Electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin V2-receptor-dependent calcium reabsorption ORIGINAL ARTICLE Electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin V2-receptor-dependent calcium reabsorption Krister Bamberga , Lena William-Olssonb , Ulrika Johanssonb, Anders Arnerc , Judith Hartleib-Geschwindnerd and Johan S€allstr€ome,f aTranslational Sciences and Experimental Medicines, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; bBioscience Renal, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; cDepartment of Clinical Sciences Lund, Lund University, Lund, Sweden; dProjects, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; eDepartment of Medical Cell Biology, Uppsala University, Uppsala, Sweden; fDepartment of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden ABSTRACT Background: The most profound effect of vasopressin on the kidney is to increase water reabsorption through V2-receptor (V2R) stimulation, but there are also data suggesting effects on calcium transport. To address this issue, we have established an isolated perfused kidney model with accurate pressure control, to directly study the effects of V2R stimulation on kidney function, isolated from sys- temic effects. Methods: The role of V2R in renal calcium handling was studied in isolated rat kidneys using a new pressure control system that uses a calibration curve to compensate for the internal pressure drop up to the tip of the perfusion cannula. Results: Kidneys subjected to V2R stimulation using desmopressin (DDAVP) displayed stable osmolality and calcium reabsorption throughout the experiment, whereas kidneys not administered DDAVP exhibited a simultaneous fall in urine osmolality and calcium reabsorption. Epithelial sodium channel (ENaC) inhibition using amiloride resulted in a marked increase in potassium reabsorption along with decreased sodium reabsorption. Conclusions: A stable isolated perfused kidney model with computer-controlled pressure regulation was developed, which retained key physiological functions. The preparation responds to pharmaco- logical inhibition of ENaC channels and activation of V2R. Using the model, the dynamic effects of V2R stimulation on calcium handling and urine osmolality could be visualised. The study thereby provides evidence for a stimulatory role of V2R in renal calcium reabsorption. ARTICLE HISTORY Received 6 June 2020 Revised 18 July 2020 Accepted 28 July 2020 KEYWORDS AVP; ENaC; kidney; vasopressin Introduction The most profound effect of vasopressin (AVP) on the kidney is to increase water reabsorption in the collecting duct medi- ated through vasopressin V2-receptors (V2R), but there are also data that indicate effects on calcium transport (1,2). The isolated perfused kidney model (reviewed in (3,4)) offers the advantage that test substances and their impact on kidney function can be studied in a more controlled way than in the intact animal. In the isolated organ model, perfusion pressure can be held constant, thereby minimising confound- ing secondary effects caused by blood pressure alterations occurring in the intact animal. Furthermore, the concentra- tion of electrolytes and pharmacological agents in the perfu- sion medium can be precisely controlled. Experiments on isolated kidney preparations can be performed using con- stant perfusion flow. In such preparations the pressure in the vascular system will change with the vessel tone in the kidney (5,6). Therefore, a pressure control system is used in many preparations in order to maintain a stable arterial pres- sure in the physiological range (3,7). In principle, these sys- tems have a pressure sensor that is connected to a feedback system that regulates the perfusion pump in order to keep the pressure constant. This introduces several technical chal- lenges. The pressure should be measured at the tip of the perfusion cannula in the renal artery, which requires a dual- lumen cannula. However, given the small size of the artery in mice and rats, it is difficult to place this relatively large can- nula in the renal artery. To overcome this, a short section of the aorta can be kept to serve as an adapter