The pathogenesis of Bartter's syndrome: Functional and histologic studies

The Pathogenesis of Battter’s Syndrome

Functional and Histologic Studies

TOSHIRO FUJITA, M.D.*

HIROSHI SAKAGUCHI, M.D.

Tokyo, Japan

MASAKATSU SHIBAGAKI, M.D

TOSHIO FUKUI, M.D.

MASAYUKI NOMURA, M.D.

SUSUMU SEKIGUCHI, M.D.

Kawasaki. Japan

From the Departments of Internal Medicine and Pathology, School of Medicine, Keio University, Tokyo, Japan; the Municipal Ida Hospital, City of Kawasaki, Kanagawa-ken, Japan. Requests for reprints should be addressed to Dr. Toshiro Fujita, the Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan. Manuscript accepted September 7, 1976.

* Present address: Section on Steroid and Mineral Metabolisr~, Hypertension-Endocrine Branch, National Heart, Lung and Blood institute, National Institutes of Health, Building 10. Bethesda, Maryland 20014.

We describe a patient with Bartter’s syndrome. In addition to the well-known characteristic findings by light microscopy, electron micrograms confirmed the presence of juxtaglomerular cell hyperplasia with polymorphous renin secretory granules and dense multivesicular bodies. Volume expansion by albumin infusion decreased plasma renin activity and aldosterone excretion, and improved the pressor response to exogenous angiotensin, suggesting that the renin-angiotensin-aldosterone system was not autonomous but that a decreased extracellular volume might be a major defect in this patient. During hypotonic saline diuresis, moreover, fractional free water clearance per fractional distal sodium delivery, CH20/CH20 I- CN,, was markedly depressed in the patient when compared with the value in the controls. Evidence presented suggests that chronic extracellular volume depletion exists as a consequence of an impaired sodium transport in the ascending limb of Henle’s loop.

In 1962, Bartter et al. [l] first reported the association of secondary

hyperaldosteronism, hypokalemic alkalosis, juxtaglomerular complex

hyperplasia and insensitivity to the pressor effects of infused angio-

tensin in two patients who had neither hypertension nor edema. Since

that time a number of similar cases have been reported [2-B]. We

describe a patient with Bartter’s syndrome who underwent extensive

studies of renal function and histology to elucidate the underlying

pathogenesis of this syndrome.

CASE REPORT

A 40 year old Japanese man was referred to our hospital for evaluation of hypokalemia. He had felt mildly fatigued for about five years. Ten months before admission he sought medical attention, when he had muscle weak- ness, polydipsia and polyuria. Subsequently, paralysis developed and he saw his physician. During the diagnostic evaluation, hypokalemia (1.9 meq/liter)

and alkalosis were discovered. The patient denied ingestions of laxatives,

diuretics, licorice or any other medication, and he had no nausea, vomiting

or diarrhea. Physical examination on admission revealed a well nourished

man who appeared to be in good health; he was 170 cm tall and weighed 76

kg. Blood pressure, with the patient in the supine position, was 1 IO/74 mm Hg; upon standing it quickly decreased to 94170 mm Hg without symptoms. Other findings of physical examination were within norrnal limits.

The serum sodium was 140 meq, potassium 2.2 meq, chloride 98 meq

and carbon dioxide 34 meq/liter; the creatinine was 1.01 mg, blood urea ni- trogen 18 mg and uric acid 7.7 mg/ 100 ml. Endogenous creatinine clearance

was 90 ml/min, para-aminohippurate (PAH) clearance 483 ml/min and the

September 1977 The American Journal of Medicine Volume 63 467

PATHOGENESIS OF BARTTER’S SYNDROME-FUJITA ET AL.

Na 140

(mEq/O 135

- 130

i F

3.5 K

3.0 (mEq/O)

2.5 -

PRA 30

(ng/ml/hr) 20

10

Urine 45

AER 30 (ilglday)

15

Urine

Na (mEq/day)

Urine

K (mEq/day)

Intake

Intake

80 BODY WEIGHT -

(kg) 75 z=r , , ) , , 1 5 10 15 20 25

Days

F/gure 1. Effects of alterations of sodium intake.

