—  SPECIALTY CONFERENCE  —

Nephropathology

Case 3 - Bartter's Syndrome

Mark Haas
Johns Hopkins Hospital
Baltimore, Maryland


Click on each slide thumbnail image for an enlarged view
Clinical History:
A 4 year old African-American girl was admitted to the hospital with a one week history of vomiting, anorexia, and lethargy. There was a history of polydipsia without reported polyuria or dysuria. Physical exam was remarkable for height 93 cm (<5th percentile, height age 2.75 years), weight 11.5 kg (<5th percentile), hypotonia, and evidence of dehydration. There was no edema. Blood pressure was 100/60. Family history was positive for hypertension, but otherwise negative for renal disease.

During the first week of hospitalization, laboratory work-up showed (normal range for age listed in parentheses for abnormal values only): serum sodium 125-135 (138-145), potassium 1.8-2.8 (3.4-4.9), chloride 82-92 (97-110), and bicarbonate 27-32 (21-28) mmol/L. BUN was 7 mg/dl and serum creatinine 0.4 mg/dl. Total serum calcium was 9.4 mg/dl and magnesium 2.1 mg/dl. Total serum protein was 7.3 g/dl and albumin 4.4 g/dl. Urinalysis showed 3+ protein, with no glucose, RBC, or WBC. A 24 hour urine collection showed specific gravity 1.010 (1.015-1.025), osmolality 224 mosmol/kg (300-900), protein 290 mg (50-80), protein/creatinine ratio 1.1 (<0.2), sodium 54 mmol, potassium 25 mmol, chloride 50 mmol (15-40), calcium 71 mg. Renal ultrasound showed low-normal kidney sizes with increased echogenicity but no apparent nephrocalcinosis. ANA, anti-double-stranded DNA, and Sjogren's SS-A and SS-B antibodies were negative, and serum C3 and C4 were normal. Serum aldosterone was 167 ng/dl (normal <40), plasma renin activity 1542 mU/ml (normal <100), and serum 6-keto-prostaglandin F1a (a product of renal prostaglandin metabolism) 13 pg/ml (normal <10).

The clinical impression was probable Bartter's syndrome, although this did not explain the proteinuria. Largely for this reason, an open renal biopsy was performed.


Case 3 - Figure 1 - Photomicrograph of two glomeruli from the mid-portion of the cortex. The juxtaglomerular apparati appear hyperplastic. PAS stain, x 200.
Case 3 - Figure 2 - Photomicrograph of two additional glomeruli. The glomerulus at the upper left is near, but not directly beneath, the renal capsule. The deeper glomerulus appears enlarged with mild to moderate mesangial hypercellularity accompanied by mild increase in mesangial matrix. H&E stain, x200.
Case 3 - Figure 3 - Direct immunofluorescence for C1q. Glomerulus shown is representative of non-sclerotic glomeruli within the specimen. Staining for C1q is present in the mesangial regions. FITC-conjugated anti-human C1q on cryostat section, x400.


Case 3 - Figure 4 - There is irregular effacement of the podocytes. Glomerular basement membranes appear normal in thickness. An electron dense deposit is identified in the mesangium, subjacent to the glomerular basement reflection.
Case 3 - Figure 5 - Electron micrograph of a cell within the juxtaglomerular apparatus. The JGA cells contain prominent rough endoplasmic reticulum and Golgi complexes. Rounded or rhomboidal secretory granules are located adjacent to Golgi complexes. Uranyl acetate and lead citrate stain, total magnification x78, 750 (original magnification x35, 000, print x2.25).

Renal Biopsy Findings

Light Microscopy:
The specimen contained 2 portions of renal cortical tissue, one a wedge biopsy and the other a needle biopsy. In excess of 50 glomeruli were present. There were several clusters of atrophic, sclerotic glomeruli, most but not all in the subcapsular cortex. Included in these clusters were occasional immature-appearing glomeruli resembling fetal glomeruli. Of the remaining glomeruli, approximately half showed a mild to moderate increase in cellularity, limited to mesangial areas, with an accompanying mild increase in mesangial matrix. Some glomeruli appeared mildly to moderately enlarged. No crescents or areas of segmental necrosis or sclerosis were seen. Many of the glomeruli showed a hyperplastic juxtaglomerular appartatus (JGA).

There was tubular atrophy and interstitial fibrosis limited to areas with atrophic glomeruli, and mild overall. Mild focal interstitial infiltrates of small lymphocytes were present, limited to foci of tubular loss. A significant number of tubules, some in small clusters, contained large proteinaceous casts. Small arteries and arterioles were generally unremarkable.

