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Effect on renal function of essential fatty acid supplementation in cystic fibrosis
Effect on renal function of essential fatty acid supplementation in cystic fibrosis B. Strandvik, MD, PhD, U. Berg, MD, PhD, A. Kallner, MD, PhD, a n d E. Kusoffsky, MD From the Department of Pediatrics, Huddinge University Hospital, Karolinska Institute, and the Department of Clinical Chemistry, Karolinska Hospital, StOckholm, Sweden Changes in renal hemodynamics, sodium homeostasis, renal acidifying capacity, and aldosterone excretion were studied before and after long-term intravenous essential fatty a c i d supplementation for a period of 3 year s in 11 patients with cystic fibrosis. The mean (_+ SD) giomerular filtration rate was high at the start of the study (133 _+ 18 ml/min/1.73 m 2 b o d y surface area) and decreased significantly (p < 0.05) to within normal values after I year of essential fatty a c i d supplementation. The urinary elimination of an oral sodium l o a d initially was very low (3,6 _+ 2.5 mmol/hr/!.73 m ~ b o d y surface a r e a vs control subjects' values of 7.9 _+ 2.0; p <0.001) and increased during treatment but was not normalized (/3 < 0.05 vs control subjects' values). Free water c l e a r a n c e and distal tubular sodium delivery, which were significantly decreased before treatment (p < 0.01 and p < 0.001 vs control subjects' values, respectively) d i d not increase significantly. The mean urinary aldosterone excretion did not significantly differ from that in control subjects before and after treatment. The acidifying c a p a c i t y was disturbed, indicating a low renal b i c a r b o n a t e threshold, and was c h a n g e d during treatment in only 2 of 10 patients. These d a t a indicate that essential fatty a c i d d e f i c i e n c y may contribute to the renal disturbances in cystic fibrosis. (J PEDIATR1989;115:242-50) In patients with cystic fibrosis, an essential fatty acid deficiency is present in various blood lipid fractions and in erythrocyt e membranes and platelets. 18 Essential fatty acids are important precursors to the eicosanoid system and are constituents of the ph0sPholipids in all membranes. 9 Changes in the phospholipid pattern influence membrane transport, both by influencing the prostanoid system and by directly affecting membrane fluidity?~ 11 We have suggested that an increased turnover of arachidonic acid can be closely related to the basic defect in C F and might be the cause of the E F A D in these patients? 2-t4
In rats with EFAD, an increased sodium permeability has been noted in lecithin membranes, 15 and rats with E F A D have hematuria. 16 Increased sodium reabsorption in the proximal renal tubule, ~7'~8 as well as in the respiratory epithelium, ~9,2~has been reported in CF. Some patients with C F have an increased glomerular filtration rate, ~8
Supported by grants from the Swedish Medical Research Council (Nos. 8558 , 5361, and 6864), Karolinska Institutet, Sigurd and Elsa Golje's Memorial Fund, and the Swedish Medical Association. Submitted for publication Aug. 23, 1988; accepted Feb. 21, 1989. Reprint requests: B. Strandvik, MD, PhD, Department of Pediatrics, Huddinge Hospital, S-141 86 Huddinge, Sweden.
although this has been an inconsistent finding.:1 Hematuria and morphologic glomerular changes are not uncommon in advanced stages of the disease. 22 It thus seems that the renal disturbances--although not clinically import a n t - a r e extensive and might be at least partly related to the EEAD in these patients. To test this hypothesis, we
242
BSA CF EFAD GFR PAH
Body surface area Cystic fibrosis Essential fatty acid deficiency Glomerular filtration rate Para-aminohippuric acid
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Renal function in cystic fibrosis
243
Ratio ~8:2/18:0 2.0
Ratio 18:2//1B:0
4.c
.
.
.
.
.
.
.
2
.
.
.
.
.
.
.
.
.
.
.
.
2,o.
,
/!
,
0
1
2
3
years
,
~
~- . . . .
4
IL
Fig. t. Mean ratio (_+2 SD) of linoleic acid to stearic acid in the triglyceride fraction of fasting serum in 10 patients with CF during 3 years of interrupted essential fatty acid Supplementation (Intralipid). Means (_+ 2 SD) for the control group are indicated by horizontal lines. For further explanation, see text,
investigated the renal function in patients with C F before and after long-term intravenous supplementation with essential fatty acids. METHODS Patients. Eleven patients (six female) with C F were studied. Their ages ranged from 5 to 26 years (mean 14 yeai's). Clinical scores according to Shwachman and Kulczycki2s were 56 to 94 (mean 78) at the start of the study. All patients had abnormal sweat test results (chloride >80 mmol/L), pulmonary symptoms, and pancreatic insufficiency. They Were in good clinical condition without antibiotic treatment at the time of the investigations. None of the patients had any signs of renal diseasel and all patients had normal oral glucose tolerance test results. All patients had normal height and weight for age and were on a normal diet without extra sodium chloride. Fatty acid Supplementation was given intravenously at regular intervals for 3 years. During the first year, nine of the patients received 10 ml of 10% lipid emulsion (Intralipid) per kilogram of body weight intravenously every fortnight; the other two patients received the preparation on 3 consecutive days once a month. The treatment was interrupted for psychologic reasons after the first year. After a 1-year intermission, treatment was accepted for another year by 7 of the 11 patients, but just for 2 consecutive days every second month. The extra supplementation of linoleic acid was 10.5 to 16.5 gm/kg b0dyweight during the first year, corresponding to 30 to 50 mg linoleic acid per kilogram of body weight per day. During the third year, corresponding values were 5 to 7 gm/kg body weight and
1.0,
,
0
//
,
1
2
3
years
[
~ . . . .
-~
I L
F i g . 2 . Mean ratio ( _+2 SD) of linoleic acid to stearie acid in the phospholipid fraction of fasting serum in 10 patients with CF during 3 years of interrupted essentia! fatty acid supplementation. For explanation, see Fig. 1 and text.
15 to 20 mg/kg body weight per day, respectively. Renal function was studied when the patients were hospitalized 2 to 4 weeks after the latest infusion of fat emulsion. All the studies were performed before and after the first year of fatty acid supplementation. Renal hemodynamics, r e n a l sodium handling, and diluting capacity were also studied at the end Of 3 years of treatment. Fifteen hospitalized children in the same age range, without CF and with no clinical evidence of malabsorption or infection, and 10 healthy adults from the medical staff were studied as reference groups for the serum fatty acid analysis and urinary aldosterone excretion. Sampling and analyses were Carried out under conditions identical with those of the patients. Informed consent was given by the patients and parents. The study was approved by the Karolinska Institute Committee of Ethics. Laboratory studies. Serum samples for lipid analysis were drawn by venipuncture in the morning, 12 hours after the latest meal. Sampling was performed before lntralipid treatment was given to the patients with CF. The serum triglycerides and phospholipids were separated by thinlayer chromatography (silica ge I plates developed with chloroform, methanol, and water 80:30:5) after Folch extraction. 24 The compounds were eluted from the gel and hydrolYzed, and the relative amounts of fatty acids in the fractions were determine d by gas-liquid chromatography (stationary phase SP 2310/2% SP 2300 on 100/120 porous
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The Journal o f Pediatrics August 1989
T a b l e I. Renal h e m o d y n a m i c s a n d urinary sodium excretion ( m e a n + SD) before and after Intralipid t r e a t m e n t for 1 year and 3 years and control subjects
Renal test
GFR (ml/min/1.73 m 2 BSA)
RPF (ml/min/1.73 m E BSA)
FF (%)
Basal urinary Na excretion (mmol/ hr/1.73 m 2 BSA) Renal response to oral Na load (mmol/hr/1.73 m 2 BSA)
n
Before
10 7 6 10 7 6 10 7 6 10 7 3r 10 7 3*
133 • 18 128 _+ 17 120 _ 19 642 • 79 651 • 60 628 _+ 85 20.7 + 1.8 20.3 • 1.8 20.3 _+ 2.2 3.6 • 2.5 4.1 • 2.9 3.4, 1.3, 2.8 3.4 _+ 2.1 3.8 +_ 2.3 3.9, 2.3, 0.9
After I yr
After 3 yr
Control subjects (n = 25)
110 • 19
112 • 10
111 _+ 14 601 • 67 589 • 126 616 • 203 18.7 _+ 2.8 21.8 • 4.0 18.6 • 3.2 4.7 • 2.7 7.1, 5.1, 10.3 6.6 • 3.6 8.2, 7.9, 11.0
7.9 _+ 2.0 5.0 + 3.1 4.8, 6.8, 9.1 15.9 • 1.5 10.9 • 4.7 10.9, 12.6, 17.6
c, Control subjects; B, before Intralipid treatment; NS, not significant; RPF, renal plasma flow; FF, filtration fraction. *Three patients receiving only 1 year of Intralipid treatment.
