Improving treatment and imaging in ADPKD
van Gastel, Maatje Dirkje Adriana
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progression in experimental PKD
Maatje D.A. van Gastel
Xiaofang Wang
Belle M. Melsert
Megan Constans
Hong Ye
Dorien J.M. Peters
Ron T. Gansevoort
Vicente E. Torres
Esther Meijer
In preparationABSTRACT
Background: Vasopressin, involved in the pathophysiology of polycystic kidney disease
(PKD), increases in response to an increase in plasma osmolality, of which sodium is the most important osmole. Blockade of the vasopressin V2 receptor by an antagonist has shown to ameliorate the rate of disease progression. However, aquaretic side-effects are common and can lead to treatment discontinuation. We hypothesized that lowering sodium intake decreases the rate of disease progression in PKD and improves the reno-protective efficacy of vasopressin V2 receptor antagonists. As less sodium intake leads to less osmolar excretion, it could also lower the aquaretic side-effects of vasopressin V2 receptor antagonists.
Methods: We studied the effect of sodium restriction in two murine models of PKD, a
rapidly progressive and a slowly progressive model. Mice of the tamoxifen-inducible kidney epithelium-specific Pkd1-deletion rapid progressive mouse model were treated for 3 weeks (from day 21 to 42 of age) with either a low sodium (LS: 0.39 g/kg) or high sodium (HS: 15.21 g/kg) diet, with or without the addition of 0.1% mozavaptan (Shanghai PI Chemicals). As slowly progressive model the hypomorphic Pkd1R3277C/R3277C-mouse on a 129s6 background was used, who received low (0.39 g/kg), normal (3.90 g/kg) or high (15.21 g/kg) sodium diets with or without the addition of 0.1% tolvaptan (Otsuka Pharma-ceutical) during 13 weeks (from day 28 to 119 of age).
Results: A low sodium diet did not ameliorate the rate of disease progression in the
rapidly progressive model of Pkd1, but resulted in better disease outcome in the slowly progressive model. We found no effect on disease progression with the vasopressin V2 receptor antagonists in both models, in contrast to previous animal and human studies. We did observe increased water intake after vasopressin V2 receptor antagonist treat-ment in both studies (both P<0.001), and found that a low sodium diet reduced aquaresis by 23.2% and 20.6% compared to normal and high sodium intake in the slowly progressive model of Pkd1.
Conclusions: Treatment with a vasopressin V2 receptor antagonist did not decrease
dis-ease progression in both murine models of PKD. Long-term treatment with a low sodium diet ameliorated disease progression in a slowly progressive mouse model of PKD, and reduced the aquaretic side-effects of treatment while using a vasopressin V2 receptor antagonist.
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INTRODUCTION
Vasopressin is known to play a pathophysiological role in autosomal dominant polycys-tic kidney disease (ADPKD). In a rat model that phenotypically resembles human AD-PKD it has been shown that vasopressin directly regulates cyst growth. Blockade of the vasopressin V2 receptor using tolvaptan was found to ameliorate the rate of disease progression, with lower rates of total kidney growth and glomerular filtration decline in 1,445 patients with ADPKD1. Blocking the vasopressin V2 receptor downstream inhibits AQP2 phosphorylation, and thus the ability of the kidney to retain water. This resulted in aquaretic side-effects in the majority of patients on tolvaptan, and discontinuation of treatment in 8.3% in the TEMPO 3:4 trial1.
Lowering the vasopressin concentration in patients with ADPKD might be of interest, as this might directly ameliorate the rate of disease progression, but potentially can also im-prove tolvaptan treatment efficacy and lower the aquaretic side-effects observed while using this drug. Vasopressin is secreted in response to an increased plasma osmolality and lowered blood volume2,3. Plasma osmolality, in turn, is predominantly dependent on the plasma osmolality and fluid intake. A recent phase 2 study in patients with ADPKD, indeed confirmed that a low osmolar with high fluid dietary intervention indeed lowered copeptin concentrations in ADPKD4. However, as different elements are studied at once, it remains unclear which part of the dietary intervention was responsible for this low-ered copeptin concentration.