Immunofluorescence (IF):
IF was performed on a portion of renal cortex with 20 glomeruli. There was granular deposition of IgG (1+, 0-4+ scale) and C1q (2+) in the mesangium of all except atrophic glomeruli; this was global in some glomeruli and segmental in others. No specific staining was noted for IgA, IgM, C3, or C4. No tubular basement membrane deposits were noted.

Electron Microscopy (EM):
EM was performed on a portion of renal cortical tissue with 2 glomeruli. Both glomeruli showed hyperplasia of the JGA on 1-micron section, with 1 showing mild mesangial hypercellularity. Ultrastructurally, many of the cells within the JGA showed prominent rough endoplasmic reticulum and Golgi complexes. These cells also contained moderately electron-dense secretory granules of varying sizes; most were roundish although several had a rhomboidal shape. The rhomboidal granules were seen directly adjacent to Golgi complexes. Each glomerulus showed a mild increase in mesangial matrix with a mild-moderate number of generally small mesangial electron-dense deposits, some near the junction of the mesangial matrix and glomerular capillary basement membrane (paramesangial). No subendothelial, subepithelial, or tubular basement membrane deposits were seen, and there were no tubulo-reticular inclusions noted. There was extensive but not complete effacement of the epithelial foot processes.

Renal Biopsy Diagnosis:

  • Hyperplasia of the juxtaglomerular apparatus with focal atrophic and immature-appearing glomeruli, consistent with Bartter's syndrome
  • Mild focal mesangial proliferative glomerulonephritis, most consistent with C1q nephropathy
Comment:
The JGA hyperplasia, while not a specific finding, is the most consistently observed histologic feature associated with Bartter's syndrome, and the presence of atrophic and immature-appearing glomeruli has also been described in this condition. The mesangial proliferative glomerulonephritis with immune complex deposits is not a finding that has been described in association with Bartter's syndrome and most likely represents a separate lesion that could account for the proteinuria. The IF findings are most consistent with C1q nephropathy; the lack of IgA, IgM, and C3 deposits, together with the negative serologies, argue against lupus nephritis.

Molecular Genetic Testing and Follow-Up
Molecular genetic analysis was performed on genomic DNA extracted from the patient's whole blood. Specific primers for the CLCNKB chloride channel gene that by PCR amplify exons 1-2 and 6-9 were used to test for deletion mutations in this gene. Amplified DNA resolved on a 1% agarose gel showed absence of the CLCNKB PCR products, indicating a homozygous deletion.

The patient was treated with indomethacin (80 mg tid), KCl (60 mEq tid), and NaCl (500 mg qid). On this regimen the patient's growth improved modestly. At age 10, her height was 127 cm (3rd percentile), weight 29 kg (30th percentile), and blood pressure 120/80. Serum Na was 137, K 2.7, Cl 93, and HCO3 25 mmol/L; an increase in KCl dose corrected the hypokalemia. BUN was 7 mg/dl and creatinine 0.6 mg/dl. Serum aldosterone (46 ng/dl) and plasma renin activity (396 mU/ml) remained elevated, though less severely than at age 4. The patient continued to have proteinuria, with a benign urine sediment. Random urine protein/creatinine ratio was 7.9 (normal <0.2).

Discussion

Clinical Features of Bartter's Syndrome
Forty years ago, Bartter et al.1  reported two cases of a newly described syndrome characterized clinically by hypokalemia, metabolic alkalosis, hyperaldosteronism, growth retardation, and normal blood pressure with a blunted pressor response to exogenous angiotensin II, and pathologically by hyperplasia of the juxtaglomerular apparatus (JGA). Since this initial description, over 100 cases of what has been termed Bartter's syndrome have been described. Additional features that have been noted in most or all of these patients include polyuria, impairment of urinary concentrating ability, and increased levels of renin, angiotensin II, and prostaglandins.2-4 

Clinically, there are two major forms of Bartter's syndrome.reviewed in refs. 3 and 4  Neonatal (also called antenatal) Bartter's syndrome, the more severe form, typically presents with polyhydramnios that is felt to be the result of intrauterine polyuria. In addition to the features noted above, infants with neonatal Bartter's syndrome typically have very high levels of urinary calcium excretion, and develop nephrocalcinosis with markedly hyperechoic medullary pyramids noted on renal ultrasound. These patients have severe failure to thrive and tend to be markedly growth retarded, and may develop renal insufficiency that is thought to be related, at least in large part, to the nephrocalcinosis. Classic Bartter's syndrome usually presents during the first few months or years of life with failure to thrive, dehydration, and growth retardation. These patients also have all of the features noted in the previous paragraph, but in contrast to patients with the neonatal form tend to have at most mild hypercalciuria and do not develop significant nephrocalcinosis.