T a b l e !1. Free water clearance a n d distal t u b u l a r sodium delivery ( m e a n _ SD) in C F patients a n d control subjects at start of study and after 1 year and 3 years of Intralipid t r e a t m e n t of the patients with C F
Renal test
Cn,o (ml/min/l.73 m 2 BSA)
CH,o + CN, (ml/min/1.73 m 2 BSA)
Uosm (mOsm/L)
n
10 7 5 10 7 5 10 7 5
Before
91. 8.2 9.1 9.6 8.7 9.5 61 60 60
_+ 2.5 • 2.4 _+ 2.7 _+ 2.6 _+ 2.5 + 2.9 _+ 16 • 18 • 20
After 4 yr
After 3 yr
Control subjects (n = 25)
12.0 _+ 1.4 8.9 +_ 2.8 10.5 • 2.2 13.4 • 1.2 9.4 _+ 2.8 11.0 _+ 2.2 65 • 13 63 • 8 62 + 8
C, Control subjects; B, before treatment; NS, not significant; Crr2o,clearance of water; CN,, clearance of sodium; Uosm, urine osmolality.
polymer support C h r o m o s o r b W [ A W ] , J o h n s - M a n v i l l e [available from S i g m a C h e m i c a l Co., St. Louis, Mo.] ). A standardized sodium intake of 2 m m o l / k g body weight was given 4 days before the renal tests. U r i n a r y sodium excretion during 6 hours was studied after an oral sodium load of 95 m m o l / 1 . 7 3 m 2 body surface area. TM During the oral sodium test, clearances of inulin a n d P A H 2~ were determined. U r i n a r y a n d blood samples were analyzed for inulin according to the a n t h r o n e method 26 and for P A H by a modification of the technique described by S m i t h et al: 27 T h e concentration of sodium was determined with a flame photometer and osmolality by the freezing point techniquel Diluting capacity was evaluated as free water clearance.
On a separate day, a short oral a m m o n i u m chloride load was performed for testing acidifying capacity. 2s,29 T h e patients were given water orally, 5 m l / k g body weight every 30 minutes, to m a i n t a i n a c o n s t a n t urine flow rate. U r i n e was collected by spontaneous voiding at 6 0 - m i n u t e intervals. In the middle of each urine sampling period, an arterialized capillary blood sample was taken. A f t e r two urine control periods, an oral a m m o n i u m chloride load of 150 m m o l / m 2 B S A was given. Blood and urine samples were t a k e n for another 5 hours a n d immediately analyzed for p H with a p H meter (Radiometer, Copenhagen, D e n m a r k ) . T h e data were plotted on a S i g g a a r d - A n d e r s e n curve fiomogram giving Pco/, s t a n d a r d bicarbon~tte concentration, and blood total CO2 c o n c e n t r a t i o n ? ~
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Significance ( p v a l u e ) C vs t yr
C vs B
< 0.01 < 0.01 < 0.01 NS NS NS NS NS NS < 0.001 < 0.01
C vs 3 yr
NS
B vs t yr
B vs 3 yr
< 0.05
<0.05
NS NS
NS NS
NS NS
NS NS
NS NS
< 0.01 <0.05
< 0.001 < 0.001
< 0,001
NS < 0.05
< 0.01
< 0,05
Significance ( p value) C vs B
< < < < < <
0.01 0.01 0.05 0,001 0.001 0.01 NS NS NS
C vs t yr
C vs 3 yr
B vs t yr
B vs 3 yr
NS
< 0.01 NS < 0.05
NS NS
< 0.0l
NS NS
NS
Twenty-four-hour urinary aldosterone excretion was determined by a radioimmunoassay? ~ The results of renal function in C F were compared with those of control subjects previously reported, ~5'29 Statistical analysis. The Mann-Whitney U test, Wilcoxon matched-pairs ranked-signs test, and Student paired t test were used for statistical analyses. RESULTS
General data. The patients had normal growth during the study period, no improvement in comparison with the 2 years before (data not shown). Lung function did not improve, and the number of infections was not reduced. The clinical score also did not change. No obvious clinical effect could thus be seen as a result of treatment.
NS
NS
Serum lipid analysis Fatty acid pattern in the serum triglyceride fraction. The relative amounts of saturated fatty acids in the triglyceride fraction were slightly increased and changed little during the study. The mean (_+ S E M ) molar percentage of linoleic acid was significantly decreased (p < 0.01) in the patients, in comparison with healthy control subjects, at the start of the study (7.9 _+ 1.1% and 16.0 +_ 0.6%, respectively). During the study, molar percentage of linoleic acid increased significantly after 1 and 3 years (p < 0.05, respectively), but no significant changes were noted in the concentration of other fatty acids. The ratio of oleic acid to linoleic acid; which was significantly increased initially (p < 0.001), did not change significantly (from 6.4 to 4.5 after 1 year to 3.5 after 3 years; control subjects 2.7).
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Ratio
U NaV, m m o l / h r / 1 . 7 3 m ~ B S A 20 i load
10.
0
1 ~
2
3
=_ . . . .
_~ I L
years
Fig. 3. Mean ratio (_+2 SD) of arachidonie acid to stearic acid in the phospholipidfraction in 10 patients with CF. For explanation, see Fig. 1 and text.
