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The relation between age and renal concentrating capacity in sickle cell disease and hemoglobin C disease
CLINICA CHIMICA ACTA
5or
T H E RELATION B E T W E E N AGE AND RENAL CONCENTRATING CAPACITY IN SICKLE CELL DISEASE AND HEMOGLOBIN C DISEASE*
L. W. STATIUS VAN EPS, H. SCHOUTEN, C. CH. T E R H A A R ROMENY-XVACHTER ANn L. W. LA P O R T E - W I J S M A N with technical assistance of A. M. S M O R E N B U R G - S T R U Y K E R B O U I ) I E R
St. Elisabeth Hospital, Department of Inte#'~ml Medicine and Clinical Chemistry, lI'ilh, mstad, Curafao (Netherlands Antilles) (Revised manuscript re~:eived November t 7, x969)
SUMMARY
I. Renal Concentrating Capacity (RCC) and Glomemlar Filtration Rate (GFR) were investigated in groups of subjects of different ages suffering from sickle cell anemia (SS(+F)), sickle cell trait (AS), sickle cell hemoglobin C disease (SC), hemoglobin C disease (CC) and hemoglobin C trait (AC). The RCC of SS(+F) patients aged IO years and older falls into a very narrow range (mean 434 mOsm: S.D. ~21), in those younger than IO years tile RCC is much more variable ranging from 460-687 mOsm. AS subjects, on tile other hand, show a significant progressive decrease in RCC with age. The youngest patients have an ahnost normal concentrating capacity and the oldest subjects are unable to concentrate better than to the fixed maximum of the anemia group. SC patients also demonstrate a declining RCC, but on a lower level than in the AS subjects, for all age groups; the RCC of the elder ones show the same fixed maximum as in SS(+F). CC and AC subjects have a normal RCC. In none of the groups investigated was a correlation found between RCC and GFR. 2. In three AS patients urine was collected simultaneously from the right and left kidney during hydropenia or vasopressin infusion. Tile results indicate that the concentrating defect affects both kidneys in the same degree.
INTRODUCTION
in the first case history of sickle cell disease a slightiy increased diuresis and urine of low specific gravity was already observed 1. This concentrating defect has * This work was supported b y a research grant of the Netherlands Foundation for the advancement of Tropical Research (WOTRO).
C!in. Chim. Acta, 2 7 (I97 o) 5Ol-5XI
502
STATIUS VAN EPS el al.
been confirmed by others ~-s. An inborn abnormality of the hemoglobin molecule is responsible for the sickling phenomenon 9, but the hereditary nature of the renal concentrating defect has been a matter of dispute. The sickle cell trait is also attended with defective renal concentration to a minor and varying degree. The hyposthenuria becomes increasingly more severe with advancing age in both groups of AS and SS(+F) patients, although with a quantitative difference; with increasing age, the subjects with sickle cell trait were found to have normal and impaired maximum of urinary concentration ~,7. The concentrating defect was restored to normal in infants with SS(+F) alter multiple transfusions with hemoglobin A blood ~,~9. This improvement is progressively lost with age and was not related to renal hemodynamics TM. Observations regarding renal concentrating ability in hemoglobin C disease, hemoglobin C trait and sickle cell hemoglobin C disease are almost completely lacking. In the present study, a strictly standardized renal concentration test~, ~ has been employed to investigate concentrating capacity in patients with hemoglobin SS(+F), AS, SC, CC and AC of different ages to clarify the pathogenesis of the concentrating defect. Furthermore, in three patients with sickle cell trait the maximal RCC of the individual kidneys were examined separately. MATERIALS AND METHODS
3t patients with SS(+ F), 29 subjects with AS, t6 subjects with AC, 9 patients with CC and I4 with SC were investigated with the renal concentration test and the results compared with those of 54 subjects with hemoglobin AA. In most cases the concentration test was combined with a z4-h endogenous creatinine clearance test. Apart from actual diseases of the kidney care was taken to avoid: anemias other than consequent to the hemoglobinopathy, fever, hypertension, abnormal basal metabolism, disturbances of calcium metabolism and cirrhosis of the liver. In 4 SS(+F) patients the concentration test was repeated a second time to evaluate the reproducibility of the test. In 7 patients the test was repeated with the subject receiving a subcutaneous injection of xo Units vasopressin half an hour before the first urine collection. In three patients with AS, being subjected to cystoscopy and ureteral catheterization, because of idiopathic hematuria (ultimately diagnosed as a complication of their sickle cell disease) maximal urinary osmolality of the right and left kidney were examined separately. In 2 patients catheterization was performed during maximal hydropenia at the end of the concentration test. The third patient was normally hydrated. She received an intravenous infusion of vasopressin (Sandoz), 2 mU/min, urine being collected in divided portions from the right and the left kidney before, during and after the period of infusion. The chemical methods employed h,,ve been described previously TM. Correlation between maximal urinary osmolality and age and between creatinine clearance and age was tested with the two-sided Kendall's rank correlation test (significance !evel 0.05). Maximal urinary concentration of tile individual groups, each subdivided in two age groups~the SS(+F) patients were divided in those younger than Io yrs and those Io yrs and older; the other groups were divided in Clin. Chim. dcta, 27 (x97 o) 5 o l - 5 t t
SICKLE CELL DISEASE S T U D I E S
503
subjects younger than 30 yrs and those 30 yrs and older~were analysed statistically with the two-sided Wiicoxon's two sample test (significance level o.o5).* RESULTS
.....
Maximtml different Tables
I-V.
in 54 control TABLE
urinary
groups
and
creatinine
subjects
These findings are compared subjects
with hemoglobin
with
clearance abnormal
and
other
data
hemoglobins
with the maxima
of urinary
of
Sex
Age
Hemoglobin g/o
M.I. R.M. M.A. W.J. I.P. F.A,
f f f m f m
M.H. M.M. M.M. M.C, M.V. M.V.
(Hb
Relic. ~!o
o
concentration
A A I L as s h o w n i n Fig. I.
S S AND S S - ~ - F I
Fetal hemogiobin o: o
Serum creatinine mg/l
Creatinine clearante ml/min
Serum osmolality ~fi()sm!kg H 2 0 before during hydropenia
. . . . . . . . . . . ~75 .......... 13o zg,~ ....... tzo 288 -115 z86 20o -z94 .....
_
Maximum concentrattng capacity,
mOsm/kg H~f)
z z 4 4 4 5
7.6 9.1 8.6 8.z 8.o 6.9
'3.3 28.0 28.7 12.1 8.7 33.5
I z.o 8.6 24.o 6.6 8.6 26.o
z.5 2. 5 3.5 4.0 3.5 z.5
f f f f f f
5 5 7 to iI lz
,%6 8.8 9.7 9.5 8.6 8.Z
-.-...... 5.9 ~
16.o 9.3 14.6 (~.3 5 .8 Io.~
. . . . . . . . . . 4.o 14 ° 5.o It5 . . . . . . . . . 4"O I ZO 4.o t7o
. . . . . . z,~8 207 287 z99 . . . . . ......... ,o~'.' ....
612 55o .17o 47.] 4 J7 .t28
C.D. M.K. J.C. H.M. R.M. C.M.
m f m m m f
1,1 15 15 15 16 16
7-4 7.5 o.6 8.8 6.9 7.8
~ ~ -'9 o 14.8 J3.o -~
3.7 o ~.8 o
5.0 5 .o t,. 5 6.0
290 287 zo3 3°9
442 300 45o 456
O
4"5
125
284
290
4()I
o
4 .o
105
293
311
43 °
L.G, P.S. E.I. I.H. S.G. S.A.
f m f f f m
17
9.1
I1.4
17.o
4.5
17o
. . . . .
463
18
9.2
16.8
9.O
6.0
130
. . . . . .
452
I8 I9 2o 22
8.8 9.J 7.5 I I.Z
xo.o 12.o
I3.O 6.9
4.0 4.0
22o 145
.......... --
IO.I
12.6
4"5
140
17-3
o
4.5
225
300
303
436
A.R. A.E. E.P. iA.l. IA.P. M L.Ep... ..
f f m m f f ¢.
23 z5 25 27 36 38 5°
7.8 6.2 8.6
27.8 I8.3 21.7
o o o
4.5 4.0 11. 5
195 I95 95
8.6
I 1.6
10. 4
5.0
155
8.6 7 .8 7.8
8.2 3.5 15. 3
5.6 zo.o o
't.° 5.5 5,5
200 i6o 13o
287 3.~o ~ -. . . . . . 284 294 289 297 . . . . 288 299
406 422 4z3 452 435 408 424
4.5
I55
29I
472
Mean
the
are listed in
I
P A T I E N T S W I T H S I C K L E CELL ANEMIA
Patient
osmolality,
of patients
8. 3
...............................
I3O lz5 ~4o 175
...... --zg~ 32o
--
. . . . . . .