for the perfu- sion cannula (7). Another solution is to measure the pressure anywhere in the perfusion system at the level of the kidney. The problem with this approach is that the pressure fall in the fine perfusion cannula is significant and will be flow- dependent. Consequently, the kidney will be perfused with a CONTACT Johan S€allstr€om johan.sallstrom@mcb.uu.se Department of Medical Cell Biology, Uppsala University, Box 571, Husargatan 3, SE-751 23 Uppsala, Sweden � 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UPSALA JOURNAL OF MEDICAL SCIENCES 2020, VOL. 125, NO. 4, 274–280 https://doi.org/10.1080/03009734.2020.1804496 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1804496&domain=pdf&date_stamp=2020-10-22 http://orcid.org/0000-0002-0538-6083 http://orcid.org/0000-0002-2604-2183 http://orcid.org/0000-0003-1386-090X http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1804496 http://www.tandfonline.com different pressure than measured. Thus, obtaining a constant perfusion pressure using a feedback system is associated with non-trivial technical problems. In this report, a new approach for perfusion pressure control is presented. In prin- ciple a basal pressure-flow calibration curve is constructed at the start of the experiment. The system can thereby auto- matically correct the flow and maintain the desired pressure at the tip of the perfusion cannula. Using this system, the stability of the kidney in terms of electrolyte handling, glom- erular filtration rate (GFR), and urine production was opti- mised, whereupon the effects of vasopressin (AVP) on renal calcium handling were studied. The most well-described hormonal regulator of renal cal- cium handling is parathyroid hormone (PTH), which pro- motes active calcium reabsorption (8). The parathyroid gland, discovered in Uppsala in 1877 by Ivar Sandstr€om (9), secretes PTH in response to low serum calcium concentrations. Several lines of evidence demonstrate that also AVP stimu- lates calcium reabsorption. Earlier studies in Brattleboro rats with diabetes insipidus demonstrated that when long-term administration of the selective V2R agonist desmopressin (DDAVP) was ceased, the excretion of calcium increased (1). In a more recent clinical trial, patients with central diabetes insipidus (CDI) and nephrogenic diabetes insipidus (NDI) were challenged with DDAVP administration (2); CDI patients demonstrated a reduced excretion of calcium, whereas no change was observed in the NDI patients. Several channels have been identified for calcium transport along the nephron (10), and mechanistic studies have indicated a stimulatory role of AVP both in the cortical thick ascending limb (cTAL) (11) and the collecting duct (12). The specific effects of AVP on calcium handling in the kidney are, however, not fully understood. The isolated kidney model provides unique pos- sibilities to explore the effects of AVP given that it is possible to precisely control the concentration in the perfusate, which is not possible in vivo where systemic variations in hormone levels occur. Materials and methods Male Sprague-Dawley rats (CDVR IGS, Charles River, Germany) were used. The animals were anaesthetised using isoflurane, the jugular vein was catheterised, and the abdomen opened via a midline incision. The right kidney was dissected free and the ureter was catheterised using PE10 polyethylene tubing. In order to prevent clotting, a bolus injection of 1500 IU/kg bw heparin (Heparin LEO, 5000 IE/mL, LEO Pharma AB, Malm€o, Sweden) was administered via the jugu- lar vein before the kidney was perfused. To avoid leakage of perfusion fluid, branches of the renal artery such as the suprarenal artery were ligated. A blunt needle was intro- duced into the right renal artery through the mesenteric artery and secured by silk thread, whereupon the perfusion was started. The renal vein was cut open and the kidney removed and placed in the perfusion system, where it was kept throughout the experiment. All experiments were approved by the local animal ethics committee (reference number N23/13). Perfusion system A recirculating perfusion system, modified from a previously