Pathophysiology and Molecular Genetics
The pathophysiology of Bartter's syndrome (both clinical forms noted above) results from a primary defect in NaCl reabsorption in the thick ascending limb of Henle's loop (TALH), which is normally responsible for reabsorbing ~30% of filtered NaCl. Figure 1 illustrates the ion transport pathways in the TALH. Na and Cl, together with K, are transported from the lumen into the epithelial cell cytoplasm by the electroneutral Na-K-2Cl cotransporter (NKCC2, the "2" referring to the cotransporter isoform present in the TALH) in the apical cell membrane. Of note, NKCC2 is the site of action of the loop diuretics furosemide and bumetanide. The Na taken up via the cotransporter is actively transported out of the cells across the basolateral membrane, in exchange for K, by the Na,K-ATPase (Na-K "pump"). The majority of the Cl taken up by the cotransporter exits the cell (driven by the electrical potential difference across the basolateral membrane) through basolateral Cl channels termed CLCNKB. A smaller amount of Cl exits via an electroneutral K-Cl cotransporter (KCC) in the basolateral membrane, the driving force for this being the chemical gradient for K across the plasma membrane. Much of the K taken up by the cotransporter is promptly "recycled" back into the lumen via apical K channels termed ROMK, the energy for this also supplied by the chemical gradient for K. The electrogenic transport of Cl across the basolateral membrane and of K across the apical membrane creates the characterisitic lumen-positive electrical potential of the TALH, which serves to drive additional lumen-to-blood transport of cations (Na, Ca, and Mg) via the paracellular pathway. As NKCC2 accounts for nearly 100% of apical membrane NaCl influx and ROMK for essentially 100% of apical K recycling, complete inhibition of either of these pathways will essentially shut down NaCl reabsorption by the TALH. Inhibition of CLCNKB will severely reduce but not completely inhibit NaCl reabsorption, noting the presence of a second, minor pathway for Cl exit across the basolateral membrane (KCC).


Figure 1. Ion transport pathways in the TALH. At the apical (lumenal) membrane, Na, K, and Cl are transported into the cells by the Na-K-2Cl cotransporter NKCC2. Na and Cl are then transported across the basolateral membrane, Na by Na,K-ATPase and Cl by CLCNKB Cl channels and (to a lesser extent) the K-Cl cotransporter, KCC. K taken up via NKCC2 is recycled back across the apical membrane via ROMK K channels. This electrogenic K efflux plus basolateral Cl efflux via CLCNKB creates a lumen-positive electrical potential that drives lumen-to-blood transport of Ca, Mg, and additional Na.

Molecular genetic studies in the mid-to-late 1990s5-7  identified 3 classes of genetic defects responsible for Bartter's syndrome: loss-of function mutations in the gene for NKCC2 on chromosome 15 (sometimes referred to as type I Bartter's syndrome), in the gene for ROMK on chromosome 11 (type II), and in that for CLCNKB (including large deletions such as seen in this case) on chromosome 1 (type III). NKCC2 and ROMK mutations tend to be associated with the neonatal form of Bartter's syndrome, while CLCNKB mutations are, as in the case presented, most often associated with classic Bartter's. It has been hypothesized that the less severe presentation associated with CLCNKB mutations may be related to the small but significant amount of TALH NaCl reabsorption that persists in the absence of basolateral Cl channel function (via KCC). In all cases the pattern of inheritance is autosomal recessive. More recently mutations in a fourth gene called BSND (on chromosome 1 but distinct from the CLCNKB gene), encoding a protein termed barttin that is highly expressed in both the TALH and the dark cells of the inner ear, have been found in patients with a form of neonatal Bartter's associated with sensorineural deafness.8  Barttin appears to be a regulatory protein that both activates and increases plasma membrane expression of CLCNKB, as well as Cl channels in more distal portions of the nephron, thus accounting for the very severe salt wasting associated with this (type IV?) form of Bartter's syndrome.9 