3o1
617 537 540 558 46o t~ 7
458 444 ,tO-*,--
* T h e s t a t i s t i c a l a n a l y s e s h a v e b e e n c a r r i e d o u t b y t h e f o u n d a t i o n M a t h e m a t i s c h C e n t r u m (Mathe m a t i c a l Centre), 2e B o e r h a v e s t r a a t 49, A m s t e r d a m , O., T h e N e t h e r l a n d s ( H e a d of t h e Statistical D e p a r t m e n t : Prof. Dr. J, H e m e l r i j k ) . W e g r a t e f u l l y a c k n o w l e d g e t h e v a l u a b l e aid a n d assistance of Dr. J. O o s t e r h o f f a n d Drs. E. A. v.d. V e l d e of t h i s I n s t i t u t e in t h e c o u r s e of t h i s i n v e s t i g a t i o n a n d in t h e p r e p a r a t i o n of t h i s m a n u s c r i p t .
Clin. Chim. Acta, 27 (x97 o) 5Ol-511
504
STATIUS VAN EPS e/~ a l .
Normal subiects (AA) The group of 54 normal subjects had a mean renal concentrating capacity of lO58 mOsm/kg H~O (S.D. ~ 128). In 37 of these subjects the mean 24-h creatinine clearance was I3o ml/min (S.D. _+29). Neither maximum urinary osmolality nor creatinine clearance showed any significant decline with age (p = 0.55 and o.17 resp.).
Homozygote sickle cell anemia (SS(+F)) RCC in SS(+ F) patients was found to be significantly depressed when compared to normal subjects both in patients younger and older than IO yrs (p < o.oooi). Except for the patients under IO yrs of age, subjects with Hb SS(+F) have RCC's which fall into a very narrow range with a mean of 434 mOsm (S.D. 21) and a range of 390 to 463 mOsm. Patients younger than IO yrs attain higher urinary osmolalities than older patients (range 460-687 mOsm). For this reason the group as a whole exhibits a significant decrease with age (p = o.oooi). However, when SS(+ F) subjects TABLE II SUBJECTS WITH SICKLE CELL TRAIT ( H b AS)
Patient
Sex
Age
Hemoglobin g%
Serum creatinine mg/!
Cveat. ch'arance mllmin
M.M. N.I. E.R. V.A. E,U. C.F.
m m f f m
3 7 8 15 18 I9
11.5 13.7 i2.o I t.9 13. 9 17.8
4.0 ~ -6.0 6.0 8. 5
85 ---95 13o 13o
B.M. E.M. S.C. E.H. s.J. J.L.
f m m f m f
20 21 24 25 25 27
14.2 15.o 17. 3 11.2 15.o 14.1
6.o 8.o 9.0 6.o 9.5 _.
16o 15o I45 16o 135 125
E.A. R.F. M.K. S.P. S.A. J.G.
f m f f f m
3I 35 45 46 47 47
15.2 14.4 13.o 14.1 15,1 15.7
9.0 9.0 6.5 9.0 9.0 11.5
i2o Io 5 15 ° Io5 95 85
P.A. M.B, M.P, L.N. A.H. P.F.
f f m f f m
51 51 54 54 56 57
12.1 14.2 i6.2 14.8 13. 5 15,o
7,5 6,o 13,o 7.5 7.o II,O
lO5 lO 5 55 It5 I3O 80
M.T. P.N. P.T. J.M, I.C.
m m m m f
58 59 64 70 77
19.2 14.2 15.4 13.2 14.2
12. 5 13.o 9.0 17.5 8.0
95 80 I1O 35 60
14.4
9.o
I1o
Mean
f
Clin. Chim. Aaa, 27 (197o) 5o1-5IX
Serum osmolality mOsmlkg HlO before during hydropenia _ --
Maximum concentrating capacity mOsm /kg HmO
282 303 29I
289 303 294
786 771 895 840 874 827
295
294
900
292 298
298 306 _ 304 __
737 698 768 690 845
__ 302 300 --290
57 ° 703 652 798 618 535
305 304 299 -__ 302
709 517 596 675 542 598
298
__ 306 305 w 297
481 523 509 424 431
294
300
673
--
2q8
297 290 296 285 292 292 290 297 294 297 295
SICKLE CELL DISEASE STUDIES
505
IO yrs and older only are considered, no decrease with advancing age can any longer be observed (p = 0.35). We have called this fixation of concentrating power around the value of 434 mOsm a fixed maximum. The mean creatinine clearance of 16o ml/min (S.D. ~ 3 5 ) of the whole group was significantly higher than for normal subjects. The group as a whole exhibits a depression ix'. creatinine clearance with advancing age (p = o.o44), but not the subgroup IO yrs and older (p -- 0.25).
Sickle cell trait (AS) In AS subjects RCC was significantly depressed when compared with normals, both for subjects younger and older than 30 yrs. Subjects with AS display a different pattern when compared with the normal and the S S ( + F) group: there is a significant progressive decline with ege (p --= e.ooo~). The younger patients, under 30 yrs of age, have an ahnost normal concentrating capacity, whereas the oldest AS patients attain the fixed maximum of S S ( + F ) group. The AS subjects display a significant depression of the creatinine clearance with age (p = 0.0025). Maximum osmolality and creatinine clearance are influenced in the same direction with increasing age, but, with the ~.ge factor constant, there is no correlation between maximal osmolality and creatinine clearance. Hemoglobin C disease (CC) and hemoglobin C trait (AC) Subjects with CC and AC have a normal RCC for all age groups: no decrease with age was observed (p = 0.96 and o.47), notwithstanding the fact that for AC subjects a significant decline with age for GFR was observed (p = 0.0058). CC subjects did not exhibit a similar decline in GFR (p = 0.59). T A B L E ill SUBJECTS WITit tlEMOGLOBIN C TRAIT (Hb AC)
Patient
Sex
Age
l~emoglobin Ot g/o
Serum creatinine mg/l
Creat. clearance ml/min
Serum osmolality mOsm/kg HtO before during hydropenia
Maximum concentrating capacity
mOsmikg H~O
R.J. A.C. W.J. E.B. A.D. J.N.
m m f m f f
14 17 I8 20 24 24
14.7 15.4 12.5 12.o I4.X 15.o
8.5 Io.5 5.5 1o.o 7.5 6.0
155 13o 175 Io5 1°5 14o
287 299 280 288 298 29I
288 3 °0 286 289 296 ---
II63 1138 1228 lO73 916 992
S.O. B.H. V.S. H.S. C.H. P.S.
f f f f f m
25 27 28 42 46 5°
13.o II.6 11.1 17.o 13. 5 17.5
7.5 6.0 5.5 io.o 8.o t2.5
11o 14 ° 90 50 55 95
288 293 29t 294 293 295
294 287 ~ 29o ~ 299
895 1o52 860 1o64 922 111o
~TIS. iC,O. C.F. L.A.
m f f f
53 54 55 63
12.5 13.7 14.o 13.o
9.5 9.0 lO.5 11.o
125 90 75 65
303 300 297 302
315 304 297 307
ioi~ II4! 959 1oo3
13.8
8.5
lO5
294
296
1°33
Mean
Clin. Chim. Acta, 27 (197 o) 5 o t - 5 1 t
STATIUS VAI~EPS et al.
506 TABLE
IV
SUBJECTS WITH HOMOZYGOTE HEMOGLOBIN C DISEASE
(Hb CC)
Patient
Sex
Age
Hemoglobin g%
Serum creatinine mg /l
Creat. clearante rot/rain
Serum osmolality mOsm/kg H20 before during hydropenia
Maximum concentrating capacity mOsm lkg HsO
D.V. E.V.
m f
13.o 12.2 . i1.o 9.7
7.5 9.0
lO5 ito . z3 o 14 °
296 297
299 294
295 287
298 298
1137 Ii88 942 I179 Io41
I3.2 12.1 11.o tt.o
7.5 9.0 7,0
7.0
1o5 95 lO 5 Io5
294 294 29I 296
297 294 293 306
II73
11,7
7.4
11o
294
297
IlO8
T.J.
f
N.M. V.W.
f f
15 22 26 27 30
f f f f
43 43 45 51
J.C. A.S.
~ :
M.A. E.A. Mean
.
. 6.5 5.5
.