filtration fraction 18 per cent. The maximum rate of secretory transport for PAH (TmPAH) was 74 mg/min (normal, 68.5 to 90.2 mg/min). Urinary volume varied from 2,800 to 3,500 ml/24 hours. There was l-l- proteinuria without glycosuria, lysozymuria or any abnormality of the urinary sediment. The pH of urine was 7.1 and decreased to 5.1 when metabolic acidosis was induced by ammonium chloride loading. After deprivation of fluids for 14 hours and the intramascular in- jection of 2.5 U pitressin tannate, urinary osmolality was 360 mOsm/kg water whereas the serum potassium was 2.2 meq/liter. The concentrating ability of the kidney did not improve even after potassium repletion (potassium level of 4.0 meq/liter), 10 months later, when the maximum urinary osmolality remained at 420 mOsm/kg water. Plasma volume (MA) was 35.4 and 38.2 ml/kg (normal, 30 to 48 ml/kg). An intravenous pyelogram disclosed bilateral mild ureteral di- latation and an enlarged bladder. Electrocardiography revealed changes characteristic of hypokalemia. Serum cal- cium, magnesium, phosphorus, albumin and transaminase levels were all within normal limits. Urinary excretion of 17-hydroxycorticosteroids and 17-ketosteroids was normal

as was serum thyroxine concentration and resin triiodothy- ronine uptake.

The patient was treated with the administration of 100 meq potassium chloride and 100 mg spironolactone daily. Sub- sequently, the serum potassium increased to 4.0 meq/liter and plasma carbon dioxide became normal.

Our patient was maintained on a metabolic ward. Urine was collected for 24-hour periods, except during infusion studies. Serum and urinary electrolytes were measured by flame photometer on a Technicon@ Autoanalyzer. Urinary lysozyme was assayed by the lysoplate method of Osserman and Lawlor [9]. Aldosterone excretion rate (AER) was measured by the modification method of Langan et al. [lo], and plasma renin activity was measured by radioimmunoassay.

The pressor response to the intravenous administration of angiotensin was assessed in the following way. The patient was kept supine for 5 hours before and during the test period. Blood pressure was determined with a sphygmomanometer

468 September 1977 The American Journal of Medicine Volume 63


PRA ”

(ng/ml/hr ) ]IJ

Urine 313~ I I

AER 20 (II g/day) ,.

Figure 2. Effects of albumin infusion.

until the pressure became stable for 15 minutes. Valine angiotensin H (Hypertensine, Ciba) was then infused intrave- nously for five consecutive 15 minute periods at rates of 5, 10, 20, 50 and 100 ngfkg body weightlmin.

Renal tubular function was studied in the patient and in four

healthy control subjects during hypotonic saline diuresis. Each subject received an oral water load of 20 ml/kg body weight over a 40 minute period, followed by the intravenous administration of 0.45 per cent saline solution, initially at a rate of 5 ml/min and subsequently adjusting to exceed urinary flow by 5 ml/min. Free water clearance (C,,) was calculated by the following formula:

U Osm V CHpO = v - -

P (V = volume of urine, UOsm

OSlTl

= urinary osmolality, POsm = plasma osmolality)

Fractional sodium delivery to the distal nephron and the distal tubular sodium reabsorption was approximately calculated from (CH20 + CNa)/lOO ml glomerular filtration rate and (CH20/CH20 + C,,), respectively [ 111.

An open biopsy of the kidney was performed. The speci- men was fixed in 10 per cent neutral formalin and stained with

hematoxylin and eosin, and periodic acid-Schiff stains. Ma- terial for electron microscopy was fixed in 1 per cent osmium tetroxide.

RESULTS

Results of Plasma Renin Activity (PRA) and Urinary Aldosterone Excretion (Figures 1, 2 and 3). Initially,

PRA was markedly increased to 18.2 ng/ml, with the patient on a daily sodium intake of :!50 meq and potassium of 75 meq, compared with normal values (0.5 to 2.0 ng/ml/hour). With a low sodium intake (17 meq/day) PRA further increased from 14.3 to 32.2 ng/ml. When the patient’s sodium intake was increased to 420 meq, PRA was suppressed to 4.0 ng/ml. Ex- pansion of the extracellular fluid volume by the intravenous administration of albumin was associated with the suppression of PRA from 16.0 ng.lml in the control period to 6.2 ng/ml on the fifth day of albumin infusion.