Sodium is one of the main determinants of plasma osmolality, and sodium intake was shown to be associated with vasopressin concentration (measured as its surrogate co-peptin) cross-sectionally in a healthy population cohort5.
In this line of reasoning, where a lowered sodium intake lowers vasopressin concentra-tion, a sodium restricted diet might slow disease progression in ADPKD. It, furthermore, might positively affect the treatment efficacy of a vasopressin V2 receptor antagonist, as less agonist (vasopressin) while receiving the same dose of antagonist (tolvaptan) could hypothetically have a positive effect on tolvaptan efficacy.
Furthermore, lowering sodium intake in combination with vasopressin V2 receptor an-tagonism very likely affects urine production, as vasopressin V2 receptor anan-tagonism blocks the urine concentrating capacity of the kidney. In normal physiology, a broad range of sodium (and other osmolar) intake can be compensated by increasing the urine sodium concentration6. As concentration of the urine is no longer possible while using a vasopressin V2 receptor antagonist, an increased intake of sodium will lead to increased
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urine production.
We hypothesized that a sodium restricted diet leads to less disease progression in AD-PKD, that it increases the renoprotective efficacy of a vasopressin V2 receptor antagonist and lowers its aquaretic side-effects. We investigated these hypotheses in two mouse models that resemble human ADPKD, one with rapidly and the other with slowly pro-gressive disease.
METHODS
Study design
The experiment under study was performed in two murine models of polycystic kidney disease (PKD), referred to as the UMCG (University Medical Center Groningen, Gro-ningen, the Netherlands) and MC (Mayo Clinic, Rochester, MN, USA) studies. We first investigated the effects of sodium restriction and addition of vasopressin V2 receptor antagonist mozavaptan (OPC-31260, Shanghai PI Chemical Ltd.) on disease progression in a rapidly progressive model of PKD in the UMCG study. After observing no beneficial effects of both sodium as well as mozavaptan treatment, we decided to repeat the study in a more slowly progressive model of PKD, where we investigated the effects of sodium modification and addition of vasopressin V2 receptor antagonist tolvaptan (OPC-14041, Otsuka Pharmaceutical Co., Ltd.), the MC study.
The UMCG study; rapidly progressive PKD
In this study the tamoxifen-inducible kidney epithelium-specific Pkd1 deletion mouse model (tam-KspCad-CreER72;Pkd1lox2-11/lox2-11) was used. Upon administration of tamoxifen to these mice, a genomic fragment containing exons 2-11 of the Pkd1 gene is specifically deleted in renal epithelial cells and cysts are formed. This model has been described pre-viously7-9. We administered tamoxifen per gavage (43 μg dissolved in ethanol, adminis-tered in a volume of 36 μL sunflower oil; Sigma-Aldrich, St Louis, MO) for three consecu-tive days to mice, starting at postnatal day 10. Thirty tam-KspCad-CreER72;Pkd1lox2-11/lox2-11 mice did not receive tamoxifen and served as healthy controls. Sodium diets, low (LS) and high (HS) consisted of respectively 0.39 g Na+/kg (0.1% NaCl) and 15.21 g Na+/kg (4.0% NaCl). Half of the Pkd1 mice received mozavaptan (1 g/kg – 0.1%) in their ground rodent chow, by homogenous mixing. Sodium as well as drug treatment was started at day 21 of age and lasted three consecutive weeks. Animals were sacrificed on day 42 of age. Spot urine was collected on three occasions during the last 24 hours prior to sacrifice. All ex-periments were approved by the local animal experimental committee of the Groningen University Medical Center and by the Commission Biotechnology in Animals of the Dutch 144
Ministry of Agriculture.