I137 lO3O
1148
Sickle cell hemoglobin C disease (SC) Patients with SC disease have an RCC with a mean of 537 mOsm (range: 403 to 698 ), which is definitely impaired in relation to normal subjects for both age groups (p < o.oooi). When compared with the AS group RCC of SC patients younger than 30 yrs is distinctly lower than in AS subjects of the same age (p < o.ooox). For subjects older than 30 yrs the difference between the two groups is less pronounced (p -- 0.039). The results in the SC group (Fig. I) suggest a progressively declining RCC with advancing age, comparable to the results in AS subjects. This decline could not be established statistically. This result, however, is not contradictory to the TABLE
V
PATIENTS WITH SICKLE CELL HEMOGLOBIN C DISEASE ( H b SC)
Patients
Sex
Age
R.S. R.F. S.T. I,C. S.F. I.G. M.J.
m f m f f f m
6 8 t5 19 I9 x9 ao
A.W. F.J. F.M. P.H.
f m m f
34 36 38 4°
M.C.
m
52
L.B. C.D.
f f
55 65
Mean
Hemoglobin g%
Serum creatinine mg /l
7.7
. . . . . 283 297 --289 291 292 293
640 698 52I 493 492 596 057
4.5 6.5 7,5 6.5 7.5
lO.7 I5.I 12.5 11.o 12.o 9.1 11,8
9.5 8.0 8.5 8,5 8.0 5.5 1o.o
85 i45 I2o 1o5 125 I2O 70
-294 303 288 287 295 308
-303 302 298 299 309 315
49o 517 526 547 522 41o 403
11.8
7.5
Ix5
293
3ot
537
.
.
.
Maximum concentrating capacity mOsm lkg ~H~O..
lO.5 13.3 12.5 I4.8
Clin. Chim, Acta. 27 (197 o) 5Ol-511
.
Serum osmolality mOsmlkg HsO before during hydropenia
. . I25 14o 12o 55 19o
.
.
Creat. clearance ml /min . .
SICKLE CELL DISEASE STUDIES
507
1400
.-~
1200, ,
"t" looo,
•
a'=o
C •
o•
•
•
:-:~ ..
o
.. oo •
800,
o*
m'o
qlo
•
'qb
O E
%%
•
600, i
°lu, I~oIb
4oo,
AS
AA
I-m
W
~
~
W
V
V
W
~
~
SS÷F. I
W
W
W
V
w
w
g
..ii 140¢
w
u
I
g
II I
~
1200,
10oo, O
': ..~. Se
i
o
O°
O e
e
.,11 8 0 0 ,
eoo,
x ~t
°O /
•
400,
X 200 y rso
SC
AC 20 4O 6o
0
CC
20 40 e •
0
20 40
60 8o
AOE Fig. x. Relationship between renal concentrating capacity and age in normal subjects (AA), sickle cell trait (AS), sickle cell anemia (SS( ~ F)), hemoglobin C trait (AC), sickle cell hemoglobin C disease (SC) and hemoglobin C disease (CC).
existence of a decline; tile small tail probability (p = 0.054 ) still permits tile possibility of a relationship. As in AS subjects, the oldest SC patients have concentrating capacities, which fall into the range of the fixed maximum of the SS(+ F) group. The results of creatinine clearance estimations in SC patients did not establish a correlation with advancing age (p -- o.48).
Serum osmolality in subiects with abnormal hemoglobins Sermn osmolality was estimated before and alter x9 h thirsting, in x9 control subjects with AA the results were a mean of 994 mOsm (range: z82-3o5) and e99 mOsm (range 286--308) respectively". The increase in serum osmolality by dehydration in different groups with abnormal hemoglobin was compared with the results of AA subjects (Wilcoxon's two-sample test). None of the groups investigated exhibited abnormal serum osmolality values before thirsting. For the SS(+F) group as a whole and for the AS subjects 30 yrs and older the increase in serum osmolality during dehydration was systematically larger than in normal subjects (p = o.o19 and p = o.049 respectively). The number of SC patients was too small for division into two groups for statistical analysis.
Reproducibility Four SS(+F) patients were subjected to the renal concentration test for a second time, yielding the following results: 425 (411), 438 (445), 64r (648) and Clin. Chim. Acta, 2 7 (t97 o) 5 o t - s t x
STATIUS VAN EPS ¢t al.
508 TABLE VI
MAXIMAL R E N A L C O N C E N T R A T I N G C A P A C I T Y O F S E P A R A T E K I D N E Y S IN PATII~NTS W I T H H E M O G L O B I N A S
P. aticnt
Sex
M.L. A.d.M. A.S.
Age
f f f
I~ 64 25
Hemoglobin g%
Maximal osmolality mOsm/kg H20
Right
Lelt
S•dium mequiv /l R L
Potassium mequiv /1 R L
Cteatinine mg /l R L
14.8 I3.3 I2.9
792
806 560 5oz
I6O x48 21o
66 45 26
2700 I6IO 415
560
457
16o I62 2IO
64 50 30
426 (417) mOsm, the second estimation in parentheses. These results demonstrate a good reproducibility of the test and are comparable with similar results found in normal subjects tI. In ? patients with sickle cell disease the renal concentration test was repeated with vasopressin being given parenterally. The results do not show any significant difference in RCC ¢¢ith or without vasopressin.
SICKLE-CELL TRAIT 10 11 ' I
' tl ' ;al 'tg'
Vasopressin
;
'g:
'IDI
R
'XlII:'
A$=Q,25y 12 hours
,J-
'
2mU/rain I
500-
400.
oo..ii "J It 1OO-
I
mOsm
kg H20 O • "'" Osmolality right Kidney =,,---.e----.e Osmolality left Kidney -T
-~ r
=
v
Osmolality Serum
Fig. 2. Evaluation of concentrating powers of individual kidneys in a patient with sickle cell trait.
Concentrating function of individual kidneys The results of urine collection from individual kidneys in 3 AS patients are given in Table VI. In Fig. 2, the results of the osmolality estimations are graphically reproduced. The data concerning this patient in Table VI are those from the third urine portion during vasopressin infusion. From these observations it can be concluded that the concentrating defect of sickle cell disease---at least of sickle cell t r a i t - affects both kidneys in a comparable degree. Clin. Chim. Acta, 27 (x97o) 5oi-5i I
2640 i72o 420
SICKLE CELL DISEASE STUDIES
509
DISCUSSION
Sickle cell nephropathy is characterized by defective renal concentration 2-7 and acidification~3,~L notwithstanding a normal glomerular filtration rate. Previous investigations3,6: have made apparent that aging is a contributing factor in the renal disorder. In young SS children the hyposthenuria appeared to be reversible and RCC could be normalized by transfusions of normal bloodS: °. After the age of io, however, this capacity for improvement decrea~s rapidly to become negligible in subjects older than 15 yrs. The results of the present investigation, undertaken to clarify the relationship between RCC and age, indicate that the group of homozygote SS patients display a division in subjects younger than to yrs and those IO yrs and older. Under xo yrs of age renal concentrating capacity is variable, but in patients older than Io yrs concentrating capacity is restricted to a very narrow range of 434 mOsm/kg H~O (S.D. +21), which has been called a fixed maximum by us. "Fixed", because this osmotic ceiling is scarcely affected by blood transf,,sions, as mentioned before, nor improved by reduction in GFRL breathing pure oxygen:, or increasing urea excretion ts, as occurs in normal subjects; nor can the osmotic ceiling be raised with ADH or hydrochlorothiazide (personal observation), as occurs in patients with diabetes insipidus. In the sickle cell trait group young subjects concentrate almost normally, but at about the age of 7o the fixed maximum of the SS group is reached. The SC group displays a comparable pattern as the AS group. However, maximal concentrating capacity is more depre~ed in the former, for comparable age groups, and the fixed maximum is reached at an earlier age. Possibly an explanation can be found in the SC patients having a higher percentage hemoglobin S than AS subjects. The increase in blood viscosity, caused by C hemoglobin t6 may also be a contributing factor. Hemoglobin C itself, however, most probably does not influence renal concentrating capacity, as our subjects with Hb CC and AC could all concentrate normally. As the abnormality is not dependent of GFR nor ADH, its Fathogenesis sho~ald be looked for elsewhere. The human kidney possesses two different groups of nephrons. 86% consist of cortical nephrons with short loops of Henle restricted to the outer medullary zone. They are most probably responsible for the so-called minimum medullary osmolality of about 4oo mOsm, as suggested by Wijdeveld tT. The remaining 14% are juxta-medullary nephrons with long loops of Henle extending into the inner medullary zone and renal papilla. They are responsible~with the accompanying vasa recta~for further increase in osmolality during thirsting up to about I2OG mOsm. Observations in different animal species support this concept. Animals with the relatively longest Mops of Henle, e.g. desert rats, can produce urine of the highest osmolality--about 3000 m O s m ~ h e r e a s an animal like the beaver, which possesses only short looped nephrons and lacks an inner medullary zone, cannot concentrate higher than 400 to 5o0 mOsm ~s. When, as a result of structural or functional alterations in the kidney, the function of the long loops of Henle and the vasa recta has been disturbed, concentrating capacity can be expected to be xeduced to about 400 mOsm corresponding .. Clin. Chim. Acta, 27 (I97 o) 5oI-SIx
~I0
STATIUS VAN EPS e[ a[.
