressed in both Figures 2a and 2b also failed to show any significant difference.
8OO
700
5OO
5O0
400
3O0
20O
~OO
O
WL
PI cos~
P2
WL
PI corrlcrs
P2
WL
Pl controls
P2
(a) urinary cAMP nmol/ml 7
5
5
4
3
2
I
O
WL
El
WL
P.2
coses
Pl carriers
P2
W[-
PI controls
P.2
(b) urinary output of cAM P nmol/(1.73 m = h) 4O0
30O
2OO
IOO
\ o
Wk
PI cases
P2
WL
P.I r ¢orri¢ s
P2
WL
Pl controls
P.2
(c) Figure 1 Urine from patients with congenital nephrogenic diabetes insipidus, carriers and controls for 1 h following a water load (WL), and during the first (P1) and second (P2) h after pitressin. (a~ concentration mosmol/kg, (b~ content cAMP (nmol/ml), (c) cAMP excretion rate (nmol/(1.73 rn2h))
DISCUSSION These results clearly show that the present protocol is incapable of discriminating between normal subjects and carriers of X-linked congenital nephrogenic diabetes insipidus. They also show that whilst urinary concentration of cAMP following pitressin is significantly less in patients with the disease than in controls, total urinary output is similar. Previously reported studies in control subjects show a conflicting experience. Some have shown an apparent increase in urinary cAMP excretion in pitressin stimulated as opposed to basal states (Fichman and Brooker, 1972; Bell et al., 1974), whilst other have been unable to demonstrate such rises (Chase et al., 1969; Hardman, 1971 ; Raij et al., 1974). A similar conflict of experience has occurred in patients with pitressin-sensitive diabetes insipidus (Chase et al., 1969; Bell et al., 1974; Raij et al., 1974). The correlations that we have found suggest that the bulk of cAMP in the urine derives from glomerular filtration, both in the water-loaded and stimulated states. This conforms to previously reported observations (Broadus et al., 1970) that two-thirds of urinary cAMP in the basal state comes from the plasma. No study has yet shown cAMP excretion in the urine to be increased in response to pitressin in a manner similar to the 10-, 20-, or 30-fold increases found following stimulation with parathormone, which has served as such a useful diagnostic test for pseudohypoparathyroidism (Chase et al., 1969), and it is salutary that all attempts to treat nephrogenic diabetes insipidus with exogenous cAMP have been unsuccessful (Jones eta[., 1972; Bell et al., 1974). The literature shows a lack of convention in the units of expression for cAMP and it is apparent from our data that both raw concentration and creatinine-related values could give rise to misleading interpretations. In view of the lack of consistent and reproducible control
Cyclic Adenosine MonopJiosphate Excretion in Urine
77
data, it must be concluded that determination of urinary output of c A M P in response to pitressin has no place at present :in the investigation and diagnosis of patients and carriers with nephrogenic diabetes insipidus.
urinary cAMP nmoI/mt
Y=O " 0 0 6 2 X - 0 " 1 7
60
S`0
4-0
x
costs
x
3'0
'
x
x ~E
2-0 x
, x
¢orciers controls
x
= 47
x
= 0-84
l'O
0
200
400
600
800
t000
urinary concentration mosmol/kg
(a) u r i n a r y cAMP nmol/ml
Y= 3-38× * 0 . 3 2
7.0
6`0
5.0
•,
4.0
x
•
•
eeses
*
corricrs
×
co,treYs
3
Cyclic Adenosine Monophosphate Excretion in Urine of Patients and Carriers of Congenital Nephrogenic Diabetes Insipidus W. S. UTTLEY, B. ATKINSONand A. ADAMS
Department of Child Life a~d Health, University of Edinburgh, Royal Hospital fi~r Sick Children, Sciennes Road, Edinburgh EH9 i l l , Scotland D. SItIRLING
Department of Medicine, Western General Hospital, Edinburgh EH4 2X~J, Scotland Urinary excretion of cyclic adenosine monophosphate (cAMP) is assessed in response to pitressin stimulation in three patients with nephrogenic diabetes insipidus, four carriers and seven controls. There is no significant difference in cAMP excretion between these groups when corrected for surface area, nor is there any significant increase in excretion after pitressin stimulation. There is very close correlation between urinary cAMP and both urinary concentration and urinary creatinine excretion. Urinary cAMP afl:er pitressin stimulation does not discriminate between carriers of nephrogenic diabetes insipidus and control subjects.