described method (13), was used with a constant perfusion pressure (90 mmHg) at 37 �C. All parts of the perfusion sys- tem up to the kidney were water-mantled and connected to a constant temperature circulating bath, ensuring constant temperature in the renal parenchyma, which is important given that temperature affects kidney function (14). The kid- ney was perfused through the renal artery by a computer- controlled peristaltic pump (Minipuls III, Gilson Inc., Middleton, WI, USA). The perfusate was returned to the reser- voir by another peristaltic pump (Pumpdrive 5001, Heidolph Instruments GmbH, Schwabach, Germany). The returning per- fusate was filtered using a glass filter (Porosity 3 or 4, ROBU Glasfilter-Geraete GmbH, Hattert, Germany) followed by a high-flow syringe filter (Acrodisc 32 mm, 5 mm Supor mem- brane, Pall Life Sciences, Ann Arbour, MI, USA). Constant per- fusion pressure was maintained by using a microcomputer (described below) that automatically adjusts the peristaltic pump. The reservoir containing 200 mL perfusion buffer was constantly gassed with 95% O2 and 5% CO2. The pH in the reservoir was monitored by an electrode and adjusted to physiological levels (pH 7.40) if necessary by addition of small amounts of HCl (1 mol/L). The perfusion buffer was a modified Krebs–Henseleit buf- fer with added sodium pyruvate (0.3 mmol/L), sodium l-lac- tate (2 mmol/L), alfa-ketoglutarate (1 mmol/L), malate (1 mmol/L), urea (6 mmol/L), and 20 amino acids using a stock solution prepared according to Taft (4). At the day of the experiment, bovine serum albumin (BSA) (bovine serum albumin lyophilised powder �96%, Sigma-Aldrich, St. Louis, MO, USA), calcium chloride (2.5 mmol/L), FITC-sinistrin (11.25 mg/L; Fresenius-Kabi, Linz, Austria), DDAVP (400 ng/L; MinirinVR , Ferring L€akemedel AB, Sweden) were added. Pressure control system A microcontroller was constructed using the Arduino UNO device (www.arduino.cc). The pressure signal from the trans- ducer is fed into the controller via a bridge amplifier. The pressure sensor was placed at the level of the kidney. The output from the microcontroller determines the speed of the peristaltic pump. A computer programme on the con- troller was developed that continuously calculates the differ- ence between the desired perfusion pressure and the actual perfusion pressure, and applies a correction to the pump rate using a proportional–integral–derivative (PID) regulator (Arduino PID library) in order to maintain the user-set pres- sure level. Before an experiment was commenced, a calibra- tion function was activated on the controller which runs the pump through a number of pre-defined flow rates. The pres- sure at each level was recorded by the controller, which gen- erates a calibration curve, using a second order polynomial fit, that is used in the consecutive experiment. This proced- ure thus allows for a flow-independent control of the perfu- sion pressure. Flow data from the experiments were collected and stored using a Powerlab data acquisition UPSALA JOURNAL OF MEDICAL SCIENCES 275 http://www.arduino.cc system (ADInstruments, Bella Vista, NSW, Australia) con- nected to a personal computer. Ten-minute mean values of the collected perfusion data were calculated for fur- ther analysis. Experimental series Three experimental series were performed encompassing five groups. In the first series, the system was optimised by eval- uating the effect of 7.5% versus 6% BSA in the perfusate in eight kidneys per group. Previous studies (13,15) have dem- onstrated that an elevated albumin concentration contrib- utes positively to stability and reabsorptive capacity of the isolated kidney. The albumin concentration considered most beneficial regarding reabsorption and stability was chosen. The second series was performed in order to study the effect of V2R stimulation using DDAVP in six kidneys using 7.5% BSA (using the previously performed 7.5% BSA experimental group as control). Finally, in a third series, the response to the epithelial sodium channel (ENaC) inhibitor amiloride (amiloride hydrochloride hydrate, Sigma-Aldrich, St. Louis, MO, USA; dissolved in water and administered