to the fixed m a x i m u m observed by us in the sickle cell anemia group. As the long loops of Henle belong to only 14% of the total nephron population, complete loss of function of these glomeruli will not impressively depress GFR. There are reasons to assume that structural and functional changes do occur in the inner medulla of the sickle cell kidney. Perillie and Epstein 8 have shown in vitro that the sickling phenomenon was enhanced by concentrated solutions of saline, notwithstanding optimal hemoglobin oxygenation and a normal pH. An increase in sickling was first observed at an osmolality of 600 mOsm. These high osmolalities do exist in vivo in the renal medulla. Sickle cell erythrocytes will undergo sickling when passing through the vasa recta of a hyperosmotic medulla during hydropenia. As the viscosity of SS blood increases progressively from 3o0 to IZOO mOsmL this will also contribute to a decrease in circulation in these vessels, which are possibly already constricted because of the hydropenia ~°. This will create hypoxemia with concomitant acidosis, both factors promoting further sickling and further increase in blood viscosity ~°, creating a vicious circle. These changes will interfere with counter-current multiplication, regardless whether the primary effect is accomplished by ultra-filtration of water as a result of hydrostatic pressure in the vasa recta st or by an active sodium pump mechanism in the ascending limb of Henle's loopS~,~a. In the former event the disturbance will be caused by a depression in hydrostatic, pressure, in the latter by an impaired energy supply to an active sodium pump mechanism. It is obvious that these functional abnormalities will gradually develop into structural lesions, as both reduced blood flow and sickling will promote intravascular thrombosis. Formation of micro-thrombi in the vasa recta will cause partial or complete obstruction of these vessels, leading to infarction and scarfing of the medulla and/or to the formation of collaterals. These collaterals will lack the highly specialized spatial configuration of the vasa recta, parallel to the venous vasa recta and long loops of Henle, necessary for counter-current multiplication. Pathological lesions have indeed been found in the renal medulla of sickle cell anemia patients s4-s6, as well as dilated capillaries ~-~. In recent observations with micro-rSntgen-angiography in SS kidneys*~, s8 impressive changes in vasa recta course and structure were observed, ranging from spiralling and dilatation of the vessels with the loss of the normal architecture to a complete obliteration and absence of vasa recta in the renal medulla. Alterations of the vasa recta in the inner medulla, therefore, are most probably the cause of the hyposthenuria in SS patients, whose kidneys become functionally comparable to the beaver kidney. This conception--namely, that the sickle cell kidney is characterized by impairment or loss of inner medullary functions~is comparable with the abnormalities observed in sickle cell nephropathy. This concerns both concentrating function and acidification function and offers an explanation for the observations made by Hatch et al. ~9 during osmotic diuresis. ACKNOWLEDGEMENTS
The authors are greatly indebted to Drs. C. A. Winkel, A. E. Hart and R. A. Martinez of the Department of Pediatrics and Dr. B. Vinke of the Department of Clin. CAirn. Acta, 27 (i97o) 5oi-5I I
SICKLE CELL DISEASE STUDIES
5II
Mcdicine for t h e i r c o o p e r a t i o n in this i n v e s t i g a t i o n a n d to Dr. E. D. A. S i n d r a m , Drs. M. A. Robles a n d Drs. E~ H. Denswil for t h e d e t e r m i n a t i o n of t h e a b n o r m a l hemoglobins. T h e y also g r a t e f u l l y a c k n o w l e d g e r e s i d e n t s a n d nursing staff of t h e St. E l i s a b e t h H o s p i t a l for t h e i r u n f a i l i n g assistance, E r w i n L u c i a for p r e p a r i n g t h e g r a p h s a n d t h e l a b o r a t o r y technicians for t h e i r v a l u a b l e t e c h n i c a l aid. In t h e course of t h e s e studies a n d in t h e p r e p a r a t i o n of t h e m a n u s c r i p t s t i m u l a t i n g h e l p a n d e n c o u r a g e m e n t was e n c o u n t e r e d in discussions w i t h Professor F r a n k l i n H . E p s t e i n , of Y a l e U n i v e r s i t y , Dr. P. G. D. B. W i j d e v e l d , U n i v e r s i t y of N i j m e g e n , Dr. E. J. D o r h o u t Mees, U n i v e r s i t y of U t r e c h t a n d Dr. E. Zeppenfeldt, Heerlen, The Netherlands. REFERENCES I J. B. HERRICK, Arch. lnter,~a! ,lled., 6 (I9IO) 517. Z C. J. D. ZARAFONETIS, W. A. ST~IGER, L. MOLTHA.~, J. MCMASTER AND V. F. COLVILLE, J. Lab. Clin. Med., 44 (I954) 959. 3 H. G. KEITEL, D. THOMPSON AND H. A. ITANO, J. Clin. Invest., 35 (I96O) 998. 4 Cx. F. WHITTEN AND A. A. YOUNES, J. Lab. Clin. Med., 55 (I96O) 4oo. 5 M. F. LEVITT, A. D. HAUSER, M. S. LEVY AND D. POLIMEROS, Am. J. Med., 29 (I90O) 61 t. 0 L. SCHLITT AND H. G. KEITEL,
Pediatrics, 26 (196o) 249.
7 Y. F. FRANCIS AND H. G. WORTHEN, J. Nat. Med. Assoc., 6o (I968) 266. 8 P. E. PERILLtE AND F. H. EPSTEIN, J. Clin. Invest., 42 (1963) 57° 9 L. PAULING0 H. A. ITANO, S. J. SINGER AND I. C. WELLS, Science, t to (I949) 543. IO L. W. STATIUS VAN EPS, H. SCHOUTEN, L. W. LA PORTE-WIJSMAN AND A. M. ~TRUYKER
BOUDIER, Clin. Chim. Acta, t 7 (1967) 449. I I L. W . STATIUS VAN EPS, C. CH. TER HAAR-WACHTER, ft. SC~OUr~N ^ND A. M. STRUVKER
BOUDIER, Clin. Chim. Acta, i 4 (1966) 637. 12 L. W. STATIUS VAN EPS, C. CH. TER HAAR ROMENY-WACHTER, L. W. LA PORTE-WIJSMAN, H. SCHOUTE~? ,,.ND A. M. STRUVKER BOUDIER, Abstracts I11. Intern. Congr. Nephrology,
September 25-3o, ~966, Washington I~.C., U.S.A. I3 J. P. A. Q. GoossEss, L. W. STATIU~,VAN Eps, H. SCHOUTEN AND A. L. GtTERSON, Annual Meeting American Society of Nephrology, Los Angeles, California, 2967 (Abstracts). 14 H. Ho PING KONG AND G. A. O. ALLEYNE, Lancet, ii (1968) 954. 15 CH. F. WHITTEN, Am. J. Diseases Children, 115 (I968) 262. 16 S. CHARACHE, C. L. CONLEY, D. F. WAUGH, R. J. UGORH'Z AND J. R. SPURRELL, J. Clin. Invest., 46 (1967) I795. 17 P. G. A. B. WlJDEVELD, Ned. Tijdschr. v. Geneesk., Io8 (I964) to79 and xt33. I8 B. SCHMIDT-NIELSEN AND R. O'DELL,
Am. J. Physiol., 200 (I961) I I I 9 .
19 J. FOURMAN AND G. C. KENNEDY, J. Endocrinol., 35 (1966) t73. 20 J. W. HARRIS, Progr. Hematol., 2 (I959) 47. 2x A. F. LEVER, The Vasa Recta and Countercurrent Multiplication, Acta Med. Scand., I78 (t965) Suppl. 434. 22 H. WIRZ, in: A. A. G. LEWIS AND G. E. W. WOLSTENHOLME(Eds.),Ciba Foundation Symposium on the Kidney, Churchill, London, I954, P. 38. 23 C. W. GOTTSCHALKAND M. MYLLE, Am. J. Physiol., 196 (T959) 927. 24 F. K. MOSTOFi, C. F. VORDER BRUEGGE AND L. W. DIGGS, Arch. Pathol., 63 (I957) 336. 25 J. BERNSTEIN AND CH, F. WHITTEN, Arch. Pathol., 7° (196o) 407 .
26 L. W. DIGGS, Am. J. Clin. Pathol., 44 (I965) x. 2 7 L. W. STATIUS VAN EPS, Renal concentration defects in sickle cell disease and other hemoglobinopathies in Curacao, Netherlands Antilles. Paper read at the session on Geographic Pathology of Renal Diseases, I V. Inter. Congr. Nephrology, Stockholm, June 26, 2969. 28 C. PINEDO-VEELS, J. DE KONING, G. DS VRIES AND L. W. STATIUS VAN EPs, Micro-R6ntgenangiography of sickle cell kidneys. Abstract, 3rd Annual Meeting, American Society of Nephrology, Washington, D.C., December x-z, I969. 29 F. E. HATCH, J. W. GULBERTSON AND L. W. DXGGS,J. Clin. Invest., 46 (I967) 336.