Mayo Clinic study; slowly progressive PKD
Here the Pkd1RC/RC mice on a 129s6 background were used. Mice were treated with a low (0.39g/kg, 0.1% NaCl), ‘normal’ (3.9 g/kg, 1.0% NaCl) or high (15.21 g/kg, 4.0% NaCl) sodium diet. Half of the groups received tolvaptan mixture (1 g/kg – 0.1%), that contains tolvap-tan and hydroxypropylcellulose at a ratio of 2:1, leading to a concentration of 0.066% of tolvaptan, This was added to their ground rodent chow, by homogenous mixing. Sodium as well as drug treatment was started at day 28 of age and lasted 13 weeks. Animals were put in a metabolic cage for 24 hours in the week prior to sacrifice and were sacrificed at day 119 of age. To additionally test urine production with and without tolvaptan use in 24 hours, three mice with and three mice without tolvaptan treatment were kept in a 24-hour metabolic cage with measurement of urine collection every three hours. The experimental protocol was approved by the Mayo Clinic Institutional Animal Care and Utilization Committee.
Experimental protocol of both studies
Littermates of the same sex were sorted into different treatment groups. They were weighed on the day of the start of the experiment and twice weekly to monitor their health. Water intake was monitored throughout the experiment by weighing all water bottles of all cages before and after replacement. At the day of sacrifice, the animals were weighed and anesthetized with isoflurane gas (0.5% isoflurane with a flow of 0.6 L/ min) or intraperitoneal ketamine (60 mg/kg) and xylazine (10 mg/kg), for the UMCG and MC study respectively. Blood was obtained by cardiac puncture. Both kidneys were re-moved and their weight was measured on a precision scale. Half of the right kidney was placed into formaldehyde, to be embedded in paraffin for histomorphometry.
Chemical determinations
Plasma urea, creatinine, glucose and electrolytes were measured with a Modular P (Roche) or pHOx-Ultra Analyzer (Nova), for the UMCG and MC study respectively. For the UMCG study urinary osmolality was calculated as 2 * (urinary sodium concentration + urinary potassium concentration) + urinary urea concentration10 and plasma osmolality was calculated as: 1.9 * (plasma sodium concentration + plasma potassium concentra-tion) + plasma glucose concentration + plasma urea concentration * 0.5 + 511. Plasma co-peptin was measured by ELISA (ELISA Kit for Coco-peptin (CPP); BioConnect Diagnostics)12. Histomorphometric analysis
Transverse tissue section (4 μm), including cortex, medulla and papilla, were stained with hematoxylin-eosin to measure total area of cysts. The total area of the cysts and of 145
fibrosis was quantified using Aperio Image Scope software (version 12.1.0.5029; Aperio Technologie Inc., Vista, CA). Cyst ratios were expressed as a percentage of total tissue. The investigator performing these measurements was blinded to disease and treatment status.
Statistical analysis
Differences between two groups were tested using a non-parametric independent sam-ple test (Mann-Whitney). Data are expressed as medians with interquartile ranges. Anal-yses were performed with IBM SPSS Statistics version 22.0.
RESULTS
Effects of sodium restriction on disease progression and mozavaptan treatment in rapidly progressive PKD
In the UMCG study, 125 mice (77 male and 48 female) were included. The percentage males and body weight at the start of the experiment was similar in all groups (Figure 1, Table 1). Two-way ANOVA analysing the effects of sex, diet and mozavaptan treatment on the outcome measures revealed that the associations for the outcome parameters did not differ between male and female mice. Sodium restriction did not ameliorate the rate of disease progression in this rapidly progressive model of PKD (Figure 1, Table 1), with no significant differences between the cyst ratios, plasma creatinine, total kidney adjusted for body weight (TKW/BW%)-ratio (P all NS). Body weight was significantly low-er in high sodium treated mice (P<0.001). No difflow-erences wlow-ere obslow-erved between any of the treatment groups regarding plasma copeptin concentration (Table 1). When compar-ing all mozavaptan treated with all not mozavaptan treated Pkd1 mice we observed a significantly lower body weight at sacrifice and lower TKW (Table 1). After adjustment for body weight the TKW was no longer significantly different between mozavaptan and not mozavaptan treated animals (P=0.8, Table 1). Plasma sodium was higher after moza-vaptan treatment (P<0.001 and P=0.04 respectively), and all urine analyses were signifi-cantly different (Table 1). The effect of a low sodium diet on the aquaretic side-effects of mozavaptan was analysed through measurement of water intake throughout the whole experiment. Mozavaptan treatment led to an increased water intake (Table 1, Figure 3). The water intake while on mozavaptan was not significantly different between low ver-sus high sodium diet in the mozavaptan treated group (P=0.2). However, water intake was lower while on a low sodium diet in untreated Pkd1 mice, as well as in the healthy controls, with reduced water intakes of 14.2% and 8.8%, respectively (both P<0.001).