to the fluid reservoir, 5 mmol/L) in the presence or absence of DDAVP was assessed in six kidneys per group. Urine was collected and the volumes quantified gravimet- rically throughout the experiment every 10 min. In the mid- dle of each 10-min period, a sample (0.5 ml) was drawn from the reservoir. Samples of urine and perfusate were analysed for FITC-sinistrin using a spectrophotometer (Paradigm Detection Platform, Beckman Coulter). Electrolytes and glu- cose were analysed using a blood gas analyser (ABL700, Radiometer, Copenhagen, Denmark; or ABX Pentra 400, Horiba Medical, Kyoto, Japan) and osmolality using a micro osmometer (Model 3 MO or 2020; Advanced Instruments, Norwood, MA, USA). Calculations and statistics All parameters were calculated for each 10-min urine collec- tion period. GFR was calculated as the renal clearance of FITC-sinistrin that has similar properties as inulin but better solubility (16). Fractional reabsorption (FR) for sodium, potas- sium, calcium, chloride, and glucose was calculated as fol- lows: FR ¼ Filtered amount�Excreted amount Filtered amount (1) The filtered amount was calculated as GFR multiplied by the perfusate concentration of the electrolyte, whereas the secreted amount was calculated as urine flow multiplied by the urine concentration of the electrolyte. The results are reported as mean values ± standard error of the mean. Single comparisons were performed using Student’s t test. Multiple comparisons were performed using two-way ANOVA and, when appropriate, followed by Fisher’s post hoc test. p < 0.05 was considered statistically significant. Calculations were performed using R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria) and Excel (Microsoft, Redmond, WA, USA). Results Functional optimisation of the isolated perfused kidney Renal perfusion was stable throughout the experiment and was not affected by altering the albumin concentration (Figure 1). GFR decreased throughout the experiment (approximately 50%) in both groups (Figure 1). When using 7.5% BSA versus 6% BSA, GFR tended to be lower, but more stable over time, but the difference did not reach statistical significance. The diuresis was significantly affected by the albumin concentration; kidneys perfused with 7.5% BSA had a diuresis that was more stable over time throughout the experiment and with less than half the urine output com- pared with kidneys perfused with 6% BSA (Figure 1). Altering the BSA concentration had a large impact on electrolyte han- dling (Table 1). The fractional sodium, chloride, calcium, and glucose reabsorption was significantly increased when using 7.5% BSA. Given that 7.5% BSA contributes to a more stable 4 0 5 0 6 0 7 0 8 0 P e rf u si o n ( m L /m in ) G F R ( µL /m in ) 0 5 0 0 1 0 0 0 1 5 0 0 D iu re si s (µ L /m in ) 0 30 60 90 120 150 180 0 1 0 0 2 0 0 3 0 0 BSA 6% BSA 7.5% * Time (minutes) Figure 1. Perfusion, glomerular filtration rate (GFR), and diuresis in kidneys per- fused with 6% or 7.5% bovine serum albumin (BSA), respectively. �p < 0.05 between groups, repeated measures ANOVA. 276 K. BAMBERG ET AL. preparation, this concentration was used for the consecutive experimental series. Effect of desmopressin (DDAVP) on calcium reabsorption In the control group, the urine osmolality gradually fell to about 200 mOsm/kg during the experiment, whereas in DDAVP-treated kidneys the osmolality increased to about 350 mOsm/kg and was sustained at that level (Figure 2). The effect on calcium reabsorption was particularly pronounced and displayed a clear temporal correlation to the changes in urine osmolality, whereas sodium reabsorption was not sig- nificantly changed (Figure 2). When the change in osmolality and urine flow up to the first hour of the experiment was plotted against the corresponding change in calcium reabsorption, the reduction in urine osmolality and the increase in urine flow during the experiment was correlated to a reduced calcium reabsorption (Figure 3). The goodness- of-fit of the linear regression was better for urine flow than osmolality. Addition of DDAVP to the perfusate did not affect GFR or renal perfusion compared with perfusate containing