Clin. Ch~m. c,a. 27 (x97o) 5o~-5z t
5or
T H E RELATION B E T W E E N AGE AND RENAL CONCENTRATING CAPACITY IN SICKLE CELL DISEASE AND HEMOGLOBIN C DISEASE*
L. W. STATIUS VAN EPS, H. SCHOUTEN, C. CH. T E R H A A R ROMENY-XVACHTER ANn L. W. LA P O R T E - W I J S M A N with technical assistance of A. M. S M O R E N B U R G - S T R U Y K E R B O U I ) I E R
St. Elisabeth Hospital, Department of Inte#'~ml Medicine and Clinical Chemistry, lI'ilh, mstad, Curafao (Netherlands Antilles) (Revised manuscript re~:eived November t 7, x969)
SUMMARY
I. Renal Concentrating Capacity (RCC) and Glomemlar Filtration Rate (GFR) were investigated in groups of subjects of different ages suffering from sickle cell anemia (SS(+F)), sickle cell trait (AS), sickle cell hemoglobin C disease (SC), hemoglobin C disease (CC) and hemoglobin C trait (AC). The RCC of SS(+F) patients aged IO years and older falls into a very narrow range (mean 434 mOsm: S.D. ~21), in those younger than IO years tile RCC is much more variable ranging from 460-687 mOsm. AS subjects, on tile other hand, show a significant progressive decrease in RCC with age. The youngest patients have an ahnost normal concentrating capacity and the oldest subjects are unable to concentrate better than to the fixed maximum of the anemia group. SC patients also demonstrate a declining RCC, but on a lower level than in the AS subjects, for all age groups; the RCC of the elder ones show the same fixed maximum as in SS(+F). CC and AC subjects have a normal RCC. In none of the groups investigated was a correlation found between RCC and GFR. 2. In three AS patients urine was collected simultaneously from the right and left kidney during hydropenia or vasopressin infusion. Tile results indicate that the concentrating defect affects both kidneys in the same degree.
INTRODUCTION
in the first case history of sickle cell disease a slightiy increased diuresis and urine of low specific gravity was already observed 1. This concentrating defect has * This work was supported b y a research grant of the Netherlands Foundation for the advancement of Tropical Research (WOTRO).
C!in. Chim. Acta, 2 7 (I97 o) 5Ol-5XI
502
STATIUS VAN EPS el al.
been confirmed by others ~-s. An inborn abnormality of the hemoglobin molecule is responsible for the sickling phenomenon 9, but the hereditary nature of the renal concentrating defect has been a matter of dispute. The sickle cell trait is also attended with defective renal concentration to a minor and varying degree. The hyposthenuria becomes increasingly more severe with advancing age in both groups of AS and SS(+F) patients, although with a quantitative difference; with increasing age, the subjects with sickle cell trait were found to have normal and impaired maximum of urinary concentration ~,7. The concentrating defect was restored to normal in infants with SS(+F) alter multiple transfusions with hemoglobin A blood ~,~9. This improvement is progressively lost with age and was not related to renal hemodynamics TM. Observations regarding renal concentrating ability in hemoglobin C disease, hemoglobin C trait and sickle cell hemoglobin C disease are almost completely lacking. In the present study, a strictly standardized renal concentration test~, ~ has been employed to investigate concentrating capacity in patients with hemoglobin SS(+F), AS, SC, CC and AC of different ages to clarify the pathogenesis of the concentrating defect. Furthermore, in three patients with sickle cell trait the maximal RCC of the individual kidneys were examined separately. MATERIALS AND METHODS
3t patients with SS(+ F), 29 subjects with AS, t6 subjects with AC, 9 patients with CC and I4 with SC were investigated with the renal concentration test and the results compared with those of 54 subjects with hemoglobin AA. In most cases the concentration test was combined with a z4-h endogenous creatinine clearance test. Apart from actual diseases of the kidney care was taken to avoid: anemias other than consequent to the hemoglobinopathy, fever, hypertension, abnormal basal metabolism, disturbances of calcium metabolism and cirrhosis of the liver. In 4 SS(+F) patients the concentration test was repeated a second time to evaluate the reproducibility of the test. In 7 patients the test was repeated with the subject receiving a subcutaneous injection of xo Units vasopressin half an hour before the first urine collection. In three patients with AS, being subjected to cystoscopy and ureteral catheterization, because of idiopathic hematuria (ultimately diagnosed as a complication of their sickle cell disease) maximal urinary osmolality of the right and left kidney were examined separately. In 2 patients catheterization was performed during maximal hydropenia at the end of the concentration test. The third patient was normally hydrated. She received an intravenous infusion of vasopressin (Sandoz), 2 mU/min, urine being collected in divided portions from the right and the left kidney before, during and after the period of infusion. The chemical methods employed h,,ve been described previously TM. Correlation between maximal urinary osmolality and age and between creatinine clearance and age was tested with the two-sided Kendall's rank correlation test (significance !evel 0.05). Maximal urinary concentration of tile individual groups, each subdivided in two age groups~the SS(+F) patients were divided in those younger than Io yrs and those Io yrs and older; the other groups were divided in Clin. Chim. dcta, 27 (x97 o) 5 o l - 5 t t
SICKLE CELL DISEASE S T U D I E S
503
subjects younger than 30 yrs and those 30 yrs and older~were analysed statistically with the two-sided Wiicoxon's two sample test (significance level o.o5).* RESULTS
.....
Maximtml different Tables
I-V.
in 54 control TABLE
urinary
groups
and
creatinine
subjects
These findings are compared subjects
with hemoglobin
with
clearance abnormal
and
other
data
hemoglobins
with the maxima
of urinary
of
Sex
Age
Hemoglobin g/o
M.I. R.M. M.A. W.J. I.P. F.A,
f f f m f m
M.H. M.M. M.M. M.C, M.V. M.V.
(Hb
Relic. ~!o
o
concentration
A A I L as s h o w n i n Fig. I.
S S AND S S - ~ - F I
Fetal hemogiobin o: o
Serum creatinine mg/l
Creatinine clearante ml/min
Serum osmolality ~fi()sm!kg H 2 0 before during hydropenia
. . . . . . . . . . . ~75 .......... 13o zg,~ ....... tzo 288 -115 z86 20o -z94 .....
_
Maximum concentrattng capacity,
mOsm/kg H~f)
z z 4 4 4 5
7.6 9.1 8.6 8.z 8.o 6.9
'3.3 28.0 28.7 12.1 8.7 33.5
I z.o 8.6 24.o 6.6 8.6 26.o
z.5 2. 5 3.5 4.0 3.5 z.5
f f f f f f
5 5 7 to iI lz
,%6 8.8 9.7 9.5 8.6 8.Z
-.-...... 5.9 ~
16.o 9.3 14.6 (~.3 5 .8 Io.~
. . . . . . . . . . 4.o 14 ° 5.o It5 . . . . . . . . . 4"O I ZO 4.o t7o
. . . . . . z,~8 207 287 z99 . . . . . ......... ,o~'.' ....
612 55o .17o 47.] 4 J7 .t28
C.D. M.K. J.C. H.M. R.M. C.M.
m f m m m f
1,1 15 15 15 16 16
7-4 7.5 o.6 8.8 6.9 7.8
~ ~ -'9 o 14.8 J3.o -~
3.7 o ~.8 o
5.0 5 .o t,. 5 6.0
290 287 zo3 3°9
442 300 45o 456
O
4"5
125
284
290
4()I
o
4 .o
105
293
311
43 °
L.G, P.S. E.I. I.H. S.G. S.A.
f m f f f m
17
9.1
I1.4
17.o
4.5
17o
. . . . .
463
18
9.2
16.8
9.O
6.0
130
. . . . . .
452
I8 I9 2o 22
8.8 9.J 7.5 I I.Z
xo.o 12.o
I3.O 6.9
4.0 4.0
22o 145
.......... --
IO.I
12.6
4"5
140
17-3
o
4.5
225
300
303
436
A.R. A.E. E.P. iA.l. IA.P. M L.Ep... ..
f f m m f f ¢.
23 z5 25 27 36 38 5°
7.8 6.2 8.6
27.8 I8.3 21.7
o o o
4.5 4.0 11. 5
195 I95 95
8.6
I 1.6
10. 4
5.0
155
8.6 7 .8 7.8
8.2 3.5 15. 3
5.6 zo.o o
't.° 5.5 5,5
200 i6o 13o
287 3.~o ~ -. . . . . . 284 294 289 297 . . . . 288 299
406 422 4z3 452 435 408 424
4.5
I55
29I
472
Mean
the
are listed in
I
P A T I E N T S W I T H S I C K L E CELL ANEMIA
Patient
osmolality,
of patients
8. 3
...............................
I3O lz5 ~4o 175
...... --zg~ 32o
--
. . . . . . .
