Parathyroid Hormone (PTH) Homeostasis in Bartter Syndrome
Parathyroid Hormone (PTH) Homeostasis in Bartter Syndrome
Parathyroid hormone (PTH) gland calcium sensing receptor (CASR) regulates PTH secretion. CASR
 is also expressed in nephron thick ascending limb (TAL). Bartter syndrome (BS), a
 normotensive hypokalemic tubulopathy, may be due to mutations in different TAL channels,
 including the potassium channel ROMK. Mutations in CASR may also cause BS through its effects
 on ROMK function. However, it is unknown whether ROMK mutations exert any effects on CASR
 function and PTH physiology. Preliminary data from our center shows that PTH levels were
 specifically elevated in type II (where ROMK is mutated) and not in type IV (where another
 gene, Barttin is defective) BS, without a common explanation. We assume that the mutation in
 ROMK may cause a dysregulation of PTH secretion via possible interaction with CASR.

 The purpose of this study is: to investigate the PT-gland function and regulation in BS.

 Methods: Patients with BS type II and IV and normal controls will undergo a standard protocol
 of controlled ionic hypo- and hypercalcemia, during which PTH secretion, phosphate balance
 and calcium excretion will be followed. Calcium Vs PTH response curves will be generated and
 compared.

 Expected impact and benefit: the results of this study will help understand the mechanisms of
 PTH regulation beyond CASR.
The parathyroid glands play a pivotal physiological function by maintaining blood calcium
 levels, specifically blood ionized calcium concentrations, within a very narrow range. They
 do so by modulating the minute-to-minute release of parathyroid hormone (PTH) into the
 circulation. Such changes have almost immediate effects on calcium excretion in the urine and
 on calcium efflux from bone and, if sustained for hours or days, affect renal vitamin D
 metabolism and ultimately the efficiency of intestinal calcium absorption. The capacity of
 chief cells of the parathyroid to detect small changes in blood ionized calcium levels,
 modify PTH release accordingly, and initiate these adaptive responses is mediated by a
 calcium-sensing receptor (CaSR) located at the cell surface.

 The importance of the CaSR in parathyroid tissue extends beyond its traditional role as a
 modifier of calcium-regulated PTH secretion to involve other key components of parathyroid
 gland function that are frequently abnormal in clinical disorders characterized by excess
 parathyroid gland activity such as hyperparathyroidism. These include disturbances in the
 control of PTH gene transcription and hormone synthesis and the development of parathyroid
 gland enlargement due to tissue hyperplasia.

 PTH acts mainly on renal proximal tubule phosphorus (Pi) reabsorption and bone osteoclast
 calcium and Pi resorption. CASR is also expressed in nephron thick ascending limb (TAL),
 where it interacts with luminal potassium ROMK channel. Mutations in several TAL channels and
 proteins (including ROMK, NKCC2, ClCKb, Barttin and CASR) cause Bartter syndrome (BS), a
 normotensive hypokalemic tubulopathy. Whereas the effects of CASR mutations on ROMK function
 in the kidney have been described , it is unknown whether ROMK mutations exert any effects on
 CASR function or PTH regulation. We describe here a group of children and adolescents with
 type II BS (due to mutations in ROMK) with abnormal PTH homeostasis.

 Preliminary Data:

 We compared laboratory data of 12 children with type II BS (4M, 8F) and 17 children (7M, 10F)
 with type IV BS (d/t mutations in the Barttin gene, a beta subunit of the ClCKb basolateral
 chloride channel in the TAL ), followed in our center over the past 10 years. A total of 86
 and 105 datasets of blood and urine analyses (average datasets/pt: 7.3±4.1 and 6.9±2.9), for
 type II and IV BS respectively, were analyzed.Potassium levels were normal in all BS-II
 children without additional salt supplementation, whereas BS-IV children were usually mildly
 hypokalemic. Estimated GFR remains normal in all children. There was no hypomagnesemia.
 Average PTH values were significantly higher in BS-II (102±39 Vs 46±24 pg/ml in BS-IV,
 p<0.001) and were above upper normal limit in 93% of cases Vs 13% in BS-IV (p<0.001). Levels
 of 25(OH) vitamin D were not different. Total serum calcium was mildly decreased (within the
 normal range) and serum Pi increased in BS-II, both in absolute values or when normalized for
 age (PiSDS). The threshold for phosphate excretion (TpGFR) was slightly higher in BS-II.
 There was no difference in the degree of hypercalciuria between groups. Based on these
 preliminary data we concluded that the elevated PTH levels only in type II BS are not related
 to a decrease in GFR or vitamin D levels or decreased serum calcium or hypercalciuria. The
 elevated Pi levels are associated with a decrease in phosphate excretion, but are not
 correlated with PTH levels. The possibility that a mutation in ROMK may cause a dysregulation
 of PTH secretion via possible interaction with CASR should now be investigated.

 Methods:

 The investigative protocol has been submitted to the local Committee for Human
 Experimentation. Informed consent will be obtained from affected children (or young adults)
 and their parents.

 We expect to recruit 5 patients from each BS subgroup. In addition we will recruit 5 normal
 volunteers to serve as an additional control group.

 Subjects will be evaluated during 2-day admissions to the General Pediatric Ward as
 previously described . On the first day of study, 2-h I.V. infusions of sodium citrate will
 be done to gradually lower blood ionized calcium concentrations to a level of at least 0.2
 mmol/L below preinfusion values; the dose of sodium citrate ranges usually from 28-118
 mg/kg/h. Blood samples for measurements of ionized calcium and PTH will be obtained 30, 15,
 and 0 min before and every 10 min during sodium citrate infusions. The following day, 2-h IV
 infusions of 10% calcium gluconate will be done to gradually raise blood ionized calcium
 concentrations to a level at least 0.2 mmol/L above preinfusion values. The dose of calcium
 gluconate usually ranges from 2-8 mg/kg/h. Blood samples for measurements of ionized calcium
 and PTH will be obtained as described previously for infusions of sodium citrate. The average
 of measurements obtained 30, 15, and 0 min before starting each infusion will be used to
 determine basal values for blood ionized calcium and serum PTH for each day of study.

 Blood ionized calcium levels will be monitored during calcium infusions using a
 calcium-specific electrode (Radiometer ICA-II, Copenhagen, Denmark); blood samples will be
 collected anaerobically, and measurements will be obtained immediately thereafter. Serum
 samples for PTH determinations will be separated by centrifugation immediately after
 collection, snap frozen on solid CO2, and stored at -70 oC until assay. Ionized calcium
 levels will be monitored after stopping calcium infusions until values returned to baseline
 levels.

 The sigmoidal curve that describes the relationship between blood ionized calcium and serum
 PTH levels will be determined for each study subject using the combined results obtained
 during sodium citrate and calcium gluconate infusions. According to the four parameter model,
 the set point for calcium-regulated PTH release represents the ionized calcium concentration
 at which serum PTH levels are midway between the maximum value achieved during hypocalcemia
 and the minimum value attained during hypercalcemia, as reported previously .

 Results obtained during calcium gluconate infusions will be separately analyzed to assess the
 inhibitory effect of increasing blood ionized calcium concentrations on PTH release. To
 improve the linear fit of the data, serum PTH levels, expressed as the natural logarithm (ln)
 of percent preinfusion values, will be plotted against the corresponding blood ionized
 calcium concentration at each 10-min interval as previously described .

 Statistical Analysis:

 Linear regression analysis will be done using the method of least squares, and slope and
 y-intercept values will be compared using the t statistic. A mono-exponential curve fitting
 algorithm of the form y =A e-2kt + B will be also used to examine the curvilinear
 relationship between blood ionized calcium and serum PTH levels during I.V. calcium
 infusions; these results will be presented as mean values with 95% confidence intervals.
Inclusion Criteria:

 -  Bartter syndrome

 -  Normal Vitamin D status

 Exclusion Criteria:

 -  Age < 14 yrs