The pathophysiological mechanisms by which a defect in TALH NaCl reabsoprtion leads to the multiple abnormalities that comprise Bartter's syndrome are complex.2-4  To briefly summarize, this defect results in increased delivery of NaCl to distal and collecting tubules, which promotes increased K and acid secretion in the latter (where such secretion is linked to apical Na uptake). However, distal NaCl reabsorption cannot fully compensate for the TALH defect, resulting in salt wasting and volume depletion. This promotes renin secretion and associated increases in circulating levels of angiotensin II and aldosterone, stimulating further K and acid secretion in collecting tubules leading to pronounced hypokalemia and alkalosis. Lack of NaCl reabsorption at the level of TALH is also associated with impaired Ca reabsorption at this site that may not be compensated more distally (especially in neonatal Bartter's), as well as decreased accumulation of salt in the medullary interstitium leading to impaired urinary concentrating ability. The specific trigger for the increased renal synthesis of prostaglandins that is characterisitic of Bartter's syndrome remains unclear, although hypokalemia may play a role in this, either directly or indirectly. Angiotensin II and bradykinin (which is also elevated in Bartter's syndrome) may also be involved as both are known stimulators of renal prostaglandin synthesis. Prostaglandins (particularly PGE2) further potentiate renin release (indeed, treatment of patients with indomethacin, an inhibitor of prostaglandin synthesis, significantly reduces the hyperreninemia of Bartter's syndrome), and also potentiate the impairment of urinary concentrating ability.

Pathologic Findings
The most consistent pathologic finding in Bartter's syndrome is JGA hyperplasia. By EM, there are epithelioid cells associated with afferent and (to a lesser extent) efferent arterioles that contain prominent Golgi complexes and secretory granules, some with a rhomboidal appearance.10  Bartter et al1  and others have also described an increased number of atrophic, immature-appearing glomeruli in children with classic Bartter's, although this is not a specific or consistent finding. As noted above, nephrocalcinosis is seen in neonatal Bartter's, and this may be associated with varying degrees of interstitial fibrosis.

There are no specific glomerular changes associated with Bartter's syndrome and proteinuria is uncommon in children with Bartter's, although some cases show enlarged glomeruli and in one such case in the original report of Bartter et al1  there was mild proteinuria. There are rare reports of children with probable congenital Bartter's and focal-segmental glomerulosclerosis.11,12  These reported cases are quite rare, and as such may simply represent the presence of two independent diseases. The mild mesangial proliferative glomerulonephritis in the patient presented above is felt to be most consistent with C1q nephropathy; the renal biopsy findings observed are consistent with those described by Iskandar and coworkers13  in pediatric cases of C1q nephropathy, and there is no clinical, serologic, or strong pathologic evidence for SLE. It is felt that the Bartter's and C1q nephropathy most likely represent independent lesions, although both are rare conditions. In support of this, while the symptoms associated with Bartter's syndrome improved when the patient was treated with KCl and indomethacin, her proteinuria has worsened.

Differential Diagnosis and Treatment
A number of conditions may produce findings resembling those of congenital Bartter's syndrome.2-4  Not surprisingly, chronic, high-dose loop diuretic use is among these.14  Conditions leading to severe gastrointestinal KCl loss, such as severe vomiting, laxative abuse, cystic fibrosis, and congenital chloride diarrhea (a rare autosomal recessive disorder) may mimic Bartter's syndrome, although these patients (in contrast to those with Bartter's) have low urine Cl excretion, often <10 mmol/day.4,15,16  Gentamicin may produce a tubulopathy with many features of Bartter's syndrome, although there is also prominent hypomagnesemia17  which is very uncommon in Bartter's. Gitelman's syndrome, a congential disorder caused by mutations in the thiazide-sensitive Na-Cl cotransporter of the distal tubule, is associated with hypokalemia, metabolic alkalosis, and usually hyperreninemia, but with very low serum Mg levels, only mildly impaired urinary concentration and growth rate, and with little or no increase in prostaglandins.3,4  Mutations in the calcium-sensing receptor (CaSR), a receptor essential for regulating parathyroid hormone secretion, can produce a Bartter's-like syndrome but with low serum calcium levels (which tend to be normal in true Bartter's syndrome) and autosomal dominant inheritance.18  Rare cases of Sjogren's syndrome19  and one of medullary sponge kidney20  have been reported with hypokalemia and other findings resembling those of congenital Bartter's syndrome.

Treatment for Bartter's syndrome is aimed at correction of hypokalemia and at minimizing the effects of increased prostaglandin production, and is generally very difficult.3,4  Large amounts of oral KCl are typically required, and may be administered together with spironolactone (aldosterone antagonist) or another K-sparing diuretic. Oral NaCl is often needed to minimize hypovolemia. Some but not all studies on the use of ACE inhibitors reported improvements in hypokalemia and lower aldosterone levels.3  Inhibitors of prostaglandin synthesis such as indomethacin have been shown to reduce levels of renin and aldosterone, and may lessen growth retardation, although height usually remains below the 10th percentile.4  Growth hormone therapy may be beneficial, but is still under investigation.3 

References

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  2. Gill JR Jr. Bartter's syndrome. Ann Rev Med 31: 405-419, 1980
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  20. LaGreca G, Bazzato G, Biasioli S, et al. Bartter's syndrome associated with sponge disease. Kidney Int. 7: 192, 1975abstract