The ratio of linoleic acid to stearic acid was decreased significantly at the start and increased significantly during the first year of treatment (p < 0.01) (Fig. 1). However, the mean ratio at 3 years did not significantly differ from that at the start of the study and was lower than at 1 year of treatment (p = 0.05). Fatty acid pattern in the serum phospholipid fraction. In the serum phospholipid fraction, the molar percentage of saturated fatty acids did not change significantly during the study. Initially the molar percentage of palmitoleic acid was significantly increased (p < 0.01), as was that of oleic acid (p < 0.001). The molar percentages of linoleic, 7-1inoleic, and araehidonic acids were significantly decreased compared with those of healthy control subjects (p<0.001, <0.01, and <0.001, respectively), being 16.8 +_ 2.7% versus 24.3 _+ 0.9%, 0.4 _+ 0.2%versus 1.0 + 0.2%, and 5.2 + 1.1% versus 9.5 +_ 0.4%, respectively. No significant change was found during the study period. The ratio of oleic acid to linoleic acid, which was significantly increased initially (p <0.001), also did not improve significantly, from 1.5 to 1.0 at 1 year and to 0.8 at 3 years (control ratio 0.6). The ratio of linoleic acid to stearic acid increased significantly during the first year (p <0.05) and did not change significantly during the third year with the lower amount of essential fatty acid supplementation (Fig. 2). The ratio of arachidonic acid to stearic acid showed a similar pattern (Fig. 3). Renal studies. All patients with cystic fibrosis were not included in all renal studies because of allergic reactions to the inulin test dose or technical errors. The number of patients studied is given in each part of the results (see Tables I and II). Renal hemodynamics. Mean glomerutar filtration rates,
~"
O
1
2
3
4
5
6 hrs
0
1
2
3
4
5
6 hrs
Fig. 4. Mean (_+SD) hourly urinary sodium excretion (UNaV) before and after an oral sodium load of 95 mmol/1.73 m2 BSA in patients with CF before (straight lines) and after 1 year (n = 10) (left panel, dotted line) and 3 years (n = 7) (right panel, dotted line) of treatment with interrupted essential fatty acids (Intralipid). Shaded area repesents control values. (Data from Berg U. Kidney Int 1981;20:753-8.)
clearances of PAH, and filtration fractions before and after Intralipid treatment are listed in Table I. Before treatment, GFR was significantly increased compared with that of the control subjects (p <0.01). The G F R decreased significantly to within normal values after 1 year of fatty acid supplementation, and did not change in those six patients who were studied after another year of Intralipid treatment. Two of three studied patients who did not get Intralipid in the third year had an increase in GFR between 1 and 3 years. In the third patient, the determination of GFR after 1 year failed, but the GFR was very high both before treatment and after 3 years, being 160 and 158 ml/min/1.73 m 2 BSA, respectively. The clearance of PAH and FF before treatment did not differ significantly from that of control subjects and did not change significantly after fatty acid supplementation (Table I). Urinary sodium excretion. The basal urinary sodium excretion in the CF group was significantly decreased in comparison with control values (p <0.001) (Table I). Although sodium elimination gradually improved, the values were still significantly lower than in the control subjects after both 1 year and 3 years of essential fatty acid supplementation (p <0.01 and <0.05, respectively). The renal response to an oral sodium load (mean urinary sodium excretion during the third to the sixth hour) was absent before treatment, with significantly lower values than those in the control subjects (p <0.001). The response improved significantly after 1 year of Intralipid treatment (p <0.05). After 3 years, the renal response had further
Volume l 15 Number 2
increased but was still significantly less than that of the control subjects (p <0.01) (Fig. 4). Diluting capacity. Diluting capacity was determined as free water clearance when the lowest urine osmolality was reached. Because GFR decreased after Intralipid treatment, free water clearance was not related to filtered load but to BSA. Free water clearance and distal tubular sodium delivery were significantly decreased before treatment in comparison with those of the control subjects (Table I1). Although no significant increases in free water clearance or distal tubular sodium delivery were noted after treatment, the free water clearance had increased to normal at 3 years. The distal tubular sodium delivery also tended to increase but remained less than control values (p <0.05). Acidifying capacity. In response to ammonium chloride, a sharp fall in the urinary pH was noted in the control subjects when the total COz concentration was depressed from 25 to 21 mmol/L (Fig. 5). A further decrease to <18 mmoi/L did not influence urinary pH. In the patients with CF, the total CO2 concentration had to be depressed to <21 mmol/L before a fall in urinary pH was found (i.e., the response curves were displaced to the left). All CF patients could finally acidify the urine to pH values <5.6. After 1 year of Intralipid treatment, the response curves in two patients were normalized and a slight improvement was seen in two other patients. The median improvement of serum molar concentration of arachidonic acid in these four patients was 8.5% (range 3% to 9.5%) in comparison with 0.3% (range - 3 % to 2%) in the other six patients (p <0.05). Other relationships to the serum fatty acid pattern were not found. Urinary aldosterone excretion. Mean urinary aldosterone excretion before treatment was 45 + 30 mmol/24 hours (mean _+ SD), not significantly different from that of the control subjects (31 + 20 retool/24 hours). No significant change was seen after 1 year of treatment, the mean value being 51 _+ 33 mmol/24 hours. No correlation was found between urinary sodium excretion and aldosterone excretion or changes in the two measurements. DISCUSSION The EFAD observed in patients with CF includes reduced levels of linoleic acid and, in several cases, of arachidonic acid in the phospholipid fraction. 2'8 Phospholipids predominate in cell membranes, so a disturbed composition might influence cell membrane transport via changes in membrane fluidity and membrane-bound enzymes?Z,33 An increased sodium permeability in lecithin membranes has been reported in rats with EFAD? 5 It is reasonable to suggest that increased sodium reabsorption
Renal function in cystic fibrosis
247
4.0 5.0 6.07.0"
8.0. 12-
1'6
2"0
2 ~.
2"8
Total CO2 ,mmol/I Fig. 5. Relationship between urinary pH and blood total CO2 concentration in 10 patients with cystic fibrosis. Shaded area indicates range of response curves of 10 healthy children.(Data from Berg U, Aperia A, Broberger O. Acta Paediatr Scand 1971;60:521-7.) in the respiratory epithelium~9,29 and in the kidneysTM of patients with CF might be explained by similar mechanisms. Fatty acid supplementation improved the fatty acid pattern in the serum phospholipids in our patients and in one other study?4 The parallelism between that improvement and the normalization of GFR and improved renal sodium elimination suggests that the EFAD directly or indirectly causes these renal disturbances in CF. Such an explanation would also account for the reported differences in GFR in CF, 18,2~ because some centers have recommended a fat-restricted diet, as we did previously. On the other hand, the effect of essential fatty acid supplementation on the disturbed acidifying capacity was less marked; this difference in effect on the different renal transport systems has not been explained. One possible explanation could be that the defective bicarbonate reabsorption is related to abnormal chloride transport that is less dependent on the membrane-bound carbonic anhydrase; such abnormal chloride transport might be closer to the basic defect in CF and may not be influenced (or only marginally so) by EFAD. ~3,]4 Our patients were relatively healthy and did not want to miss school or work for the Intralipid treatment. Thus we could give only a moderate extra supply of essential fatty acids, which seemed to be insufficient to normalize serum levels of essential fatty acids, especially during the third year. Nevertheless, this modest supplementation was associated with significant changes in renal function, even during the third year, which might be explained by a loading of essential fatty acids in fat storage during the earlier, more intensive treatment. 8 Another explanation might be found in changes in prostaglandin metabolism. A more intensive supplementation with essential fatty acids would possibly cause more pronounced changes and more clearly differen-
248
Strandvik et al.