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Effects of sodium restriction on disease progression and tolvaptan treatment in slowly progressive PKD
120 Pkd1 mice (62 male and 58 female) were included in the MC study, with equal distri-bution of sexes across all groups. Body weights were similar in all groups at the start, as well as the end of the experiment with no significant differences between any of the groups. Two-way ANOVA analysing the effects of sex, diet and tolvaptan treatment on the outcome measures revealed that sex influenced the majority of outcomes of the variables, but no significant interactions were observed. Sodium restriction ameliorated disease progression, with lower total kidney volumes, both adjusted and unadjusted for body weight (P=0.03 and P=0.02, respectively) and borderline significantly lower cyst ratios (P=0.07). It did not ameliorate the rate of disease progression on plasma markers, as there was no difference in plasma urea or creatinine concentration (P=0.5, P=0.8 and P=0.9, respectively). Body weight was not significantly different. Tolvaptan treatment had no effect on the body weight at sacrifice. It had no effect on plasma creatinine, or TKW as well as TKW adjusted for body weight (Table 2). Plasma sodium was significantly lower after tolvaptan treatment in the low sodium treated group. Tolvaptan led to a significant increase in water intake in all sodium treated groups (all P<0.001). Sodium restriction led to a reduction in urine production of 20.6% on a low compared to a high sodium diet. Without tolvaptan treatment, urine production was on average reduced with 30.5% on a low compared to a high sodium diet. Analysing urine production throughout the course of the experiment, one can appreciate that polyuria reduces after long-term tolvaptan treatment (Figure 3). When analysing urine production during 24 hours in mice with and without tolvaptan (Figure 4), we discov-ered that tolvaptan treatment only leads to increased urine production throughout the night (P=0.005), whereas no difference was observed throughout the day (P=0.3).
DISCUSSION
We studied the effect of low sodium intake and this combined with vasopressin V2 re-ceptor antagonists in two models of Pkd1 mice, models for human ADPKD with a muta-tion in the PKD1 gene. A low sodium diet ameliorated the rate of disease progression measured as total kidney growth and borderline significantly cyst ratio in the murine model of slowly progressive PKD. A low sodium diet also led to reduced aquaresis and urine production in this model, i.e. less aquaretic side-effects of tolvaptan treatment. No treatment effect of the vasopressin V2 receptor antagonists was observed on the rate of disease progression in both studies.
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Sex stratified quartiles of plasma copeptin concentration (pmol/L) Healthy controls Pkd1 Pkd1 +mozavaptan Low sodium High sodium Low sodium High sodium Low sodium High sodium W eight day 21 (g) 8.2 (7.1 – 9.0) 8.0 (7.7 – 8.6) 8.5 (7.5 – 9.0) 7.8 (7.0 – 8.5) 8.3 (7.8 – 8.6) 7.9 (7.2 – 8.3) W eight day 42 (g) 16.6 (16.4 – 18.2) 15.8 (14.2 – 17.3) 17.2 (14.8 – 18.0) × 14.6 (13.6 – 16.0) 14.7 (13.2 – 15.8) *× 13.2 (12.2 – 14.2) * W ater intake/mouse/ week (g) 19.4 (18.2 – 20.0) *× 22.6 (21.1 – 26.8) * 26.0 (20.0 – 28.4) 28.5 (26.6 – 37.0) 127.6 (102.4 – 130.5) * 120.5 (107.6 – 141.4) *