With an ordinary diet, the aldosterone excretion rate was 18 pg/day which was slightly above the upper normal range (2 to 12 pg/day). It was increased up to

September 1977 The American Journal of Medlclne Volume 63 469


Na 140 3.5 K

(mEdO 135 3.0(mEq/i)

- 130 2.5 -

PRA 3o

(ng/ml/hr) :i

Unne 60

AER 40

(/&day) 20

BODY WElGHT6’1

(kg) 75- , , , , , , , , , , , , 0 I 5 IO 15 20 25 30

Days

Intake

Intake

Figure 3. Effects of increased potassium intake alone and in combination wil spironolactone.

SYSTOLIC DIASTOLIC

BLOOD PRESSURE BLOOD PRESSURE

86 80-

#’ .

a B.P.

,, , , , ,1 I,, , , ,I 5 10 20 50 100 5 IO 20 50 100

Angiotensin (mp g/kg/min)

0 Normals

0 Patient (before treatment)

n Patient (after albumin infusion)

Effect of infusion of angiotensin II on blood pressure


PATHOGENESIS OF BARlTER’S SYNDROME-FUJITA ET AL.

45 bug/day with a low sodium diet and came down to 10 hg/day with a high sodium diet. Albumin infusion also suppressed the aldosterone excretion rate to 15 pg/day. When the patient’s potassium intake was increased from 75 to 175 meq daily for 10 days, the aldosterone excretion rate increased from 24 to 32 pg/day. In a

subsequent study, the administration of spironolactone,

100 mg daily, increased serum potassium from 2.6 to

3.6 meq/liter, and the aldosterone excretion rate in-

creased further to 63 pg/day.

Balance Studies. With the patient on an ordinary in-

take, urinary losses of potassium accounted for almost

all of his daily intake of potassium, despite hypokalemia

(Figure 1). When dietary sodium was restricted from 250

to 17 meq/day, urinary sodium excretion decreased as

low as 20 meq/day and the urinary potassium excretion

also decreased. Increasing the dietary sodium to 320

and further to 420 meq/day then resulted in an imme-

diate increase in the urinary sodium excretion and

negative potassium balance. These results are inter-

preted as evidence that the hypokalemia resulted from

renal potassium loss due to hyperaldosteronism, the

potassium retention induced by the low sodium diet

being due to decreased sodium delivery to the distal tubule for exchange with potassium under the influence

of aldosterone. Expansion of extracellular fluid volume

by albumin infusion depressed urinary potassium ex-

cretion and the serum potassium level was slightly el-

evated (Figure 2). When potassium chloride was given

alone and in combination with spironolactone, potas-

sium was retained and the serum potassium level rose

toward normal for the first time (Figure 3).

Angiotensin Infusion Test (Figure 4). It can be seen

that our patient showed grossly diminished pressor

sensitivity to angiotensin compared with the normal

subjects. Pressor sensitivity was improved by rapid

volume expansion witlh albumin, 125 g, infused intra-

venously over five days.

Hypotonic Saline Diuresis (Figure 5). Results in normal subjects show that from 7 to 16 per cent of the

filtered fluid is delivered distally, of which 80 per cent

is converted to free water. In the patient, on the other

hand, some 8 to 14 per cent of the filtrate is delivered

distalty, but only from 20 to 32 per cent is converted to

free water clearance. The evidence suggests that, in

the patient, sodium transport is not significantly impaired

atthe proximal tubule but that it is markedly depressed

at the diluting segment.

results of Histologic Studies. Light microscopy: Twenty-five glomeruli appeared almost normal except for three sclerosed glomeruli. But, there were mild focal increases in the mesangial cells and in the matrix of a

few glomeruli. Nearly every juxtaglomerular apparatus

appeared markedly hyperptastic and hypertrophic (Figure 6). Small arteries and particularly afferent ar-

terioles appeared moderately thickened, apparently

0

100 + r h

x 80 c

l

Controls

Patient

a l

0 l l

* + 0

l

+

.m l e l

0-u 6 8 10 12 14 16

CHZO + CNa (rnl/rnin/lOOGFR)

Figufe 5. Relationship between the fraction of fluid delivered distally which is converted to free water ( C,-&CH~O + C,,) to the fraction so delivered (CHAT +- C,Q) during hypotonic saline diuresis.

because a large number of the muscular cells have

been replaced by juxtaglomerular-type cells with

prominent nuclei (Figure 7).