tiate between primary and secondary disturbances in the kidneys of CF patients. The effect of essential fatty acid supplementation on renal function in patients with CF includes a normalization of GFR and an increase in the urinary elimination of a sodium load. The hyperfiltration found in the CF patients before treatment has been discussed previously.TM Hypoinsulinism and glucose intolerance have been reported in CF patients35; the pathogenic mechanisms of the hyperfiltration found in early insulin-dependent diabetes mellitus might be similar. The possibility that the decrease in GFR to normal values, as seen in this study, might be a consequence of the previous hyperfiltering state, causing development of focal glomerulosclerosis,36seems less plausible because our patients did not have albuminuria, which usually is seen with focal sclerosis. Furthermore, the three patients not treated with Intralipid during the third year had the same high values of GFR after 3 years as before the study. The increased sodium excretion was probably caused by a decrease in proximal sodium reabsorption, because the distal tubular sodium delivery tended to increase. Urinary aldosterone excretion was not significantly changed. The normalization of GFR might be related to the tubuloglomerular feedback induced by the increased distal sodium delivery?7 The increased urinary sodium excretion might be related to an increased intestinal absorption of sodium resulting from essential fatty acid supplementation, but this is unlikely because earlier studies have not shown impaired intestinal sodium absorption in patients with CF? 8 On the contrary, there is reason to believe that Na + and K § activated adenosinetriphosphatase, both in the intestine and kidney, is working less efficiently when the EFAD is cured; arachidonic acid may stimulate the synthesis of an adenosinetriphosphatase inhibitory enzyme,39 a suggestion that offers another reasonable explanation for the changed sodium excretion after the supplementation of essential fatty acids. The responses to ammonium chloride differentiate proximal and distal tubular acidifying defects. 4~ A proximal defect (i.e., a defect in bicarbonate reabsorption) leads to a leakage of bicarbonate into the urine at low blood total CO2 values. Thus a displacement of the response curve at the left indicates a reduced bicarbonate threshold. A distal tubular acidifying defect is indicated by an inability of the kidney to acidify the urine finally, even at very low blood total CO2 values. Our results suggest a proximal bicarbonate reabsorption defect in CF, confirming earlier studies.4~,42 At any given plasma bicarbonate concentration, more bicarbonate is excreted in the urine by normal subjects, compared with patients with CF, when secretin is
The Journal of Pediatrics August 1989
administered intravenously.43 Secretin thus seems to decrease the renal bicarbonate threshold value. However, the urinary bicarbonate excretion after secretin administration has not been found to increase in patients with CF? 2 The pancreatic response to secretin infusion is also impaired,44,3~ so the unresponsiveness to secretin seems to be a general characteristic of the disease. Blocked chloride channels have been suggested to be the primary defect in CF, 46but chloride was not measured in the urine. Chloridebicarbonate is another pathway for chloride transport; our results indicate that this transport system also might be influenced in the kidneys of patients with CF. The reduced bicarbonate threshold and the increased proximal sodium reabsorption18 indicate a general proximal tubular disturbance in CF. Renal acidification may continue at nearly normal levels i n t h e absence of intraluminal sodium ions,47 so the previously shown sodium retention does not necessarily contradict a defect in bicarbonate reabsorption. Reduced activity of carbonic anhydrase might be one hypothetical explanation, because function of this enzyme is a prerequisite for adequate bicarbonate reabsorption?s Carbonic anhydrase is a membrane-bound enzyme, and a disturbance in its action may be related to EFAD, 49 Further studies of the renal pathophysiology in CF would probably help separate primary for secondary disturbances and thus improve knowledge of basic abnormalities in the disease. Mrs. Eva Edeb/ick is gratefully acknowledged for skillful technical assistance. REFERENCES
1. Kuo PT, Huang NN, Basset DR. The fatty acid composition of serum chylomicrons and adipose tissue of children with cystic fibrosis of the pancreas. J PEDIATR1962;60:394-403. 2. Hubbard VS, Dunn GD, di Sant Agnese PA. Abnormal fatty acid composition of plasma lipids in cystic fibrosis. Lancet 1977;2:1302-04. 3. Galabert C, Filliat M, Chazalette JP. Fatty acid composition of serum lecithins in cystic fibrosis patients without steatorrhea [Letter]. Lancet 1978;1:903. 4. Rogiers V, Crokaert R, Vis H-L. Altered phospholipid composition and changed fatty acid pattern of the various phospholipid fractions of red cell membranes of cystic fibrosis in children with pancreatic insufficiency. Clin Chim Acta 1980;105:105-15. 5. Rogiers V, Mandelbaum I, Mozes N, et al. In vitro study of the incorporation and transport of nonesterified fatty acids into the phospholipids of the red blood cell membranes of cystic fibrosis patients. Pediatr Res 1982;16:761-8. 6. Rogiers V, Crokaert R, Vis HL. Long-chain non-esterified fatty acid pattern in plasma of cystic fibrosis patients and their parents. Pediatr Res 1980;t4:1088-91. 7. Rogiers V, Vereruysse A, Dab I, Crokaert R, Vis HL. Fatty acid 16htternof platelet phospholipids in cystic fibrosis. Eur J Pediatr 1984;142:305-6.
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8. Farrell PM, Minschter EH, Engle M J, Brown J, Lau S-M. Fatty acid abnormalities in cystic fibrosis. Pediatr Res 1985;19:104-9. 9. Stubbs CD, Smith AD. The modification of mammalian membran e polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim Biop'0ys Acta 1984;779:81-137. 10. Owens JS, Brachdorfer KR, Day RC, McIntyre N. Decreased erythrocyte membrane fluidity and altered lipid composition in human liver disease [Abstract]. Clin Sci 1981;60;30. 11. Roman I, Guraj P, Nowicka C, Angielski S. Regulation of Ca 2+ efftux from kidney and liver mitochondria by unsaturated fatty acids and Na + ions. Eur J Biochem 1979;102:61523. 12. Carlstedt-Duke J, Br6nneg/trd M, Strandvik B. Pathological regulation of arachidonic acid i:elease in cystic fibrosis: the putative basic defect. Proc Natl Acad Sci USA 1986;83: 9202-6. 13. Strandvik B, Br6nnegfird M, Carlstedt-Duke J. Arachidonic acid release in CF. In: Mastella G, Quinton PM, eds. Cellular and molecular basis of cystic fibrosis. San Francisco: San Francisco Press, 1988:445-50. 14. Strandvik B, Br~Snnegfird M, Gilljam H, Carlstedt-Duke J. Defective regulation of arachidonic acid release and symptoms in cystic fibrosis. Seancl J Gastroenterol 1988;23(suppl 143):1-4. 15. Moore L J, Richard sson T, DeLuca HF. Essential fatty acid and ionic Permeability of lecithin membranes. Chem Phys Lipids 1963;3:39-58. 16. Holman RT. Essential fatty acid deficiency. Prog Chem Fats Lipids 1968;9:279-348. 17. Robson AM, Tateishi S, Ingelflnger JR, Strominger DB, Klahr S. Renal function in patients with cystic fibrosis. J PEDIATR 1971;79:42-50. 18. Berg U, Kusoffsky E, Strandvik B. Renal function in cystic fibrosis with special reference to the renal sodium handling. Acta Paediatr Scand 1982;71:833-8. 19. Knowles M, Gatzy J, Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 1981;305:1489-95. 20. Boucher RC, Stutts M J, Kuowles MR, Cautley L, Gatzy JT. Na + transport in Cystic fibrosis respiratory epithelia: abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 1986;78:1245-52. 21. Spino M, Chai RP, Isles AF, et al. Assessment of glomerular filtration rate and effective renal plasma flow in cystic fibrosis. J PEDIATR 1985;107 64-70. 22. Oppenheimer EH. Glomerular lesions in cystic fibrosis: possib!e relation to diabetes mellitus, acquired cyanotic heart disease and cirrhosis of the liver. Johns Hopkins Med J 1972;131:351-64. 23. Shwachman H, Kulczycki LL. Long-term study of one hundred and five patients with cYstic fibrosis: studies made over a five- to fourteenTyear period. Am J Dis Child 1958; 96:6-15. 24. Folch J, Lees ML, Sloane Stanley GH. A simple method for the isolation and Purification of total lipids from animal tissues. J Biol Chem 1957;226:497-509. 25. Berg U. Renal function in acute febrile urinary tract infection in children: path0physiologic aspects on the reduced concentrating capacity. Kidney Int 1981 20:753-8.