Total kidney weight (mg)
235 (204 – 247) * 227 (206 – 271) * 415 (361 – 448) × 356 (320 – 399) 351 (337 – 395) *× 309 (284 - 344) * TKW/BW (%) 1.34 (1.26 – 1.43) *× 1.53 (1.43 – 1.59) * 2.46 (2.03 – 2.77) 2.52 (2.13 – 2.88) 2.42 (2.07 – 2.72) 2.44 (2.18 – 2.52) Cyst ratio (%) 4.5 (2.5 – 6.7) * 5.9 (4.3 – 9.5) * 26.8 (16.8 – 29.4) 29.6 (20.5 – 34.6) 29.3 (21.2 – 34.0) 24.3 (16.1 – 29.7)
Plasma copeptin (pmol/L)
20.4 (16.6 – 24.1) 21.3 (19.7 – 26.5) 22.6 (18.0 – 26.0) 23.6 (18.6 – 29.2) 21.6 (19.3 – 25.1) 22.2 (19.2 – 26.5)
Plasma sodium (mmol/L)
147 (146 – 148) 146 (145 – 148) 147 (143 – 150) 147 (145 – 149) 149 (147 – 156) * 153 (150 - 156) *
Plasma creatinine (μmol/L)
7.0 (7.0 – 9.0) * 6.0 (6.0 – 8.0) * 11.0 (9.0 – 13.0) 9.0 (7.0 -12.0) 12.5 (10.0 – 15.0) 10.0 (8.8 – 12.3)
Plasma urea (mmol/L)
10.0 (8.1 – 11.5) * 10.0 (9.4 – 10.9) * 19.8 (16.1 – 24.6) 19.0 (16.7 – 26.4) 16.8 (13.5 – 21.2) 19.8 (15.6 – 23.7)
Plasma osmolality (mOsm/L)
331 (329 – 335) * 328 (326 – 332) * 342 (330 – 350) 339 (334 – 343) 343 (334 – 353) 344 (339 – 357) *
Urine sodium (mmol/L)
58 (34 – 59) *× 197 (110 – 292) * 10 (10 – 14) × 120 (61 – 180) 10 (10 – 10) × 23 (12 – 32) *
Urine creatinine (μmol/L)
4.9 (2.6 – 5.5) * 2.2 (2.2 – 2.8) * 1.3 (0.7 – 1.9) 1.0 (0.5 – 1.5) 0.3 (0.1 – 0.6) * 0.3 (0.1 – 0.4) * Table 1. Sacrifice data (day 42) of the ra pidly progressive Pkd1-mice after dietary sodium modification and mozavaptan treatment
in the UMCG study
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Table 1. Continued Data are shown as median (interquartile range). Significance was tested using a Mann-Whitney U test; * states a significant diff erence between the PKD group and HC or PKD V2RA gro up of the same dieta ry intervention; × states a significant diff erence when comparing LS and HS within one modality . Abbreviations: HC: healthy control, PK D: polycystic kidney disease,
LS: low sodium, HS: high sodium, TKW/
BW : total kidney weight / body weight, S/C: sodium/creatinine, U/C: urea/creatinine.
Sex stratified quartiles of plasma copeptin concentration (pmol/L)
Healthy controls Pkd1 Pkd1 +mozavaptan Low sodium High sodium Low sodium High sodium Low sodium High sodium Urine S/C (mmol/μmol) 12.0 (10.5 – 14.8) 89.5 (56.1 – 104.3) 12.5 (7.7 – 15.8) 113.3 (75.0 – 200.0) 33.3 (16.7 – 100.0) * 69.6 (42.5 – 207.5)
Urine U/C (mmol/μmol)
422 (409 – 422)
508 (402 – 669)
496 (407 – 683)
570 (433 – 1,165)
805 (620 – 960) *
Urine osmolality (Osm/L)
1352 (1352 – 1979) * 584 (499 – 1006) 857 (495 – 978) 221 (180 – 343) * 245 (219 – 274) * 149
7
Sex stratified quartiles of plasma copeptin concentration (pmol/L) Pkd1 Pkd1 +tolvaptan Low sodium Normal sodium High sodium Low sodium Normal sodium High sodium W eight day 28 (g) 15 (13 – 16) 15 (14 – 17) 15 (14 – 16) 15 (13 – 17) 15 (13 – 17) 15 (15 – 17) W eight day 119 (g) 27 (25 – 34) 30 (28 – 34) 29 (26 – 32) 27 (25 – 33) 28 (26 – 32) 28 (25 – 31) W ater intake/mouse/ week (g) 25.1 (23.2 – 29.2) 25.8 (24.4 – 29.3) 36.1 (32.1 – 38.3) × 55.3 (55.3 – 55.3) * 58.0 (49.1 – 58.5) * 69.6 (65.9 – 82.8) *×
Total kidney weight (mg)