Electron microscopy: The juxtaglomerular apparatus

displayed large numbers of epithelioid cells which

contained abundant electron-opaque secretory granules

within the poorly discernible limiting membranes (Figure

8). The shape of the granules was irregular, because

granules touching each other tended to adhere to one

another and then merged together to form polymor-

phous granules. Some granules were almost homogeneous and others partially contained electrondense

areas, which might seem to be lysosome residue. Be-

Figure 6. Hyperplasia of juxtaglomerular complex.

Hematoxylin and eosin stain; original magnification X200, reduced by 2 1 per cent.

September 1977 The American Journal of Medlclne Volume 63 471

PATHOGENEM OF BARTTER’S SYNDROME-FUJITA ET AL.

Figure 7. Two thickened small arteries with juxtaglomerular-type cells including prominent nuclei. Hematoxylin and eosin stain; original magnification X 160, reduced by 18 per cent.

sides the granules, moreover, dense multivesicular bodies, possibly of lysosomal origin, were found in the epithelioid cells (Figure 9).

COMMENTS

The diagnosis of Bartter’s syndrome seems well established in this patient. The hyperplasia of the juxtaglomerular apparatus, the extremely high plasma renin activity, the hypokalemic alkalosis and the normal blood pressure are all in accord with the diagnosis.

The etiology of Bartter’s syndrome remains unknown. Bartter et al. [ 11 first suggested that the primary defect was vascular insensitivity to the pressor action of an-

Figure 8. Electron micrograph showing the wall of the afferent arteriole adhering to the renal corpuscle (Bs, Bow- man’s space). Epithelioid cells (E) with filled renin secretory granules lie next to smooth muscle cells (S). Original magnification X4.200, reduced by 36 per cent.

giotensin. This would lead to a compensatory overproduction of renin, secondary hyperaldosteronism and hypokalemia. Yet, it has been noted that insensitivity to angiotensin was not specific for Bartter’s syndrome but occurred whenever plasma renin activity was increased, as in patients with cirrhosis and malignant hypertension [ 121. Cannon et al. [2] demonstrated mild but persistent renal sodium wasting in a patient with Bartter’s syndrome and suggested that the syndrome was the result of a form of sodium-losing nephropathy. Data of our patient support this proposal, as do the results as Goodman et al. [4] and White [6]. In our patient, the aldosterone excretion rate increased markedly with potassium loading, in addition to the expected increase with sodium restriction and the suppression to normal levels with sodium loading. These findings demonstrate a physiologic rather than autonomous overproduction of renin and aldosterone. Moreover, plasma renin activity and urinary aldosterone excretion were both suppressed, and sensitivity to angiotensin improved with albumin infusion. In the study of White, rapid infusion of 3 liters of saline solution resulted in correction of angiotensin insensitivity and, in addition, the natriuretic response to loading with saline solution was somewhat greater in these patients than in normal subjects.

The primary site of the tubular sodium reabsorptive defect in Bar-her’s syndrome has not been established, but several investigators have reported the possibility of the existence either at the proximal tubule [2,5] or at the ascending limb of Henle’s loop [6,7]. In our case, proximal tubular mechanisms appeared intact, since the value of TmPAH was normal without glycosuria or

Figure 9. Electron micrography showing polymorphous secretory granules with the poorly discernible limiting membranes, around dense multivesicular bodies (MV). Some granules (G,) with homogeneous matrix, and others (Gp) containing electron-dense areas. Original magnification X 15,000, reduced by 36 per cent.