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26. Hilger HH, Klumper JD, Ullrich KJ. Wasserriickresorption und Ionentranspori durch die Sammelrohrzellen der Sfiugetierniere. Pflugers Arch 1958;267:218-37. 27. Smith HW, Fingelstein N, Aliminosa L, Crawford B, Garber M. The renal clearances of substituted hippuric acid derivatives and other aromatic acids in dogs and man. J Clin Invest 1945;24:388-404. 28. Edelmann CM Jr, Boichis H, Soriano JR, Stark H. The renal response of children to acute ammonium chloride acidosis. Pediatr Res 1967;1:452-60. 29. Berg U, Aperia A, Broberger O. Subclinical defects in renal regulation of acid base balance in children with recurrent urinary tract infections. Acta Paediatr Scand 1971;60: 521-7. 30. Siggaard-Anderson O. The pH-log Pco~ blood acid-base nomogram revised. Scand J Clin Lab Invest 1962;14:598604. 31. Sealey JE, Buhler FR, Laragh JH, Manning EL, Brunner HR. Aldosterone excretion. Circ Res 1972;31:367-78. 32. Engelhard VA, Glaser M, Storm DR. Effect of membrane phospholipid compositional changes on adenylate cyclase in LM cells. Biochemistry 1978;17:3191-200. 33. Colard D, Kerrbn A, Roy C. Effect of adenylate cyclase activities of unsaturated fatty acid incorporation into rat liver plasma membrane phospholipids, modulation by linoleate. Biochem Biophys Res Comm 1980;95:97-102. 34. Roscnlund ML, Selekman JA, Kim HK, Kritchevsky D. Dietary essential fatty acids in cystic fibrosis. Pediatrics 1977;59:428-32. 35. Lippe BM, Sperling MA, Dooley RR. Pancreatic alpha and beta cell functions in cystic fibrosis. J PEDIATR 1977;90: 751-5. 36. Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in ageing, renal ablation, and intrinsic renal disease. N Engl J Med 1982; 307:652-9. 37. Navar LG, Burke T J, Robinson RR, Clapp JR. Distal tubular feedback in the autoregulation of single nephron glomerular filtration rate. J Clin Invest 1974;53:516-26. 38. Strandvik B, Plantin L-O, Bergstr6m K, Broberger O, Freyschuss U. Total exchangeable sodium in cystic fibrosis. Monogr Paediatr 1981;14:36-9. 39. Schwartzman M, Ferreri NR, Carroll MA, Songu-Mize E, McGiff JC. Renal cytochrome P-450-related arachidonate metabolite inhibits (Na+-K+)ATPase. Nature 1985;314: 620-2. 40. Soriano JR, Boichis H, Edelmann CM Jr. Bicarbonate reabsorption and hydrogen ion excretion in children with renal tubular acidosis. J PEDIATR 1967;71:802-13. 41. Scott J, Elias E, Moult PJA, Barnes S, Wilis MR. Rickets in adult cystic fibrosis with myopathy, pancreatic insufficiency and proximal renal tubular dysfunction. Am J Med 1977; 63:488-92. 42. Bretscher D, Schneider A, Hagmann R, et al. Response of renal handling of sodium and bicarbonate to secretin in normals and patients with cystic fibrosis [Abstract]. Pediatr Res 1974; 8:899. 43. Oetliker O, Hadorn B, Chattas A, Schultz SM. Secretin induces renal bicarbonate loss in man. Biol Gastro-Enterol 197!;4:309-16.
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44. Hadorn B, Johansen PG, Anderson CM. Pancreozyminsecretin test of exocrine pancreatic function in cystic fibrosis and the significance of the result for the pathogenesis of the disease. Aust Paediatr J 1968;4:8-22. 45. Gaskin KJ, Durie PR, Corey M, Wei P, Forstner GG: Evidence for a primary defect of pancreatic HCO3-secretion in cystic fibrosis. Pediatr Res 1982;16:554-7. 46. Quinton PM. Chloride impermeability in cystic fibrosis. Nature 1983;301:421-2.
47. Giebisch G, Malnic G. Studies on the mechanism of tubular acidification. Physiologist 1976;19:511-24. 48. Rector FC Jr, Carter NW, Seldin DW. The mechanism of bicarbonate reabsorpton in the proximal and distal tubules of the kidney. J Clin Invest 1965;44:278-90. 49. L6nnerholm G, Wistrand PJ. Carbonic anhydrase in the human kidney: a histochemical and immunocytochemical study. Kidney Int 1984;25:886-98.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the 1989 issues of THE JOURNALOF PEDIATRICS are available to subscribers (only) from the Publisher, at a cost of $48.00 ($64.00 international) for Vol. 114 (January-June) and Vol. 115 (July-December), shipping charges included. Each bound volume contains subject and author indexes, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram, with the Journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact The C.V. Mosby Company, Circulation Department, 11830 Westline Industrial Dr., St. Louis, M O 63146-3318, USA/800-325-4177, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular Journal subscription.
In rats with EFAD, an increased sodium permeability has been noted in lecithin membranes, 15 and rats with E F A D have hematuria. 16 Increased sodium reabsorption in the proximal renal tubule, ~7'~8 as well as in the respiratory epithelium, ~9,2~has been reported in CF. Some patients with C F have an increased glomerular filtration rate, ~8
Supported by grants from the Swedish Medical Research Council (Nos. 8558 , 5361, and 6864), Karolinska Institutet, Sigurd and Elsa Golje's Memorial Fund, and the Swedish Medical Association. Submitted for publication Aug. 23, 1988; accepted Feb. 21, 1989. Reprint requests: B. Strandvik, MD, PhD, Department of Pediatrics, Huddinge Hospital, S-141 86 Huddinge, Sweden.
although this has been an inconsistent finding.:1 Hematuria and morphologic glomerular changes are not uncommon in advanced stages of the disease. 22 It thus seems that the renal disturbances--although not clinically import a n t - a r e extensive and might be at least partly related to the EEAD in these patients. To test this hypothesis, we
242
BSA CF EFAD GFR PAH
Body surface area Cystic fibrosis Essential fatty acid deficiency Glomerular filtration rate Para-aminohippuric acid
Volume l l 5 Number 2
Renal function in cystic fibrosis
243
Ratio ~8:2/18:0 2.0
Ratio 18:2//1B:0
4.c
.
.
.
.
.
.
.
2
.
.
.
.
.
.
.
.
.
.
.
.
2,o.
,
/!
,
0
1
2
3
years
,
~
~- . . . .