589 (419 – 884) × 836 (627 – 1,162) 738 (618 – 953) 680 (569 – 1,084) 808 (526 – 985) 739 (554 – 980) TKW/BW (%) 2.18 (1.51 – 2.96) × 2.62 (2.15 – 3.61) 2.61 (2.41 – 3.02) 2.51 (1.96 – 3.54) 2.64 (2.07 – 3.30) 2.73 (2.14 – 3.24) Cyst ratio (%) 44.5 (39.5 – 48.3) × 49.4 (44.9 – 54.9) 44.9 (39.9 – 54.4) 40.8 (36.2 – 51.5) 46.2 (39.8 – 51.3) 42.6 (37.8 – 47.2)
Plasma sodium (mmol/L)
142 (140 – 145) 143 (141 – 144) 143 (141 – 146) 142 (138 – 145) × 146 (145 – 148) * 145 (141 – 147)
Plasma creatinine (μmol/L)
44.2 (35.4 – 53.0) 53.0 (44.2 – 53.0) 39.8 (24.3 – 64.1) 44.2 (30.9 – 66.3) 53.0 (8.8 – 79.6) 44.2 (8.8 – 53.0)
Plasma urea (mmol/L)
28 (26 – 32) 27 (18 – 30) 25 (18 – 29) 22 (20 – 25) * 25 (21 – 32) 22 (20 – 25)
Plasma osmolality (mOsm/L)
308 (304 – 316) 308 (301 – 312) 303 (302 – 311) 307 (298 – 318) 317 (305 – 320) 307 (299 – 313) Table 2. Sacrifice data (day 119) of the slowly progressive Pkd1-mice after dietary sodium modification and tolvaptan treatment in
the MC study Significance
betwee n the two study gro ups was tested using a Mann-Whitney U test.* states a significant diff erence compared to the same modality of the not V2RA trea ted group . × states a significant diff erence compared to the normal sodium group of the same treatment modality . Abbreviations: TKW/BW , total kidney weight / body weigh t-ratio; LS, low sodium; NS, normal sodium; HS, high sodium; PKD, polycystic kidney disease; V2RA, vasopressin V2 receptor antagonist treated. creatinine. 150
7
Figure 1. Effects of the sodium diets as well as addition of mozavaptan per treatment group on the development of a rapidly progressive model of polycystic kidney disease in Pkd1-mice. Data collected on the day of sacrifice (day 42). Data are expressed as median with interquartile ranges. * states a significant difference between the PKD group and HC or PKD V2RA group of the same dietary intervention; × states a significant difference when compar-ing LS and HS within one modality. Abbreviations: HC, healthy control; PKD, polycystic kid-ney disease; LS, low sodium; HS, high sodium, TKW/BW, total kidkid-ney weight / body weight.
0 5 10 15 20 Body weight * � * � gr am s 0.0 0.1 0.2 0.3 0.4 0.5 Kidney weight * * * * � � gr am s 0 1 2 3 4Kidney weight * *� % B W 0 50 100 150 Water intake � � * * * * m ou se/ w eek ( gram s) 0 10 20 30 40 Cyst ratio * * B W (% ) 0 10 20 30 40Plasma copeptin pg /m L 0 5 10 15 20Plasma creatinine * * m m ol/L 120 130 140 150 160 170Plasma sodium * * m m ol /L 0 100 200 300 400Plasma osmolality * * m Os m /L 0 2 4 6Urine creatinine* * * * mmol/L 0 50 100 150 200 250 Urine sodium/creatinine * m m ol/ m mo l 0 500 1000 1500 2000 2500Urine osmolality * * * mO sm /L HCLS HCHS PKDLS PKDHS PKDLSV2RA PKDHSV2RA 151
7
Figure 2. Effects of the sodium diets as well as addition of tolvaptan per treatment group on the development of a slowly progressive model of polycystic kidney disease in Pkd1-mice. Data collected on the day of sacrifice (day 119). Data are expressed as median with interquartile ranges. * states a significant difference compared to the same modality of the not V2RA treated group. × states a significant difference compared to the normal sodium group of the same treatment modality. Abbreviations: TKW/BW, total kidney weight / body weight-ratio; LS, low sodium; NS, normal sodium; HS, high sodium; PKD, polycystic kidney disease; V2RA, vasopressin V2 receptor antagonist treated.