only 7.5% BSA but tended to reduce the urine flow rate (Table 1). The DDAVP group exhibited a significantly increased fractional potassium reabsorption, but there was a general tendency for an increase in the fractional reabsorp- tion of all measured electrolytes (Table 1). Effect of epithelial sodium channel (ENaC) inhibition Administration of amiloride caused a pronounced increase in fractional potassium reabsorption, whereas fractional sodium reabsorption decreased (Figure 4). The changes were not affected by DDAVP. DDAVP-treated kidneys displayed an ele- vated fractional calcium reabsorption compared with the control group both during baseline (0.99 ± 0.00 versus 0.94 ± 0.02; p < 0.05) and during amiloride treatment (0.99 ± 0.00 versus 0.90 ± 0.02; p < 0.05), which agrees with observations in the previous series described above. Discussion The experiments performed demonstrate that the presented new approach for pressure control was able to maintain a functional preparation with stable perfusion for up to 3 h. By using the method, the surgical procedure is facilitated since the cannula can be placed directly in the renal artery; only two experiments failed due to problems with surgery. Using a higher concentration (7.5%) of albumin promoted fluid and electrolyte reabsorption and contributed to a more stable diuresis over time. Addition of DDAVP tended to further pro- mote reabsorption of most electrolytes, leading to an elec- trolyte reabsorption approaching in vivo values (17,18). Table 1. Three-hour mean values in kidneys perfused with 6% or 7.5% bovine serum albumin (BSA) or 7.5% BSA þ desmopressin (DDAVP; 400 ng/L). 6% BSA 7.5% BSA 7.5% BSA þ DDAVP Perfusion (mL/min) 58 ± 3 60 ± 4 54 ± 2 GFR (lL/min) 899 ± 105 684 ± 90 747 ± 96 Urine flow (lL/min) 167 ± 30 61 ± 14a 27.67 ± 7.96 FRNa 0.88 ± 0.02 0.96 ± 0.01 a 0.97 ± 0.01 FRK 0.38 ± 0.02 0.51 ± 0.02 a 0.61 ± 0.03b Urine Na/K ratio 10.33 ± 1.63 3.61 ± 0.74a 3.34 ± 1.07 FRCl 0.86 ± 0.02 0.95 ± 0.01 a 0.97 ± 0.01 FRCa 0.79 ± 0.02 0.92 ± 0.01 a 0.99 ± 0.00b FR glucose 0.96 ± 0.01 0.97 ± 0.00a 0.98 ± 0.00 Urine osmolality (mOsm/kg) 241 ± 9 230 ± 9 353 ± 9b ap < 0.05 versus 6% BSA. bp < 0.05 versus 7.5% BSA. Ca: calcium; Cl: chloride; FR: fractional reabsorption; GFR: glomerular filtration rate; K: potassium; Na: sodium. 100 200 300 400 100 200 300 400 * 0.85 0.90 0.95 1.00 0.85 0.90 0.95 1.00 * Time (min) 0.90 0.95 1.00 0 90 180 Control DDAVP 0.90 0.95 1.00 O sm o la lit y (m O sm /k g ) F R C a F R N a Figure 2. Urine osmolality, fractional calcium (FRCa), and sodium reabsorption (FRNa) in kidneys perfused with desmopressin (DDAVP; 400 ng/L) compared to control experiments. The bars represent mean values from the period 90–180 min (indicated by the line). Albumin concentration in the perfusate was 7.5%. �p < 0.05 versus control. UPSALA JOURNAL OF MEDICAL SCIENCES 277 DDAVP also reversed the fall in urine osmolality that occurred without DDAVP, which further contributed to the stability of the system. The applied experimental model is thus considered suitable for studies on electrolyte transport, which is illustrated by the prompt changes in potassium and sodium handling in response to administration of the potas- sium-sparing diuretic amiloride that inhibits ENaC. The pre- sented model also demonstrates that it is possible for researchers to design their own experimental equipment fit- ted to their needs using inexpensive and widely available programmable microcontrollers. Other recently published applications include perfusion controllers for hydrogels (19) and temperature regulators for intravital microscopy (20). We have, however, not found any recent developments regard- ing perfusion systems for kidneys. Controlled water reabsorption in the kidney according to physiological needs takes place in the collecting duct where aquaporin-2 (AQP2) channels are incorporated into the apical cell membrane through stimulation of V2R by AVP. Aquaporins increase water permeability and consequently increase fluid reabsorption to the hyperosmolar interstitium (21). The effects of AVP on calcium handling is not that well described, but there is evidence of a stimulatory