4
IL
Fig. t. Mean ratio (_+2 SD) of linoleic acid to stearic acid in the triglyceride fraction of fasting serum in 10 patients with CF during 3 years of interrupted essential fatty acid Supplementation (Intralipid). Means (_+ 2 SD) for the control group are indicated by horizontal lines. For further explanation, see text,
investigated the renal function in patients with C F before and after long-term intravenous supplementation with essential fatty acids. METHODS Patients. Eleven patients (six female) with C F were studied. Their ages ranged from 5 to 26 years (mean 14 yeai's). Clinical scores according to Shwachman and Kulczycki2s were 56 to 94 (mean 78) at the start of the study. All patients had abnormal sweat test results (chloride >80 mmol/L), pulmonary symptoms, and pancreatic insufficiency. They Were in good clinical condition without antibiotic treatment at the time of the investigations. None of the patients had any signs of renal diseasel and all patients had normal oral glucose tolerance test results. All patients had normal height and weight for age and were on a normal diet without extra sodium chloride. Fatty acid Supplementation was given intravenously at regular intervals for 3 years. During the first year, nine of the patients received 10 ml of 10% lipid emulsion (Intralipid) per kilogram of body weight intravenously every fortnight; the other two patients received the preparation on 3 consecutive days once a month. The treatment was interrupted for psychologic reasons after the first year. After a 1-year intermission, treatment was accepted for another year by 7 of the 11 patients, but just for 2 consecutive days every second month. The extra supplementation of linoleic acid was 10.5 to 16.5 gm/kg b0dyweight during the first year, corresponding to 30 to 50 mg linoleic acid per kilogram of body weight per day. During the third year, corresponding values were 5 to 7 gm/kg body weight and
1.0,
,
0
//
,
1
2
3
years
[
~ . . . .
-~
I L
F i g . 2 . Mean ratio ( _+2 SD) of linoleic acid to stearie acid in the phospholipid fraction of fasting serum in 10 patients with CF during 3 years of interrupted essentia! fatty acid supplementation. For explanation, see Fig. 1 and text.
15 to 20 mg/kg body weight per day, respectively. Renal function was studied when the patients were hospitalized 2 to 4 weeks after the latest infusion of fat emulsion. All the studies were performed before and after the first year of fatty acid supplementation. Renal hemodynamics, r e n a l sodium handling, and diluting capacity were also studied at the end Of 3 years of treatment. Fifteen hospitalized children in the same age range, without CF and with no clinical evidence of malabsorption or infection, and 10 healthy adults from the medical staff were studied as reference groups for the serum fatty acid analysis and urinary aldosterone excretion. Sampling and analyses were Carried out under conditions identical with those of the patients. Informed consent was given by the patients and parents. The study was approved by the Karolinska Institute Committee of Ethics. Laboratory studies. Serum samples for lipid analysis were drawn by venipuncture in the morning, 12 hours after the latest meal. Sampling was performed before lntralipid treatment was given to the patients with CF. The serum triglycerides and phospholipids were separated by thinlayer chromatography (silica ge I plates developed with chloroform, methanol, and water 80:30:5) after Folch extraction. 24 The compounds were eluted from the gel and hydrolYzed, and the relative amounts of fatty acids in the fractions were determine d by gas-liquid chromatography (stationary phase SP 2310/2% SP 2300 on 100/120 porous
244
S t r a n d v i k et al.
The Journal o f Pediatrics August 1989
T a b l e I. Renal h e m o d y n a m i c s a n d urinary sodium excretion ( m e a n + SD) before and after Intralipid t r e a t m e n t for 1 year and 3 years and control subjects
Renal test
GFR (ml/min/1.73 m 2 BSA)
RPF (ml/min/1.73 m E BSA)
FF (%)
Basal urinary Na excretion (mmol/ hr/1.73 m 2 BSA) Renal response to oral Na load (mmol/hr/1.73 m 2 BSA)
n
Before
10 7 6 10 7 6 10 7 6 10 7 3r 10 7 3*
133 • 18 128 _+ 17 120 _ 19 642 • 79 651 • 60 628 _+ 85 20.7 + 1.8 20.3 • 1.8 20.3 _+ 2.2 3.6 • 2.5 4.1 • 2.9 3.4, 1.3, 2.8 3.4 _+ 2.1 3.8 +_ 2.3 3.9, 2.3, 0.9
After I yr
After 3 yr
Control subjects (n = 25)
110 • 19
112 • 10
111 _+ 14 601 • 67 589 • 126 616 • 203 18.7 _+ 2.8 21.8 • 4.0 18.6 • 3.2 4.7 • 2.7 7.1, 5.1, 10.3 6.6 • 3.6 8.2, 7.9, 11.0
7.9 _+ 2.0 5.0 + 3.1 4.8, 6.8, 9.1 15.9 • 1.5 10.9 • 4.7 10.9, 12.6, 17.6
c, Control subjects; B, before Intralipid treatment; NS, not significant; RPF, renal plasma flow; FF, filtration fraction. *Three patients receiving only 1 year of Intralipid treatment.
T a b l e !1. Free water clearance a n d distal t u b u l a r sodium delivery ( m e a n _ SD) in C F patients a n d control subjects at start of study and after 1 year and 3 years of Intralipid t r e a t m e n t of the patients with C F
Renal test
Cn,o (ml/min/l.73 m 2 BSA)
CH,o + CN, (ml/min/1.73 m 2 BSA)
Uosm (mOsm/L)
n
10 7 5 10 7 5 10 7 5
Before
91. 8.2 9.1 9.6 8.7 9.5 61 60 60
_+ 2.5 • 2.4 _+ 2.7 _+ 2.6 _+ 2.5 + 2.9 _+ 16 • 18 • 20
After 4 yr
After 3 yr
Control subjects (n = 25)
12.0 _+ 1.4 8.9 +_ 2.8 10.5 • 2.2 13.4 • 1.2 9.4 _+ 2.8 11.0 _+ 2.2 65 • 13 63 • 8 62 + 8
C, Control subjects; B, before treatment; NS, not significant; Crr2o,clearance of water; CN,, clearance of sodium; Uosm, urine osmolality.
polymer support C h r o m o s o r b W [ A W ] , J o h n s - M a n v i l l e [available from S i g m a C h e m i c a l Co., St. Louis, Mo.] ). A standardized sodium intake of 2 m m o l / k g body weight was given 4 days before the renal tests. U r i n a r y sodium excretion during 6 hours was studied after an oral sodium load of 95 m m o l / 1 . 7 3 m 2 body surface area. TM During the oral sodium test, clearances of inulin a n d P A H 2~ were determined. U r i n a r y a n d blood samples were analyzed for inulin according to the a n t h r o n e method 26 and for P A H by a modification of the technique described by S m i t h et al: 27 T h e concentration of sodium was determined with a flame photometer and osmolality by the freezing point techniquel Diluting capacity was evaluated as free water clearance.
On a separate day, a short oral a m m o n i u m chloride load was performed for testing acidifying capacity. 2s,29 T h e patients were given water orally, 5 m l / k g body weight every 30 minutes, to m a i n t a i n a c o n s t a n t urine flow rate. U r i n e was collected by spontaneous voiding at 6 0 - m i n u t e intervals. In the middle of each urine sampling period, an arterialized capillary blood sample was taken. A f t e r two urine control periods, an oral a m m o n i u m chloride load of 150 m m o l / m 2 B S A was given. Blood and urine samples were t a k e n for another 5 hours a n d immediately analyzed for p H with a p H meter (Radiometer, Copenhagen, D e n m a r k ) . T h e data were plotted on a S i g g a a r d - A n d e r s e n curve fiomogram giving Pco/, s t a n d a r d bicarbon~tte concentration, and blood total CO2 c o n c e n t r a t i o n ? ~
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Renal function in cystic fibrosis
245
Significance ( p v a l u e ) C vs t yr
C vs B
< 0.01 < 0.01 < 0.01 NS NS NS NS NS NS < 0.001 < 0.01
C vs 3 yr
NS
B vs t yr
B vs 3 yr
< 0.05
<0.05
NS NS
NS NS
NS NS
NS NS
NS NS
< 0.01 <0.05
< 0.001 < 0.001
< 0,001
NS < 0.05
< 0.01
< 0,05
Significance ( p value) C vs B
< < < < < <
0.01 0.01 0.05 0,001 0.001 0.01 NS NS NS
C vs t yr
C vs 3 yr
B vs t yr
B vs 3 yr
NS
< 0.01 NS < 0.05
NS NS
< 0.0l
NS NS
NS
Twenty-four-hour urinary aldosterone excretion was determined by a radioimmunoassay? ~ The results of renal function in C F were compared with those of control subjects previously reported, ~5'29 Statistical analysis. The Mann-Whitney U test, Wilcoxon matched-pairs ranked-signs test, and Student paired t test were used for statistical analyses. RESULTS
General data. The patients had normal growth during the study period, no improvement in comparison with the 2 years before (data not shown). Lung function did not improve, and the number of infections was not reduced. The clinical score also did not change. No obvious clinical effect could thus be seen as a result of treatment.
