The β3-AR agonist BRL37344 ameliorates the main symptoms of X-linked nephrogenic diabetes insipidus in the mouse model of the disease

Abstract

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the β3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the β3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the β3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human β3-AR agonists might represent an effective possible treatment strategy for X-NDI.

Keywords: (3–6) vasopressin; BRL37344; antidiuresis; beta‐3 adrenoreceptor; kidney; nephrogenic diabetes insipidus.

FIGURE 1

A single i.p. injection of BRL37344 1 mg/kg promoted short‐lasting reabsorption of water and solutes in X‐NDI mice. 16 X‐NDI mice were individually placed in metabolic cages. For 3 days, 11 received a single i.p. injection of BRL37344 1 mg/kg (BRL), whereas 5 control animals received saline alone (CTR) (protocol 1). (A) Urine samples were collected 3 h after injection (I urine collection), 5 h after the I collection (II urine collection) and again 16 h later (III urine collection). The whole pie represents 24 h and each part of whole proportionally indicates the duration of the urine collection time windows. In particular, the black part corresponds to the current urine collection, the grey part to the sample collection already done and white part to the missing collection. In all scatter plots, data were given as mean ± SEM and each dot represents the mean effect of the injection in 3 days experiments on different parameters for each mouse. Data were expressed as a percentage of the mean value measured in 5 days preceding the experiments (baseline set ad 100%, not represented in the plot) for each mouse. The missing dots are due to the lack of urine production by some mice. (B) Urine output and urine osmolality were measured in the three time windows in CTR and BRL X‐NDI mice. CTR mice showed minimal fluctuations around their baseline (set as 100%) for urine output and osmolality analysis. Conversely, BRL37344 i.p. injection reduced urine output (≈70%) and increased urine osmolality (≈40%) during the first 3 h (I). The antidiuretic effect of BRL37344 was not seen in the later urine collections (II, III). Significant differences between CTR and BRL were tested by a two‐tailed unpaired t‐test.**p < 0.01, ****p < 0.0001. Significant differences between data measured during the monitoring (baseline) and data collected during the treatment, for both CTR and BRL, tested by two‐tailed paired t‐test, were reported in the text. (c) 24 h urine output and osmolality and water intake of CTR and BRL X‐NDI mice. No significant effect was seen in 24 h parameters of BRL mice. For graphical representation and data analysis details see above.

FIGURE 2

The effect of a single intra‐peritoneal injection of BRL37344 3 mg/kg was similar to that caused by BRL37344 1 mg/kg. 16 X‐NDI mice were individually placed in metabolic cages. For 3 days, 11 received a single i.p. injection of BRL37344 3 mg/kg (BRL), whereas 5 control animals received saline alone (CTR) (protocol 2). (A) Urine samples were collected 3 h after injection (I urine collection), 5 h after the I collection (II urine collection) and again 16 h later (III urine collection). The whole pie represents 24 h and each part of the whole proportionally indicates the duration of the urine collection time windows. In particular, the black part corresponds to the current urine collection, the grey part to the sample collection already done and the white part to the missing collection. All data of urine output and osmolality and water intake were expressed, for each mouse, as a percentage setting the average of values measured in the 5 days preceding the experiment (baseline, not represented in the plot) as 100%. In all scatter plots, data were given as mean ± SEM and each dot represents the mean effect of the injection in 3 days of experiments on different parameters for each mouse. The missing dots were due to the lack of urine production by some mice. (B) Urine output and urine osmolality in the three time windows in CTR and BRL X‐NDI mice. BRL37344 i.p. injection reduced urine output and increased urine osmolality during the first 3 h (I). The antidiuretic effect of BRL37344 was not observed in the subsequent urine collections (II, III). Significant differences between CTR and BRL were tested by two‐tailed unpaired t‐test. *p < 0.05, **p < 0.01, ****p < 0.0001. Significant differences between data measured during the monitoring (baseline) and data collected during the treatment, for both CTR and BRL, tested by two‐tailed paired t‐test, were reported in the text. (C) 24 h urine output and osmolality and water intake of CTR and BRL X‐NDI mice. No significant effect was seen in 24 h parameters of BRL mice. For graphical representation and data analysis details see above.