There is considerable evidence to suggest that cyclic adenosine monophosphate (cAMP) and the adenylate cyclase (E.C.4.6.1.t) perform as a mediator or second messenger in the action of antidiuretic hormone (ADH) on the tubules of the renal medulla. In the isolated toad bladder, cAMP mimics the action of ADH with regard to its effect on permeability (Orloff and Handler, I962). Further, adding vasopressin to homogenized rat renal medulla increases its content of cAMP. Specificity of this phenomenon is shown by the lack of a similar effect by substances such as adrenalin, glycogen and parathormone, known to stimulate adenylate cyclase (Chase and Aurbach, 1968). The mode of action has been discussed by OrIoff and Handler (I967). Two hormone-specific receptors for ADH are postulated. These are sited on the basal surface of tubular epithelial cells and one is calcium-dependent. Cyclic AMP released within the cell is thought to be responsible for changes in the apicat surface of epithelial cells by affkcting their permeability to salt and water. Speculation has arisen that a defect of the adenylate cyclase-cAMP system or its receptors may be the biochemical abnormality resulting in X-linked congenital nephrogenic diabetes insipidus (McKusick 30480) (Bell et aI., 1972; Fichman and Brooker, 1972). This condition still has a neonatal mortality of between 5 and 10~o in affected males (Royer, 1974) with a significant incidence of brain damage due to hypernatraemic episodes. It causes considerable family distress, and clearly reliable prenatal diagnosis is desirable. At present this is not possible and, furthermore, current methods of carrier-detection are not entirely reliable (Uttley and Thistlethwaite, 1972). The present investigation examines whether urinary cAMP following stimulation with pitressin can detect carriers of X-linked nephrogenic diabetes insipidus.
SUBJECTS AND METHODS Three male patients aged three, five and six years were studied. Each had a pitressin-resistant hyposthenuria and family studies had shown concentrating defects to be present. Four female carriers, average age 31 years, range 26~1:1 years, agreed to investigation. Diagnosis in three of these was based on presently accepted methods of carrier-detection (Carter and Simpkiss, 1956) and in the tburth on genetic criteria only (Uttley and Thistlethwaite, 1972). Three of these carriers were mothers of the children studied and gave their informed consent to the investigation. Control information was obtained from seven healthy colleagues (four male, three female), average 25 years, range 21-35. The protocol was designed to be reproducible in a ward situation and to be non-invasive. A water load of 500 ml/1.73 m 2 was administered to inhibit endogenous ADH release. The bladder was emptied after 30 min and a basal collection of urine over a further period of 1 h was obtained (water load). A subcutaneous injection of aqueous pitressin 5 ml/1.73 m 2 was given and urine collected over two fhrther periods of 1 h (P1 and P2). Urinary cAMP was measured by a competitive proteinbinding isotope dilution method (Brown et aL, 1972) as modified by Porter (1974). An extract of bovine adrenals was used as the specific binding protein and separation of bound and free cAMP was achieved using charcoal. The standard error of the method is 0.08~o (n = 19) at 0. I nmol/ml. Negligible cross-reactivity occurred with other cyclic nucleotides. RESULTS All subjects demonstrated peripheral vasoconstriction, confirming the potency of the pitressin used. The effect ofpitressin on urinary concentration is shown in Figure 75
U#ley, Atkinson, Adams and Shirling
76 urine concentration mmosmol/kg
l a, which demonstrates the expected clear difference between patients and other groups but fails to distinguish between carriers and controls. Urinary concentration of cAMP is shown in Figure 1b. There is no response to pitressin stimulation in the patients. Carriers and controls both show a striking increase in concentration following pitressin, but this does not discriminate between the two groups. Urinary output of cAMP in nmol/(1.73m2h) is similar in all groups and there is no change with pitressin (Figure lc). A similar result is obtained when urinary cAMP output is related to urinary creatinine. The scattergram (Figure 2a) obtained by plotting paired values for urinary cAMP (nmol/ml) and urinary concentration (mosmol/kg) shows a very close relationship between the t w o (correlation coefficient: r = 0.84, n = 47). Derivations of these observations, e.g. cAMP excretion rate against free water clearance, continue to bear a similar relationship. The scattergram (Figure 2b) shows a close relationship between urinary cAMP (nmol/mI) and urinary creatinine (mg/ml) (correlation coefficient: r = 0.87, n = 44). Comparison of the regression lines obtained for the individual groups of subjects from the data expressed in both Figures 2a and 2b also failed to show any significant difference.