NS
NS
Serum lipid analysis Fatty acid pattern in the serum triglyceride fraction. The relative amounts of saturated fatty acids in the triglyceride fraction were slightly increased and changed little during the study. The mean (_+ S E M ) molar percentage of linoleic acid was significantly decreased (p < 0.01) in the patients, in comparison with healthy control subjects, at the start of the study (7.9 _+ 1.1% and 16.0 +_ 0.6%, respectively). During the study, molar percentage of linoleic acid increased significantly after 1 and 3 years (p < 0.05, respectively), but no significant changes were noted in the concentration of other fatty acids. The ratio of oleic acid to linoleic acid; which was significantly increased initially (p < 0.001), did not change significantly (from 6.4 to 4.5 after 1 year to 3.5 after 3 years; control subjects 2.7).
246
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The Journal of Pediatrics August 1989
Ratio
U NaV, m m o l / h r / 1 . 7 3 m ~ B S A 20 i load
10.
0
1 ~
2
3
=_ . . . .
_~ I L
years
Fig. 3. Mean ratio (_+2 SD) of arachidonie acid to stearic acid in the phospholipidfraction in 10 patients with CF. For explanation, see Fig. 1 and text.
The ratio of linoleic acid to stearic acid was decreased significantly at the start and increased significantly during the first year of treatment (p < 0.01) (Fig. 1). However, the mean ratio at 3 years did not significantly differ from that at the start of the study and was lower than at 1 year of treatment (p = 0.05). Fatty acid pattern in the serum phospholipid fraction. In the serum phospholipid fraction, the molar percentage of saturated fatty acids did not change significantly during the study. Initially the molar percentage of palmitoleic acid was significantly increased (p < 0.01), as was that of oleic acid (p < 0.001). The molar percentages of linoleic, 7-1inoleic, and araehidonic acids were significantly decreased compared with those of healthy control subjects (p<0.001, <0.01, and <0.001, respectively), being 16.8 +_ 2.7% versus 24.3 _+ 0.9%, 0.4 _+ 0.2%versus 1.0 + 0.2%, and 5.2 + 1.1% versus 9.5 +_ 0.4%, respectively. No significant change was found during the study period. The ratio of oleic acid to linoleic acid, which was significantly increased initially (p <0.001), also did not improve significantly, from 1.5 to 1.0 at 1 year and to 0.8 at 3 years (control ratio 0.6). The ratio of linoleic acid to stearic acid increased significantly during the first year (p <0.05) and did not change significantly during the third year with the lower amount of essential fatty acid supplementation (Fig. 2). The ratio of arachidonic acid to stearic acid showed a similar pattern (Fig. 3). Renal studies. All patients with cystic fibrosis were not included in all renal studies because of allergic reactions to the inulin test dose or technical errors. The number of patients studied is given in each part of the results (see Tables I and II). Renal hemodynamics. Mean glomerutar filtration rates,
~"
O
1
2
3
4
5
6 hrs
0
1
2
3
4
5
6 hrs
Fig. 4. Mean (_+SD) hourly urinary sodium excretion (UNaV) before and after an oral sodium load of 95 mmol/1.73 m2 BSA in patients with CF before (straight lines) and after 1 year (n = 10) (left panel, dotted line) and 3 years (n = 7) (right panel, dotted line) of treatment with interrupted essential fatty acids (Intralipid). Shaded area repesents control values. (Data from Berg U. Kidney Int 1981;20:753-8.)
clearances of PAH, and filtration fractions before and after Intralipid treatment are listed in Table I. Before treatment, GFR was significantly increased compared with that of the control subjects (p <0.01). The G F R decreased significantly to within normal values after 1 year of fatty acid supplementation, and did not change in those six patients who were studied after another year of Intralipid treatment. Two of three studied patients who did not get Intralipid in the third year had an increase in GFR between 1 and 3 years. In the third patient, the determination of GFR after 1 year failed, but the GFR was very high both before treatment and after 3 years, being 160 and 158 ml/min/1.73 m 2 BSA, respectively. The clearance of PAH and FF before treatment did not differ significantly from that of control subjects and did not change significantly after fatty acid supplementation (Table I). Urinary sodium excretion. The basal urinary sodium excretion in the CF group was significantly decreased in comparison with control values (p <0.001) (Table I). Although sodium elimination gradually improved, the values were still significantly lower than in the control subjects after both 1 year and 3 years of essential fatty acid supplementation (p <0.01 and <0.05, respectively). The renal response to an oral sodium load (mean urinary sodium excretion during the third to the sixth hour) was absent before treatment, with significantly lower values than those in the control subjects (p <0.001). The response improved significantly after 1 year of Intralipid treatment (p <0.05). After 3 years, the renal response had further
Volume l 15 Number 2
increased but was still significantly less than that of the control subjects (p <0.01) (Fig. 4). Diluting capacity. Diluting capacity was determined as free water clearance when the lowest urine osmolality was reached. Because GFR decreased after Intralipid treatment, free water clearance was not related to filtered load but to BSA. Free water clearance and distal tubular sodium delivery were significantly decreased before treatment in comparison with those of the control subjects (Table I1). Although no significant increases in free water clearance or distal tubular sodium delivery were noted after treatment, the free water clearance had increased to normal at 3 years. The distal tubular sodium delivery also tended to increase but remained less than control values (p <0.05). Acidifying capacity. In response to ammonium chloride, a sharp fall in the urinary pH was noted in the control subjects when the total COz concentration was depressed from 25 to 21 mmol/L (Fig. 5). A further decrease to <18 mmoi/L did not influence urinary pH. In the patients with CF, the total CO2 concentration had to be depressed to <21 mmol/L before a fall in urinary pH was found (i.e., the response curves were displaced to the left). All CF patients could finally acidify the urine to pH values <5.6. After 1 year of Intralipid treatment, the response curves in two patients were normalized and a slight improvement was seen in two other patients. The median improvement of serum molar concentration of arachidonic acid in these four patients was 8.5% (range 3% to 9.5%) in comparison with 0.3% (range - 3 % to 2%) in the other six patients (p <0.05). Other relationships to the serum fatty acid pattern were not found. Urinary aldosterone excretion. Mean urinary aldosterone excretion before treatment was 45 + 30 mmol/24 hours (mean _+ SD), not significantly different from that of the control subjects (31 + 20 retool/24 hours). No significant change was seen after 1 year of treatment, the mean value being 51 _+ 33 mmol/24 hours. No correlation was found between urinary sodium excretion and aldosterone excretion or changes in the two measurements. DISCUSSION The EFAD observed in patients with CF includes reduced levels of linoleic acid and, in several cases, of arachidonic acid in the phospholipid fraction. 2'8 Phospholipids predominate in cell membranes, so a disturbed composition might influence cell membrane transport via changes in membrane fluidity and membrane-bound enzymes?Z,33 An increased sodium permeability in lecithin membranes has been reported in rats with EFAD? 5 It is reasonable to suggest that increased sodium reabsorption
Renal function in cystic fibrosis
247
4.0 5.0 6.07.0"
8.0. 12-
1'6
2"0
2 ~.