0 10 20 30 40 50 Body weight gr am s 0.0 0.5 1.0 1.5Kidney weight � gr am s 0 1 2 3 4 5Kidney weight � % B W 0 20 40 60 Cyst ratio (% ) 0 20 40 60 80 100 Water intake * * *� � m ous e/ w eek ( gr am s) 0 20 40 60 80 100Plasma creatinine mm ol/L 125 130 135 140 145 150Plasma sodium* � m m ol/L
LS NS HS LSV2RA NSV2RA HSV2RA
0 100 200 300 400Plasma osmolality m O sm /L 152
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Figure 3. Urine volume, as surro gate for water intake, throughout the rapidly progressive (left panel) and slowly progressive (right panel) experiments. Wee k 1 Wee k 2 Wee k 3 0 25 50 75 100 125 150 HC L S HC HS PKD L S PKD HS PKD L S V2 R A PKD HS V 2RA urin e vo lum e/w eek (gr ams) Wee k 1 Wee k 2 Wee k 3 Wee k 4 Wee k 5 Wee k 6 Wee k 7 Wee k 8 Wee k 9 Wee k 10 Wee k 11 Wee k 12 Wee k 13 0 100 200 300 400 500 HC L S HC HS PKD HS PKD L S V2 R A PKD NS V 2RA PKD HS V 2RA urin e vo lum e/w eek (gr ams) 153
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Figure 4. 24-hour urine production in Pkd1-mice on tolvaptan versus control treatment. The grey box indicates night time, when the lights are off.
6-9pm 9p m-12am12-3am 3-6am 6-9am 9am-12 pm 12-3p m 3-6pm 0 5 10 15 Control Tolvaptan
ur
ine
v
ol
um
e
(g
ra
m
s)
We were surprised to find no effect of V2RA treatment, as this is contrary to current lit-erature. We have three hypotheses that could explain why we did not find any treatment effects. First of all, it could be due to an insensitivity for vasopressin of the two mouse models, as also is indicated by the fact that the efficacy of vasopressin V2 receptor antag-onism has been shown repeatedly in various animal studies and is accepted as treatment for ADPKD1,13-15. Indications to support this hypothesis have previously been reported. Hopp et al. reported that enhanced hydration did not affect disease progression, nor uri-nary vasopressin excretion in a Pkd1 model very similar to our slowly progressive model, whereas in PCK-rats in that study a treatment effect and lowered urinary vasopressin concentrations were observed16, as one would expect via hydrations physiological effect on plasma osmolality and consequently vasopressin release. On the contrary, it is known of our slowly progressive Pkd1 mouse model that they have aquaporin-2 expression on the cysts, indicating that the cysts derive from the collecting duct and that the vasopres-sin V2 receptor is present on these cells, leading to believe that they are sensitive to vas-opressin. The second hypothesis is that we did not give them adequate treatment. Mice is both studies developed a phenotype of polycystic kidney disease Pkd1, shown by in-creased kidney weights and cyst ratios compared to healthy controls. We also observed the physiological effects of vasopressin V2 receptor antagonism in both models, namely an increased water intake and lowered urine osmolality. In the slowly progressive mouse model we observed the expected increase in water intake on a combined high sodium and tolvaptan treatment. This might be due to a changed bioavailability of the V2RA 154
we received compared to that previously used. The third hypothesis for not finding any vasopressin V2 receptor antagonist treatment effect could be that there was insufficient 24-hour blockade of the vasopressin V2 receptor. Mice only eat during the night, and es-pecially during the first few hours after dark. As the vasopressin V2 receptor antagonist is mixed with ground chow, this could potentially lead to insufficient drug concentra-tions throughout the day, and thus insufficient blockade of the vasopressin V2 receptor throughout the day. A study of Aihara, Yamamura et al. who treated mice with different dosages tolvaptan mixed through standard chow found that there is a time and dose dependent plasma tolvaptan concentration17. At a concentration of 0.1% the tolvaptan concentration in the morning is 146.0 ± 18.1 ng/ml, for 0.01% this is 7.2 ± 1.8 ng/ml. The effective tolvaptan dosage