3o1
617 537 540 558 46o t~ 7
458 444 ,tO-*,--
* T h e s t a t i s t i c a l a n a l y s e s h a v e b e e n c a r r i e d o u t b y t h e f o u n d a t i o n M a t h e m a t i s c h C e n t r u m (Mathe m a t i c a l Centre), 2e B o e r h a v e s t r a a t 49, A m s t e r d a m , O., T h e N e t h e r l a n d s ( H e a d of t h e Statistical D e p a r t m e n t : Prof. Dr. J, H e m e l r i j k ) . W e g r a t e f u l l y a c k n o w l e d g e t h e v a l u a b l e aid a n d assistance of Dr. J. O o s t e r h o f f a n d Drs. E. A. v.d. V e l d e of t h i s I n s t i t u t e in t h e c o u r s e of t h i s i n v e s t i g a t i o n a n d in t h e p r e p a r a t i o n of t h i s m a n u s c r i p t .
Clin. Chim. Acta, 27 (x97 o) 5Ol-511
504
STATIUS VAN EPS e/~ a l .
Normal subiects (AA) The group of 54 normal subjects had a mean renal concentrating capacity of lO58 mOsm/kg H~O (S.D. ~ 128). In 37 of these subjects the mean 24-h creatinine clearance was I3o ml/min (S.D. _+29). Neither maximum urinary osmolality nor creatinine clearance showed any significant decline with age (p = 0.55 and o.17 resp.).
Homozygote sickle cell anemia (SS(+F)) RCC in SS(+ F) patients was found to be significantly depressed when compared to normal subjects both in patients younger and older than IO yrs (p < o.oooi). Except for the patients under IO yrs of age, subjects with Hb SS(+F) have RCC's which fall into a very narrow range with a mean of 434 mOsm (S.D. 21) and a range of 390 to 463 mOsm. Patients younger than IO yrs attain higher urinary osmolalities than older patients (range 460-687 mOsm). For this reason the group as a whole exhibits a significant decrease with age (p = o.oooi). However, when SS(+ F) subjects TABLE II SUBJECTS WITH SICKLE CELL TRAIT ( H b AS)
Patient
Sex
Age
Hemoglobin g%
Serum creatinine mg/!
Cveat. ch'arance mllmin
M.M. N.I. E.R. V.A. E,U. C.F.
m m f f m
3 7 8 15 18 I9
11.5 13.7 i2.o I t.9 13. 9 17.8
4.0 ~ -6.0 6.0 8. 5
85 ---95 13o 13o
B.M. E.M. S.C. E.H. s.J. J.L.
f m m f m f
20 21 24 25 25 27
14.2 15.o 17. 3 11.2 15.o 14.1
6.o 8.o 9.0 6.o 9.5 _.
16o 15o I45 16o 135 125
E.A. R.F. M.K. S.P. S.A. J.G.
f m f f f m
3I 35 45 46 47 47
15.2 14.4 13.o 14.1 15,1 15.7
9.0 9.0 6.5 9.0 9.0 11.5
i2o Io 5 15 ° Io5 95 85
P.A. M.B, M.P, L.N. A.H. P.F.
f f m f f m
51 51 54 54 56 57
12.1 14.2 i6.2 14.8 13. 5 15,o
7,5 6,o 13,o 7.5 7.o II,O
lO5 lO 5 55 It5 I3O 80
M.T. P.N. P.T. J.M, I.C.
m m m m f
58 59 64 70 77
19.2 14.2 15.4 13.2 14.2
12. 5 13.o 9.0 17.5 8.0
95 80 I1O 35 60
14.4
9.o
I1o
Mean
f
Clin. Chim. Aaa, 27 (197o) 5o1-5IX
Serum osmolality mOsmlkg HlO before during hydropenia _ --
Maximum concentrating capacity mOsm /kg HmO
282 303 29I
289 303 294
786 771 895 840 874 827
295
294
900
292 298
298 306 _ 304 __
737 698 768 690 845
__ 302 300 --290
57 ° 703 652 798 618 535
305 304 299 -__ 302
709 517 596 675 542 598
298
__ 306 305 w 297
481 523 509 424 431
294
300
673
--
2q8
297 290 296 285 292 292 290 297 294 297 295
SICKLE CELL DISEASE STUDIES
505
IO yrs and older only are considered, no decrease with advancing age can any longer be observed (p = 0.35). We have called this fixation of concentrating power around the value of 434 mOsm a fixed maximum. The mean creatinine clearance of 16o ml/min (S.D. ~ 3 5 ) of the whole group was significantly higher than for normal subjects. The group as a whole exhibits a depression ix'. creatinine clearance with advancing age (p = o.o44), but not the subgroup IO yrs and older (p -- 0.25).
Sickle cell trait (AS) In AS subjects RCC was significantly depressed when compared with normals, both for subjects younger and older than 30 yrs. Subjects with AS display a different pattern when compared with the normal and the S S ( + F) group: there is a significant progressive decline with ege (p --= e.ooo~). The younger patients, under 30 yrs of age, have an ahnost normal concentrating capacity, whereas the oldest AS patients attain the fixed maximum of S S ( + F ) group. The AS subjects display a significant depression of the creatinine clearance with age (p = 0.0025). Maximum osmolality and creatinine clearance are influenced in the same direction with increasing age, but, with the ~.ge factor constant, there is no correlation between maximal osmolality and creatinine clearance. Hemoglobin C disease (CC) and hemoglobin C trait (AC) Subjects with CC and AC have a normal RCC for all age groups: no decrease with age was observed (p = 0.96 and o.47), notwithstanding the fact that for AC subjects a significant decline with age for GFR was observed (p = 0.0058). CC subjects did not exhibit a similar decline in GFR (p = 0.59). T A B L E ill SUBJECTS WITit tlEMOGLOBIN C TRAIT (Hb AC)
Patient
Sex
Age
l~emoglobin Ot g/o
Serum creatinine mg/l
Creat. clearance ml/min
Serum osmolality mOsm/kg HtO before during hydropenia
Maximum concentrating capacity
mOsmikg H~O
R.J. A.C. W.J. E.B. A.D. J.N.
m m f m f f
14 17 I8 20 24 24
14.7 15.4 12.5 12.o I4.X 15.o
8.5 Io.5 5.5 1o.o 7.5 6.0
155 13o 175 Io5 1°5 14o
287 299 280 288 298 29I
288 3 °0 286 289 296 ---
II63 1138 1228 lO73 916 992
S.O. B.H. V.S. H.S. C.H. P.S.
f f f f f m
25 27 28 42 46 5°
13.o II.6 11.1 17.o 13. 5 17.5
7.5 6.0 5.5 io.o 8.o t2.5
11o 14 ° 90 50 55 95
288 293 29t 294 293 295
294 287 ~ 29o ~ 299
895 1o52 860 1o64 922 111o
~TIS. iC,O. C.F. L.A.
m f f f
53 54 55 63
12.5 13.7 14.o 13.o
9.5 9.0 lO.5 11.o
125 90 75 65
303 300 297 302
315 304 297 307
ioi~ II4! 959 1oo3
13.8
8.5
lO5
294
296
1°33
Mean
Clin. Chim. Acta, 27 (197 o) 5 o t - 5 1 t
STATIUS VAI~EPS et al.
506 TABLE
IV
SUBJECTS WITH HOMOZYGOTE HEMOGLOBIN C DISEASE
(Hb CC)
Patient
Sex
Age
Hemoglobin g%
Serum creatinine mg /l
Creat. clearante rot/rain
Serum osmolality mOsm/kg H20 before during hydropenia
Maximum concentrating capacity mOsm lkg HsO
D.V. E.V.
m f
13.o 12.2 . i1.o 9.7
7.5 9.0
lO5 ito . z3 o 14 °
296 297
299 294
295 287
298 298
1137 Ii88 942 I179 Io41
I3.2 12.1 11.o tt.o
7.5 9.0 7,0
7.0
1o5 95 lO 5 Io5
294 294 29I 296
297 294 293 306
II73
11,7
7.4
11o
294
297
IlO8
T.J.
f
N.M. V.W.
f f
15 22 26 27 30
f f f f
43 43 45 51
J.C. A.S.
~ :
M.A. E.A. Mean
.
. 6.5 5.5
.