effect on calcium reabsorption from studies both in rodents (1) and in humans (2). In the present experiments, the kidneys are nat- urally exposed to the normal physiological levels of AVP before the organ was excised from the rat. When perfused without AVP, the antidiuretic effect rapidly declined, as dem- onstrated by the gradually reduced osmolality and increased urine flow. The reduced osmolality due to absence of AVP is expected, since it is the normal mechanism for regulation of fluid homeostasis in response to water loading and demon- strates that this mechanism is functional in the isolated kid- ney. The fall in osmolality was accompanied by a matched decrease in calcium reabsorption that, interestingly, had simi- lar temporal characteristics. Given that the effects of vaso- pressin require aquaporin trafficking (22), the simultaneous effects on water and calcium reabsorption indicate a com- mon tubular target and a related mechanism. Incorporation of aquaporins into the apical membrane involves a complex series of events mediated by the G-protein-coupled V2R at the basolateral membrane (reviewed in (22,23)). Among the different signalling pathways involved, calcium signalling has osmolality (mOsm/kg) F R C a −0.25 −0.20 −0.15 −0.10 −0.05 0.00 0.05 R2= 0.49 urine flow (µL/min) F R C a −0.25 −0.20 −0.15 −0.10 −0.05 0.00 0.05 0 −20−50−100−150 0 20 40 60 80 100 R2= 0.79 (A) (B) Figure 3. Correlation between change (D) in urine osmolality (A) and urine flow (B) and fractional calcium reabsorption (FRCa) in kidneys perfused with 7.5% albu- min without DDAVP. Values are calculated as the change from the first 10-min period up to 60 min into the experiment. The values are individual time points derived from the two experimental groups without added DDAVP. A straight line was fitted to the data using linear regression, with the square of the correlation coefficient (R2) indicated. 0 20 40 60 80 100 0 .9 0 0 .9 2 0 .9 4 0 .9 6 0 .9 8 1 .0 0 Time (min) F R N a Baseline Amiloride 0 .9 0 0 .9 2 0 .9 4 0 .9 6 0 .9 8 1 .0 0 * 0 20 40 60 80 100 0 .2 0 0 .4 0 0 .6 0 0 .8 0 1 .0 0 Time (min) F R K BSA 7.5% BSA 7.5% + DDAVP Baseline Amiloride 0 .2 0 0 .4 0 0 .6 0 0 .8 0 1 .0 0 BSA 7.5% BSA 7.5% + DDAVP F R K F R N a Amiloride Baseline Amiloride Baseline * * * 120 120 Figure 4. Fractional reabsorption of sodium (FRNa) and potassium (FRK) in response to amiloride (5 mmol/L) in kidneys perfused with BSA 7.5% or BSA 7.5% þ desmopressin (DDAVP; 400 ng/L). The left panel presents the time ser- ies data of fractions observed during the 120-min experiment period, whereas the right panel displays mean values at baseline (0–60 min) and at amiloride exposure (60–120 min). �p < 0.05 versus baseline. 278 K. BAMBERG ET AL. been shown to have a stimulatory role on aquaporin incorp- oration (24). The mechanisms for AVP-mediated calcium reabsorption are not clear. Data on PTH-mediated transepithelial calcium transport have been linked to the apical transient receptor potential cation channel subfamily V member 5 and 6 (TRPV5/6) in the distal convoluted tubule and the connecting tubule (25). In the collecting duct, further downstream of the tubular system, canonical transient receptor potential chan- nel member 3 (TRPC3) may instead have a particular role for calcium reabsorption (26). Interestingly, AVP has been shown to induce co-localization of AQP2 channels and TRPC3 cation channels to the apical cell membrane in the medullary col- lecting duct. Overexpression of these channels in cultured collecting duct cells was also related to increased transepi- thelial calcium flux (27). Accordingly, calcium channels stimu- lated by AVP may, besides being involved in the signalling mechanisms for aquaporin trafficking, directly contribute to calcium reabsorption. In line with this, TRPC3 knockout mice displayed an increased urinary calcium concentration com- pared to their wild-type controls when subjected to water deprivation (28). The correlation observed in the present study between change in osmolality and urine flow and change in calcium reabsorption (Figure 3), as well as their temporal relationship (Figure 2), consequently further