FIGURE 3

Multiple repeated intra‐peritoneal injections of BRL37344 1 mg/kg improved urine concentrating ability in X‐NDI mice. 16 X‐NDI mice were individually placed in metabolic cages. 11 X‐NDI mice received every 4 h 6 i.p. injections (showed by arrows) of BRL37344 1 mg/kg (BRL), while 5 received saline alone (CTR) (protocol 4). (A) Urine samples were collected every 4 h after injections (I, II, III, IV, V, VI urine collections). The whole pie represents 24 h and each part of the whole proportionally shows the duration of the urine collection time windows. The black part corresponds to the current urine collection, the grey part to the sample collection already done and the white part to the missing collection. All data of urine output, osmolarity and water intake were expressed, for each mouse, as a percentage setting the average of values measured, in the same time windows, in the 5 days preceding the experiment (baseline, not represented in the plot) as 100%. In all scatter plots data were given as mean ± SEM and each dot represents a mouse. The missing dots were due to the lack of urine production by some mice. (B) Urine output and urine osmolality in the six time windows in CTR and BRL X‐NDI mice. All BRL injections significantly reduced urine output and increased urine osmolality compared to vehicle injections in CTR mice. Significant differences between CTR and BRL were tested by two‐tailed unpaired t‐test. *p < 0.05, ***p < 0.001 ****p < 0.0001. Significant differences between data measured during the monitoring (baseline) and data collected during the treatment, for both CTR and BRL, tested by two‐tailed paired t‐test, were reported in the text. (C) 24 h urine output and osmolality and water intake of CTR and BRL X‐NDI mice. 6 BRL37344 injections promoted a huge water and solutes reabsorption: 24 h urine output and water intake were reduced by ≈30% and ≈20% respectively and in parallel, 24 h urine osmolality was increased of about ≈30% in BRL mice compared to CTR mice. For graphical representation and data analysis details see above. Significant differences between CTR and BRL were tested by two‐tailed unpaired t‐test. **p < 0.01 ****p < 0.0001.

FIGURE 4

Multiple repeated intra‐peritoneal injections of BRL37344 1 mg/kg (protocol 4) did not change X‐NDI mice's habits. To exclude the possible side effects of multiple BRL injections on the physiological activities of animals (drowsiness, lethargy, malaise, etc.), we compared the urine volume and osmolality of each collection, expressed as a percentage of urine output and osmolality of 24 h (100%), of CTR with those from BRL mice. Even though at each collection, except for the fourth, the urine volume produced by BRL‐treated mice was always statistically reduced and urine osmolality always increased compared to CTR mice, the urine production at each collection followed the same trend between CTR and BRL mice, with a peak in the night hours (10 PM–2 AM, IV collection) when the animals were more active. Significant differences between CTR and BRL were tested by two‐tailed unpaired t‐test. Data were given as mean ± SEM and each dot represents a mouse. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Multiple repeated BRL37344 1 mg/kg i.p. injections promoted NKCC2 activation in the thick ascending limb of X‐NDI mice. (A) At the end of the 6 i.p injections/24 h experiment, a quantitative reverse transcription polymerase chain reaction was performed on kidneys from X‐NDI treated with saline (CTR, n = 4) or with BRL37344 (BRL, n = 8). Relative quantification of gene expression (RQ) was performed setting the amount of NKCC2 mRNA in CTR as 1. No differences were seen in NKCC2 transcription between the two groups. The experiment was repeated three times and comparable results were obtained. In the scatter plot data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. (B) Western blotting with anti‐NKCC2 and antiphosphorylated NKCC2 antibodies was carried out using homogenates prepared from whole kidneys of CTR (n = 3) and BRL (n = 9) mice. Representative lanes were reported in the figure. The expression levels of each protein were normalized to total protein content using Stain‐free™ gels technologies. Densitometric analysis showed a two‐fold increase in pNKCC2 (active form), normalized to total NKCC2, in BRL compared to CTR mice. No differences were seen in NKCC2 abundance. The experiment was repeated three times and comparable results were obtained. In the scatter plot, data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. ****p < 0.0001 with two‐tailed unpaired Student's t‐test (C) Kidneys from CTR and BRL mice were subjected to immunofluorescence localization of NKCC2 (in green) and pNKCC2 (in green) and counterstained with Evans blue (red) (CTR = 3, BRL = 5). The number of NKCC2‐positive TAL cells and the localization of NKCC2 were similar in two groups but the fluorescence intensity of pNKCC2 was increased in BRL mice. Representative images were shown. The experiment was repeated three times and comparable results were obtained. (bar = 25 μm).