2"8
Total CO2 ,mmol/I Fig. 5. Relationship between urinary pH and blood total CO2 concentration in 10 patients with cystic fibrosis. Shaded area indicates range of response curves of 10 healthy children.(Data from Berg U, Aperia A, Broberger O. Acta Paediatr Scand 1971;60:521-7.) in the respiratory epithelium~9,29 and in the kidneysTM of patients with CF might be explained by similar mechanisms. Fatty acid supplementation improved the fatty acid pattern in the serum phospholipids in our patients and in one other study?4 The parallelism between that improvement and the normalization of GFR and improved renal sodium elimination suggests that the EFAD directly or indirectly causes these renal disturbances in CF. Such an explanation would also account for the reported differences in GFR in CF, 18,2~ because some centers have recommended a fat-restricted diet, as we did previously. On the other hand, the effect of essential fatty acid supplementation on the disturbed acidifying capacity was less marked; this difference in effect on the different renal transport systems has not been explained. One possible explanation could be that the defective bicarbonate reabsorption is related to abnormal chloride transport that is less dependent on the membrane-bound carbonic anhydrase; such abnormal chloride transport might be closer to the basic defect in CF and may not be influenced (or only marginally so) by EFAD. ~3,]4 Our patients were relatively healthy and did not want to miss school or work for the Intralipid treatment. Thus we could give only a moderate extra supply of essential fatty acids, which seemed to be insufficient to normalize serum levels of essential fatty acids, especially during the third year. Nevertheless, this modest supplementation was associated with significant changes in renal function, even during the third year, which might be explained by a loading of essential fatty acids in fat storage during the earlier, more intensive treatment. 8 Another explanation might be found in changes in prostaglandin metabolism. A more intensive supplementation with essential fatty acids would possibly cause more pronounced changes and more clearly differen-
248
Strandvik et al.
tiate between primary and secondary disturbances in the kidneys of CF patients. The effect of essential fatty acid supplementation on renal function in patients with CF includes a normalization of GFR and an increase in the urinary elimination of a sodium load. The hyperfiltration found in the CF patients before treatment has been discussed previously.TM Hypoinsulinism and glucose intolerance have been reported in CF patients35; the pathogenic mechanisms of the hyperfiltration found in early insulin-dependent diabetes mellitus might be similar. The possibility that the decrease in GFR to normal values, as seen in this study, might be a consequence of the previous hyperfiltering state, causing development of focal glomerulosclerosis,36seems less plausible because our patients did not have albuminuria, which usually is seen with focal sclerosis. Furthermore, the three patients not treated with Intralipid during the third year had the same high values of GFR after 3 years as before the study. The increased sodium excretion was probably caused by a decrease in proximal sodium reabsorption, because the distal tubular sodium delivery tended to increase. Urinary aldosterone excretion was not significantly changed. The normalization of GFR might be related to the tubuloglomerular feedback induced by the increased distal sodium delivery?7 The increased urinary sodium excretion might be related to an increased intestinal absorption of sodium resulting from essential fatty acid supplementation, but this is unlikely because earlier studies have not shown impaired intestinal sodium absorption in patients with CF? 8 On the contrary, there is reason to believe that Na + and K § activated adenosinetriphosphatase, both in the intestine and kidney, is working less efficiently when the EFAD is cured; arachidonic acid may stimulate the synthesis of an adenosinetriphosphatase inhibitory enzyme,39 a suggestion that offers another reasonable explanation for the changed sodium excretion after the supplementation of essential fatty acids. The responses to ammonium chloride differentiate proximal and distal tubular acidifying defects. 4~ A proximal defect (i.e., a defect in bicarbonate reabsorption) leads to a leakage of bicarbonate into the urine at low blood total CO2 values. Thus a displacement of the response curve at the left indicates a reduced bicarbonate threshold. A distal tubular acidifying defect is indicated by an inability of the kidney to acidify the urine finally, even at very low blood total CO2 values. Our results suggest a proximal bicarbonate reabsorption defect in CF, confirming earlier studies.4~,42 At any given plasma bicarbonate concentration, more bicarbonate is excreted in the urine by normal subjects, compared with patients with CF, when secretin is
The Journal of Pediatrics August 1989
administered intravenously.43 Secretin thus seems to decrease the renal bicarbonate threshold value. However, the urinary bicarbonate excretion after secretin administration has not been found to increase in patients with CF? 2 The pancreatic response to secretin infusion is also impaired,44,3~ so the unresponsiveness to secretin seems to be a general characteristic of the disease. Blocked chloride channels have been suggested to be the primary defect in CF, 46but chloride was not measured in the urine. Chloridebicarbonate is another pathway for chloride transport; our results indicate that this transport system also might be influenced in the kidneys of patients with CF. The reduced bicarbonate threshold and the increased proximal sodium reabsorption18 indicate a general proximal tubular disturbance in CF. Renal acidification may continue at nearly normal levels i n t h e absence of intraluminal sodium ions,47 so the previously shown sodium retention does not necessarily contradict a defect in bicarbonate reabsorption. Reduced activity of carbonic anhydrase might be one hypothetical explanation, because function of this enzyme is a prerequisite for adequate bicarbonate reabsorption?s Carbonic anhydrase is a membrane-bound enzyme, and a disturbance in its action may be related to EFAD, 49 Further studies of the renal pathophysiology in CF would probably help separate primary for secondary disturbances and thus improve knowledge of basic abnormalities in the disease. Mrs. Eva Edeb/ick is gratefully acknowledged for skillful technical assistance. REFERENCES
1. Kuo PT, Huang NN, Basset DR. The fatty acid composition of serum chylomicrons and adipose tissue of children with cystic fibrosis of the pancreas. J PEDIATR1962;60:394-403. 2. Hubbard VS, Dunn GD, di Sant Agnese PA. Abnormal fatty acid composition of plasma lipids in cystic fibrosis. Lancet 1977;2:1302-04. 3. Galabert C, Filliat M, Chazalette JP. Fatty acid composition of serum lecithins in cystic fibrosis patients without steatorrhea [Letter]. Lancet 1978;1:903. 4. Rogiers V, Crokaert R, Vis H-L. Altered phospholipid composition and changed fatty acid pattern of the various phospholipid fractions of red cell membranes of cystic fibrosis in children with pancreatic insufficiency. Clin Chim Acta 1980;105:105-15. 5. Rogiers V, Mandelbaum I, Mozes N, et al. In vitro study of the incorporation and transport of nonesterified fatty acids into the phospholipids of the red blood cell membranes of cystic fibrosis patients. Pediatr Res 1982;16:761-8. 6. Rogiers V, Crokaert R, Vis HL. Long-chain non-esterified fatty acid pattern in plasma of cystic fibrosis patients and their parents. Pediatr Res 1980;t4:1088-91. 7. Rogiers V, Vereruysse A, Dab I, Crokaert R, Vis HL. Fatty acid 16htternof platelet phospholipids in cystic fibrosis. Eur J Pediatr 1984;142:305-6.
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