8OO
700
5OO
5O0
400
3O0
20O
~OO
O
WL
PI cos~
P2
WL
PI corrlcrs
P2
WL
Pl controls
P2
(a) urinary cAMP nmol/ml 7
5
5
4
3
2
I
O
WL
El
WL
P.2
coses
Pl carriers
P2
W[-
PI controls
P.2
(b) urinary output of cAM P nmol/(1.73 m = h) 4O0
30O
2OO
IOO
\ o
Wk
PI cases
P2
WL
P.I r ¢orri¢ s
P2
WL
Pl controls
P.2
(c) Figure 1 Urine from patients with congenital nephrogenic diabetes insipidus, carriers and controls for 1 h following a water load (WL), and during the first (P1) and second (P2) h after pitressin. (a~ concentration mosmol/kg, (b~ content cAMP (nmol/ml), (c) cAMP excretion rate (nmol/(1.73 rn2h))
DISCUSSION These results clearly show that the present protocol is incapable of discriminating between normal subjects and carriers of X-linked congenital nephrogenic diabetes insipidus. They also show that whilst urinary concentration of cAMP following pitressin is significantly less in patients with the disease than in controls, total urinary output is similar. Previously reported studies in control subjects show a conflicting experience. Some have shown an apparent increase in urinary cAMP excretion in pitressin stimulated as opposed to basal states (Fichman and Brooker, 1972; Bell et al., 1974), whilst other have been unable to demonstrate such rises (Chase et al., 1969; Hardman, 1971 ; Raij et al., 1974). A similar conflict of experience has occurred in patients with pitressin-sensitive diabetes insipidus (Chase et al., 1969; Bell et al., 1974; Raij et al., 1974). The correlations that we have found suggest that the bulk of cAMP in the urine derives from glomerular filtration, both in the water-loaded and stimulated states. This conforms to previously reported observations (Broadus et al., 1970) that two-thirds of urinary cAMP in the basal state comes from the plasma. No study has yet shown cAMP excretion in the urine to be increased in response to pitressin in a manner similar to the 10-, 20-, or 30-fold increases found following stimulation with parathormone, which has served as such a useful diagnostic test for pseudohypoparathyroidism (Chase et al., 1969), and it is salutary that all attempts to treat nephrogenic diabetes insipidus with exogenous cAMP have been unsuccessful (Jones eta[., 1972; Bell et al., 1974). The literature shows a lack of convention in the units of expression for cAMP and it is apparent from our data that both raw concentration and creatinine-related values could give rise to misleading interpretations. In view of the lack of consistent and reproducible control
Cyclic Adenosine MonopJiosphate Excretion in Urine
77
data, it must be concluded that determination of urinary output of c A M P in response to pitressin has no place at present :in the investigation and diagnosis of patients and carriers with nephrogenic diabetes insipidus.
urinary cAMP nmoI/mt
Y=O " 0 0 6 2 X - 0 " 1 7
60
S`0
4-0
x
costs
x
3'0
'
x
x ~E
2-0 x
, x
¢orciers controls
x
= 47
x
= 0-84
l'O
0
200
400
600
800
t000
urinary concentration mosmol/kg
(a) u r i n a r y cAMP nmol/ml
Y= 3-38× * 0 . 3 2
7.0
6`0
5.0
•,
4.0
x
•
•
eeses
*
corricrs
×
co,treYs
3