FIGURE 6

Multiple repeated BRL37344 1 mg/kg i.p. injections promoted NCC activation in the distal convolute tubule of X‐NDI mice. (A) After 6 i.p injections/24 h, quantitative reverse transcription polymerase chain reaction was performed on kidneys from X‐NDI treated with saline (CTR, n = 4) or with BRL37344 (BRL, n = 8). NCC relative quantification indicated that the treatment with BRL did not affect NCC transcription. Comparable results were obtained from three independent experiments. In the scatter, plot data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. (B) Western blotting analysis of total and phosphorylated forms of NCC on total kidney homogenates from CTR (n = 3) and BRL (n = 9) mice revealed strong activation of NCC (pNCC) in BRL mice with no differences in NCC abundance between the two groups. Representative lanes were reported in the figure. Densitometric analysis showed the expression levels of NCC and pNCC normalized to total protein content using Stain‐free™ gels technologies. The experiment was repeated three times and comparable results were obtained. In the scatter plot data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. **p < 0.01, ***p < 0.001 with two‐tailed unpaired Student's t‐test. (C) kidney sections from CTR (n = 3) and BRL (n = 5) mice, counterstained with Evans Blue (in red), were subjected to immunofluorescence localization of NCC and pNCC (in green). BRL injections did not change the expression levels and localization of NCC but increased pNCC apical expression in DCT cells. The experiment was repeated three times and comparable results were obtained. (bar = 25 μm).

FIGURE 7

Multiple repeated intra‐peritoneal injections of BRL37344 1 mg/kg promoted AQP2 phosphorylation at Ser256 increasing its expression at the apical plasma membrane of principal cells in the collecting ducts of X‐NDI mice. (A) Real‐time RT‐PCR indicated that 6 i.p. injections of BRL37344 did not change renal AQP2 gene transcription levels (CTR = 4, BRL = 8). Comparable results were obtained from three independent experiments. In the scatter plot, data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. (B) Western blotting analysis showed no statistically significant differences in total AQP2 but a clear increase of AQP2 phosphorylation at Ser256 in total kidney lysates BRL (n = 9) versus CTR mice (n = 3). Densitometric analysis showed the expression levels of AQP2 and pAQP2 normalized to total protein content using Stain‐free™ gels technologies. Comparable results were obtained from three independent experiments. In the scatter plot, data were given as mean ± SEM and each dot represents the average of data from three experiments for each mouse. **p < 0.01, ***p < 0.001 with two‐tailed unpaired Student's t‐test. (C) Kidney sections from CTR (n = 3) and BRL (n = 5) mice, counterstained with Evans Blue (in red), were subjected to immunofluorescence localization of AQP2 and pAQP2 (in green). BRL injections clearly increased AQP2 phosphorylation and promoted its accumulation at the apical membrane of collecting ducts principal cells. The experiment was repeated three times and comparable results were obtained (bar = 25 μm).