I137 lO3O
1148
Sickle cell hemoglobin C disease (SC) Patients with SC disease have an RCC with a mean of 537 mOsm (range: 403 to 698 ), which is definitely impaired in relation to normal subjects for both age groups (p < o.oooi). When compared with the AS group RCC of SC patients younger than 30 yrs is distinctly lower than in AS subjects of the same age (p < o.ooox). For subjects older than 30 yrs the difference between the two groups is less pronounced (p -- 0.039). The results in the SC group (Fig. I) suggest a progressively declining RCC with advancing age, comparable to the results in AS subjects. This decline could not be established statistically. This result, however, is not contradictory to the TABLE
V
PATIENTS WITH SICKLE CELL HEMOGLOBIN C DISEASE ( H b SC)
Patients
Sex
Age
R.S. R.F. S.T. I,C. S.F. I.G. M.J.
m f m f f f m
6 8 t5 19 I9 x9 ao
A.W. F.J. F.M. P.H.
f m m f
34 36 38 4°
M.C.
m
52
L.B. C.D.
f f
55 65
Mean
Hemoglobin g%
Serum creatinine mg /l
7.7
. . . . . 283 297 --289 291 292 293
640 698 52I 493 492 596 057
4.5 6.5 7,5 6.5 7.5
lO.7 I5.I 12.5 11.o 12.o 9.1 11,8
9.5 8.0 8.5 8,5 8.0 5.5 1o.o
85 i45 I2o 1o5 125 I2O 70
-294 303 288 287 295 308
-303 302 298 299 309 315
49o 517 526 547 522 41o 403
11.8
7.5
Ix5
293
3ot
537
.
.
.
Maximum concentrating capacity mOsm lkg ~H~O..
lO.5 13.3 12.5 I4.8
Clin. Chim, Acta. 27 (197 o) 5Ol-511
.
Serum osmolality mOsmlkg HsO before during hydropenia
. . I25 14o 12o 55 19o
.
.
Creat. clearance ml /min . .
SICKLE CELL DISEASE STUDIES
507
1400
.-~
1200, ,
"t" looo,
•
a'=o
C •
o•
•
•
:-:~ ..
o
.. oo •
800,
o*
m'o
qlo
•
'qb
O E
%%
•
600, i
°lu, I~oIb
4oo,
AS
AA
I-m
W
~
~
W
V
V
W
~
~
SS÷F. I
W
W
W
V
w
w
g
..ii 140¢
w
u
I
g
II I
~
1200,
10oo, O
': ..~. Se
i
o
O°
O e
e
.,11 8 0 0 ,
eoo,
x ~t
°O /
•
400,
X 200 y rso
SC
AC 20 4O 6o
0
CC
20 40 e •
0
20 40
60 8o
AOE Fig. x. Relationship between renal concentrating capacity and age in normal subjects (AA), sickle cell trait (AS), sickle cell anemia (SS( ~ F)), hemoglobin C trait (AC), sickle cell hemoglobin C disease (SC) and hemoglobin C disease (CC).
existence of a decline; tile small tail probability (p = 0.054 ) still permits tile possibility of a relationship. As in AS subjects, the oldest SC patients have concentrating capacities, which fall into the range of the fixed maximum of the SS(+ F) group. The results of creatinine clearance estimations in SC patients did not establish a correlation with advancing age (p -- o.48).
Serum osmolality in subiects with abnormal hemoglobins Sermn osmolality was estimated before and alter x9 h thirsting, in x9 control subjects with AA the results were a mean of 994 mOsm (range: z82-3o5) and e99 mOsm (range 286--308) respectively". The increase in serum osmolality by dehydration in different groups with abnormal hemoglobin was compared with the results of AA subjects (Wilcoxon's two-sample test). None of the groups investigated exhibited abnormal serum osmolality values before thirsting. For the SS(+F) group as a whole and for the AS subjects 30 yrs and older the increase in serum osmolality during dehydration was systematically larger than in normal subjects (p = o.o19 and p = o.049 respectively). The number of SC patients was too small for division into two groups for statistical analysis.
Reproducibility Four SS(+F) patients were subjected to the renal concentration test for a second time, yielding the following results: 425 (411), 438 (445), 64r (648) and Clin. Chim. Acta, 2 7 (t97 o) 5 o t - s t x
STATIUS VAN EPS ¢t al.
508 TABLE VI
MAXIMAL R E N A L C O N C E N T R A T I N G C A P A C I T Y O F S E P A R A T E K I D N E Y S IN PATII~NTS W I T H H E M O G L O B I N A S
P. aticnt
Sex
M.L. A.d.M. A.S.
Age
f f f
I~ 64 25
Hemoglobin g%
Maximal osmolality mOsm/kg H20
Right
Lelt
S•dium mequiv /l R L
Potassium mequiv /1 R L
Cteatinine mg /l R L
14.8 I3.3 I2.9
792
806 560 5oz
I6O x48 21o
66 45 26
2700 I6IO 415
560
457
16o I62 2IO
64 50 30
426 (417) mOsm, the second estimation in parentheses. These results demonstrate a good reproducibility of the test and are comparable with similar results found in normal subjects tI. In ? patients with sickle cell disease the renal concentration test was repeated with vasopressin being given parenterally. The results do not show any significant difference in RCC ¢¢ith or without vasopressin.
SICKLE-CELL TRAIT 10 11 ' I
' tl ' ;al 'tg'
Vasopressin
;
'g:
'IDI
R
'XlII:'
A$=Q,25y 12 hours
,J-
'
2mU/rain I
500-
400.
oo..ii "J It 1OO-
I
mOsm
kg H20 O • "'" Osmolality right Kidney =,,---.e----.e Osmolality left Kidney -T
-~ r
=
v
Osmolality Serum
Fig. 2. Evaluation of concentrating powers of individual kidneys in a patient with sickle cell trait.
Concentrating function of individual kidneys The results of urine collection from individual kidneys in 3 AS patients are given in Table VI. In Fig. 2, the results of the osmolality estimations are graphically reproduced. The data concerning this patient in Table VI are those from the third urine portion during vasopressin infusion. From these observations it can be concluded that the concentrating defect of sickle cell disease---at least of sickle cell t r a i t - affects both kidneys in a comparable degree. Clin. Chim. Acta, 27 (x97o) 5oi-5i I
2640 i72o 420
SICKLE CELL DISEASE STUDIES
509
DISCUSSION
Sickle cell nephropathy is characterized by defective renal concentration 2-7 and acidification~3,~L notwithstanding a normal glomerular filtration rate. Previous investigations3,6: have made apparent that aging is a contributing factor in the renal disorder. In young SS children the hyposthenuria appeared to be reversible and RCC could be normalized by transfusions of normal bloodS: °. After the age of io, however, this capacity for improvement decrea~s rapidly to become negligible in subjects older than 15 yrs. The results of the present investigation, undertaken to clarify the relationship between RCC and age, indicate that the group of homozygote SS patients display a division in subjects younger than to yrs and those IO yrs and older. Under xo yrs of age renal concentrating capacity is variable, but in patients older than Io yrs concentrating capacity is restricted to a very narrow range of 434 mOsm/kg H~O (S.D. +21), which has been called a fixed maximum by us. "Fixed", because this osmotic ceiling is scarcely affected by blood transf,,sions, as mentioned before, nor improved by reduction in GFRL breathing pure oxygen:, or increasing urea excretion ts, as occurs in normal subjects; nor can the osmotic ceiling be raised with ADH or hydrochlorothiazide (personal observation), as occurs in patients with diabetes insipidus. In the sickle cell trait group young subjects concentrate almost normally, but at about the age of 7o the fixed maximum of the SS group is reached. The SC group displays a comparable pattern as the AS group. However, maximal concentrating capacity is more depre~ed in the former, for comparable age groups, and the fixed maximum is reached at an earlier age. Possibly an explanation can be found in the SC patients having a higher percentage hemoglobin S than AS subjects. The increase in blood viscosity, caused by C hemoglobin t6 may also be a contributing factor. Hemoglobin C itself, however, most probably does not influence renal concentrating capacity, as our subjects with Hb CC and AC could all concentrate normally. As the abnormality is not dependent of GFR nor ADH, its Fathogenesis sho~ald be looked for elsewhere. The human kidney possesses two different groups of nephrons. 86% consist of cortical nephrons with short loops of Henle restricted to the outer medullary zone. They are most probably responsible for the so-called minimum medullary osmolality of about 4oo mOsm, as suggested by Wijdeveld tT. The remaining 14% are juxta-medullary nephrons with long loops of Henle extending into the inner medullary zone and renal papilla. They are responsible~with the accompanying vasa recta~for further increase in osmolality during thirsting up to about I2OG mOsm. Observations in different animal species support this concept. Animals with the relatively longest Mops of Henle, e.g. desert rats, can produce urine of the highest osmolality--about 3000 m O s m ~ h e r e a s an animal like the beaver, which possesses only short looped nephrons and lacks an inner medullary zone, cannot concentrate higher than 400 to 5o0 mOsm ~s. When, as a result of structural or functional alterations in the kidney, the function of the long loops of Henle and the vasa recta has been disturbed, concentrating capacity can be expected to be xeduced to about 400 mOsm corresponding .. Clin. Chim. Acta, 27 (I97 o) 5oI-SIx
~I0
STATIUS VAN EPS e[ a[.