lysozymuria. On the other hand, we studied the site of

the renal tubular defect in a well-established patient with

Bartter’s syndrome by utilizing both clearance and di-

uretic blockade methodologies. During hypotonic saline

diuresis, expansion of extracellular fluid results in a

depression of sodium reabsorption at the proximal tu-

bule, thereby progressively increasing delivery of filtrate

to the diluting segment [ 131. It is well established that

the free water generation depends upon sodium

transport without water at the ascending limb of Henle’s

loop [ 141. Therefore, the amount of free water clear-

ance at maximum diuresis is an estimate of the sodium

reabsorptive capacity of the ascending limb of Henle’s

loop. The present results (Figure 5) demonstrated that

the rate of sodium delivery to the distal nephron (CHPO

+ C,,) was within normal limits in our patient. This

suggests that the proximal tubular reabsorption of so-

dium is not significantly impaired. CHZO i- CNa probably

underestimates the rate of delivery in the patient ex-

creting large amounts of potassium. However, during

maximum water diuresis in this patient, the fractional

potassium excretion was only 17 per cent of the frac-

tional sodium excretion. It is, therefore, safe to conclude

that the rate of sodium delivery to the distal tubule in this

patient is not significantly greater than that of the control

subjects. On the other hand, the present data indicated,

as well as those of Chaimovitz et al., that free water

formation was markedly reduced at all rates of distal

tubular delivery, further suggesting that salt wasting is

mainly a consequence of a defect of sodium transport

at the ascending limb of Henle’s loop. A similar obser-

vation was made in a patient with the syndrome studied

by Bartter et al. [20]. Consequently, defective sodium

reabsorption in this portion, in addition to severe hy-

pokalemia, impaired the ability of the kidney to develop

an adequate medullary hyperosmolality [ 151, leading

to this hyposthenuria in the patient.

The hypokalemia in Bartter’s syndrome has been

ascribed to excessive potassium wasting due to hy-

peraldosteronism. In our patient, however, hyperal-

dosteronism was not severe enough to cause hypo-

kalemia, and hypokalemia persisted even when the

aldosterone excretion was suppressed to normal levels

by the administration of albumin. There seems to be a

decrease in the ability of the kidney to preserve po-

tassium adequately. Several lines of evidence have

shown that the initiating event in this syndrome was an

inability of the ascending limb of Henle’s loop to reab-

sorb sodium adequately, so that a large amount of so-

dium was delivered to the sodium-potassium exchange

sites in the distal tubule. Aldosterone-enhanced distal tubular sodium-potassium exchange could account for

some but not all of the potassium-wasting state.

Moreover, an accelerated sodium-potassium exchange

at the distal tubule might compensate “obligatory” salt

PATHOGENESIS OF BAR?TER’S SYNDROME-FUJITA ET AL.

loss, leading to normal plasma volume and to normal

sodium balance with salt restriction in the patient.

However, Modlinger et al. [8] demonstrated that blood volume tended to increase concomitant with the de-

crease in plasma renin activity, by the administration

of large doses of propranolol in a patient with Bartter’s

syndrome. This response to propranolol suggests that

the decreased blood volume was a result and not a

cause of the hyperreninemia. Thus, although it is sure

that renal salt wasting exists in this patient, the relation

between renal salt wasting and hyperreninemia in

Bartter’s syndrome remains to be elucidated.

Histologic findings by electron miicroscopy showed

that the secretory granules increased in number, oc-

cupying all or part of the cytoplasrr of the epithelioid

cells (juxtaglomerular cells). Some granules were amorphous and in such granules the limiting mem-

branes were but poorly discernible, suggesting that the

epithelioid cells were hyperactive in secretion. Elec-

tron-dense areas within the secretory granules and

multivesicular bodies were found in the epithelioid cells,

both of which were morphologically very similar to ly-

sosomes. On the other hand, it is well known that jux-

taglomerular granules have a lysosomal nature since

they contain acid phosphatase [ 16,17-l. Thus, it is sure

that multivesicular bodies have some relation to the

secretory granules. Evidence presented suggests that

the electron-dense residual bodies may be released

from the secretory granules and then aggregate to form

multivesicular bodies. To the contrary, there is another

speculation that multivesicular bodiles may be precur-

sors of the contents of the secretory granules. Gener-

ally, the problem of a product or precursor is always

discussed but it cannot be clarified whenever we study

about granulogenesis in a continuity of the transitional

form of the secretory granules.

Another problem is the moderate thickness of small

arteries. As shown in Figure 7, the srnooth muscle cells

apparently were replaced by juxtaglomerular-type cells

with prominent nuclei but without hyaline degeneration.

The interpretation is consistent with tine proposal of Hatt

[ 181, that an increase in the number of granular cells

occurs through metaplasia of the smooth muscle cells

of the media of the afferent arteriole. Bracket-t et al. [3]

reviewed the vascular changes found on biopsy in pa-

tients with Barber’s syndrome. They suggested that the

renal arteriolar lesion resulted in decreased perfusion

through afferent arterioles which set renin release at high levels. Yet, it has been noted that a similar arteri-

olar lesion occurs in familial chloricle diarrhea. a con-

dition associated with chronic volurne depletion [ 191,

and that renin injections can cause vascular change. Thus, it is possible that prolonged excessive release of

endogenous renin might cause the vascular lesion of

the afferent arterioles in this patient.

September 1977 The American Journal of Medicine Volume 63 473


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The pathogenesis of Bartter's syndrome: Functional and histologic studies