is around 20 ng/ml. Unpublished data of this study showed that in the evening 0.01% tolvaptan results in an undetectable low plasma tolvaptan con-centration, whereas 0.1% remains right above the efficacy level with 49.3 ± 31.5 ng/ml. With our effective dosage of 0.066% tolvaptan we might have given a too low dosage to sustain an effective plasma concentration throughout the day. This hypothesis was strengthened when we analysed 24-hour urine production every three hours in mice with and without tolvaptan treatment. The latter group had significant higher urine vol-umes during the first hours at night, whereas no differences were observed later at night and throughout the day, indicating that mice only had tolvaptan and polyuria in a limited time of the day. As blockade of the vasopressin V2 receptor leads to increased plasma vasopressin concentrations18, one can appreciate that during the hours where the vaso-pressin V2 receptor is not adequately blocked, the receptor is even exposed to higher vasopressin concentrations compared to normal. Higher vasopressin concentrations are known to aggravate disease progression in PKD19.
We found a beneficial effect of sodium restriction in the slowly progressive model, but not in the rapidly progressive murine model. The rapidly progressive model might be so rapidly progressive that lifestyle modification has no influence on the course of the disease. We hypothesize that the sodium reduction did lead to less disease progression via lowering circulating vasopressin concentrations. Unfortunately, vasopressin – or co-peptin – levels cannot be expected to be different because we only obtained blood at sacrifice, while the mice were stressed and anesthetized. Of both conditions it is known that they influence vasopressin concentration, as stress is known to directly increase vasopressin20 and isoflurane has recently shown to induce stress in rats, leading to in-creased corticosterone concentrations21.
These data, first of all, indicate that sodium restriction might ameliorate the disease pro-gression of PKD. Treating patients with a low sodium diet could directly affect disease 155
progression, and is in line with recently published papers. A post-hoc analysis of the HALT-PKD clinical trials showed that salt restriction ameliorated the rate of disease pro-gression in ADPKD22, and a pilot study by Amro et al. showed that a low osmolar com-bined with increased fluid intake dietary regime causes, like we hypothesized, a decrease in copeptin concentration in patients with PKD4. Because of the short-term follow-up, unfortunately no effects on disease progression could be studied. Second, we observed that a low sodium diet lowered aquaretic side-effects of vasopressin V2 receptor antago-nism and could thus increase tolerability of its treatment. Recently a study in patients with ADPKD also showed that urine volume on tolvaptan treatment was mainly depend-ent on osmolar and sodium intake (Kramers AJKD 2018, accepted for publication). Un-fortunately, no treatment effect of V2RA was observed and we could thus not conclude whether a low sodium diet affects V2RA treatment effect positively or negatively. This needs further study.
In conclusion, we found that in a slowly progressive murine model of Pkd1, a low sodium diet ameliorated disease progression and decreases polyuria in vasopressin V2 recep-tor antagonist treated mice. Because no treatment effect of V2RA was found, no effect of sodium on this could be studied. These data suggest that prescribing a low sodium diet to AFPKD patients using tolvaptan may be warranted, because it improves polyuria, which is the most prominent reason not to start tolvaptan or to stop this treatment. Whether a low sodium would affect the renoprotective efficacy of a V2RA remains to be studied.
ACKNOWLEDGEMENTS
None
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Novel renal imaging techniques to assess total kidney and liver volume in polycystic kidney disease