to the fixed m a x i m u m observed by us in the sickle cell anemia group. As the long loops of Henle belong to only 14% of the total nephron population, complete loss of function of these glomeruli will not impressively depress GFR. There are reasons to assume that structural and functional changes do occur in the inner medulla of the sickle cell kidney. Perillie and Epstein 8 have shown in vitro that the sickling phenomenon was enhanced by concentrated solutions of saline, notwithstanding optimal hemoglobin oxygenation and a normal pH. An increase in sickling was first observed at an osmolality of 600 mOsm. These high osmolalities do exist in vivo in the renal medulla. Sickle cell erythrocytes will undergo sickling when passing through the vasa recta of a hyperosmotic medulla during hydropenia. As the viscosity of SS blood increases progressively from 3o0 to IZOO mOsmL this will also contribute to a decrease in circulation in these vessels, which are possibly already constricted because of the hydropenia ~°. This will create hypoxemia with concomitant acidosis, both factors promoting further sickling and further increase in blood viscosity ~°, creating a vicious circle. These changes will interfere with counter-current multiplication, regardless whether the primary effect is accomplished by ultra-filtration of water as a result of hydrostatic pressure in the vasa recta st or by an active sodium pump mechanism in the ascending limb of Henle's loopS~,~a. In the former event the disturbance will be caused by a depression in hydrostatic, pressure, in the latter by an impaired energy supply to an active sodium pump mechanism. It is obvious that these functional abnormalities will gradually develop into structural lesions, as both reduced blood flow and sickling will promote intravascular thrombosis. Formation of micro-thrombi in the vasa recta will cause partial or complete obstruction of these vessels, leading to infarction and scarfing of the medulla and/or to the formation of collaterals. These collaterals will lack the highly specialized spatial configuration of the vasa recta, parallel to the venous vasa recta and long loops of Henle, necessary for counter-current multiplication. Pathological lesions have indeed been found in the renal medulla of sickle cell anemia patients s4-s6, as well as dilated capillaries ~-~. In recent observations with micro-rSntgen-angiography in SS kidneys*~, s8 impressive changes in vasa recta course and structure were observed, ranging from spiralling and dilatation of the vessels with the loss of the normal architecture to a complete obliteration and absence of vasa recta in the renal medulla. Alterations of the vasa recta in the inner medulla, therefore, are most probably the cause of the hyposthenuria in SS patients, whose kidneys become functionally comparable to the beaver kidney. This conception--namely, that the sickle cell kidney is characterized by impairment or loss of inner medullary functions~is comparable with the abnormalities observed in sickle cell nephropathy. This concerns both concentrating function and acidification function and offers an explanation for the observations made by Hatch et al. ~9 during osmotic diuresis. ACKNOWLEDGEMENTS
The authors are greatly indebted to Drs. C. A. Winkel, A. E. Hart and R. A. Martinez of the Department of Pediatrics and Dr. B. Vinke of the Department of Clin. CAirn. Acta, 27 (i97o) 5oi-5I I
SICKLE CELL DISEASE STUDIES
5II
Mcdicine for t h e i r c o o p e r a t i o n in this i n v e s t i g a t i o n a n d to Dr. E. D. A. S i n d r a m , Drs. M. A. Robles a n d Drs. E~ H. Denswil for t h e d e t e r m i n a t i o n of t h e a b n o r m a l hemoglobins. T h e y also g r a t e f u l l y a c k n o w l e d g e r e s i d e n t s a n d nursing staff of t h e St. E l i s a b e t h H o s p i t a l for t h e i r u n f a i l i n g assistance, E r w i n L u c i a for p r e p a r i n g t h e g r a p h s a n d t h e l a b o r a t o r y technicians for t h e i r v a l u a b l e t e c h n i c a l aid. In t h e course of t h e s e studies a n d in t h e p r e p a r a t i o n of t h e m a n u s c r i p t s t i m u l a t i n g h e l p a n d e n c o u r a g e m e n t was e n c o u n t e r e d in discussions w i t h Professor F r a n k l i n H . E p s t e i n , of Y a l e U n i v e r s i t y , Dr. P. G. D. B. W i j d e v e l d , U n i v e r s i t y of N i j m e g e n , Dr. E. J. D o r h o u t Mees, U n i v e r s i t y of U t r e c h t a n d Dr. E. Zeppenfeldt, Heerlen, The Netherlands. REFERENCES I J. B. HERRICK, Arch. lnter,~a! ,lled., 6 (I9IO) 517. Z C. J. D. ZARAFONETIS, W. A. ST~IGER, L. MOLTHA.~, J. MCMASTER AND V. F. COLVILLE, J. Lab. Clin. Med., 44 (I954) 959. 3 H. G. KEITEL, D. THOMPSON AND H. A. ITANO, J. Clin. Invest., 35 (I96O) 998. 4 Cx. F. WHITTEN AND A. A. YOUNES, J. Lab. Clin. Med., 55 (I96O) 4oo. 5 M. F. LEVITT, A. D. HAUSER, M. S. LEVY AND D. POLIMEROS, Am. J. Med., 29 (I90O) 61 t. 0 L. SCHLITT AND H. G. KEITEL,
Pediatrics, 26 (196o) 249.
7 Y. F. FRANCIS AND H. G. WORTHEN, J. Nat. Med. Assoc., 6o (I968) 266. 8 P. E. PERILLtE AND F. H. EPSTEIN, J. Clin. Invest., 42 (1963) 57° 9 L. PAULING0 H. A. ITANO, S. J. SINGER AND I. C. WELLS, Science, t to (I949) 543. IO L. W. STATIUS VAN EPS, H. SCHOUTEN, L. W. LA PORTE-WIJSMAN AND A. M. ~TRUYKER
BOUDIER, Clin. Chim. Acta, t 7 (1967) 449. I I L. W . STATIUS VAN EPS, C. CH. TER HAAR-WACHTER, ft. SC~OUr~N ^ND A. M. STRUVKER
BOUDIER, Clin. Chim. Acta, i 4 (1966) 637. 12 L. W. STATIUS VAN EPS, C. CH. TER HAAR ROMENY-WACHTER, L. W. LA PORTE-WIJSMAN, H. SCHOUTE~? ,,.ND A. M. STRUVKER BOUDIER, Abstracts I11. Intern. Congr. Nephrology,
September 25-3o, ~966, Washington I~.C., U.S.A. I3 J. P. A. Q. GoossEss, L. W. STATIU~,VAN Eps, H. SCHOUTEN AND A. L. GtTERSON, Annual Meeting American Society of Nephrology, Los Angeles, California, 2967 (Abstracts). 14 H. Ho PING KONG AND G. A. O. ALLEYNE, Lancet, ii (1968) 954. 15 CH. F. WHITTEN, Am. J. Diseases Children, 115 (I968) 262. 16 S. CHARACHE, C. L. CONLEY, D. F. WAUGH, R. J. UGORH'Z AND J. R. SPURRELL, J. Clin. Invest., 46 (1967) I795. 17 P. G. A. B. WlJDEVELD, Ned. Tijdschr. v. Geneesk., Io8 (I964) to79 and xt33. I8 B. SCHMIDT-NIELSEN AND R. O'DELL,
Am. J. Physiol., 200 (I961) I I I 9 .
19 J. FOURMAN AND G. C. KENNEDY, J. Endocrinol., 35 (1966) t73. 20 J. W. HARRIS, Progr. Hematol., 2 (I959) 47. 2x A. F. LEVER, The Vasa Recta and Countercurrent Multiplication, Acta Med. Scand., I78 (t965) Suppl. 434. 22 H. WIRZ, in: A. A. G. LEWIS AND G. E. W. WOLSTENHOLME(Eds.),Ciba Foundation Symposium on the Kidney, Churchill, London, I954, P. 38. 23 C. W. GOTTSCHALKAND M. MYLLE, Am. J. Physiol., 196 (T959) 927. 24 F. K. MOSTOFi, C. F. VORDER BRUEGGE AND L. W. DIGGS, Arch. Pathol., 63 (I957) 336. 25 J. BERNSTEIN AND CH, F. WHITTEN, Arch. Pathol., 7° (196o) 407 .
26 L. W. DIGGS, Am. J. Clin. Pathol., 44 (I965) x. 2 7 L. W. STATIUS VAN EPS, Renal concentration defects in sickle cell disease and other hemoglobinopathies in Curacao, Netherlands Antilles. Paper read at the session on Geographic Pathology of Renal Diseases, I V. Inter. Congr. Nephrology, Stockholm, June 26, 2969. 28 C. PINEDO-VEELS, J. DE KONING, G. DS VRIES AND L. W. STATIUS VAN EPs, Micro-R6ntgenangiography of sickle cell kidneys. Abstract, 3rd Annual Meeting, American Society of Nephrology, Washington, D.C., December x-z, I969. 29 F. E. HATCH, J. W. GULBERTSON AND L. W. DXGGS,J. Clin. Invest., 46 (I967) 336.
Clin. Ch~m. c,a. 27 (x97o) 5o~-5z t