sup- ports that AVP increases reabsorption of both water and cal- cium through incorporation of these channels on the apical membrane of the cells in the collecting duct. The physio- logical relevance may be to protect against urolithiasis dur- ing water restriction, where otherwise calcium levels could be elevated to levels risking precipitation. Another indication on the possible role of this channel in pathophysiology comes from a case report where TRPC3 expression was found to be upregulated in a patient with Williams–Beuren syndrome, a rare neurodevelopmental disorder also associ- ated with hypercalcemia (29). However, TRPC3 expression was found both in the kidney and the intestinal epithelium. Thus, an increased intestinal uptake could also have contrib- uted to the elevated plasma calcium concentrations. To further assess that the perfused kidney model repli- cates in vivo physiological responses, we assessed the response to ENaC inhibition by amiloride. Administration of amiloride caused a prompt decrease in sodium reabsorption accompanied by an increased potassium reabsorption. These are the expected effects of ENaC inhibition (30), which reduces potassium excretion by hyperpolarizing the principal cells in the collecting duct, thus reducing the electrochemical driving force for potassium extrusion into the lumen. ENaC inhibition will also lower the intracellular sodium concentra- tion, thus reducing basolateral potassium entry through the Na-K-ATPase which also most likely contributes to the reduced potassium secretion (31). Given that both ENaC and TRPC3 are expressed in the principal cells (26,32), the normal response confirms that this nephron segment, where the proposed effects of AVP on calcium reabsorption occurs, is functional in this model. Fractional calcium reabsorption was higher in the DDAVP group both during control conditions and during amiloride treatment. The elevated calcium reabsorption by DDAVP supports the findings in the previous series and also indicates that the acute effect of DDAVP on calcium handling is independent of sodium transport through ENaC. Conclusion In the present report, a new model for pressure control of iso- lated perfused kidneys has been evaluated and found suitable for studies on electrolyte and fluid transport. The preparation responds to pharmacological inhibition of ENaC channels as well as activation of V2R. During V2R stimulation, calcium reabsorption was strongly promoted. Using the presented experimental model, the dynamic effects of V2R-stimulation on calcium handling and urine osmolality was visualised, which, to the best of our knowledge, has not been previously described. The study thereby provides further evidence for the stimulatory role of V2R in calcium transport. Disclosure statement K.B., L.W.O., U.J. and J.H.G. are employed by AstraZeneca. J.S. is employed at the Swedish Medical Products Agency, SE-751 03 Uppsala, Sweden. The views expressed in this paper are the personal views of the author and not necessarily the views of the Government agency. Funding The experimental study was performed 2013–2014 and was supported by AstraZeneca, Sweden. No financial support has been received from AstraZeneca for preparation of the manuscript. Notes on contributors Krister Bamberg, PhD, director, scientific lead, CKD late stage portfolio at Department of Translational Sciences and Experimental Medicine, Early Cardiovascular Renal and Metabolism BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg. Lena William-Olsson, BSc, in vivo biologist at Department of Bioscience Renal, Early Cardiovascular Renal and Metabolism BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg. Ulrika Johansson, BSc, senior researcher at Department of Bioscience Renal, Early Cardiovascular Renal and Metabolism BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg. Anders Arner, MD, PhD, researcher at the Department of Clinical Sciences Lund, Lund University and professor emeritus at the Department of Physiology and Pharmacology, Karolinska Institutet. Judith Hartleib-Geschwindner, PhD, senior director and global project lead at Department of Projects, Early Cardiovascular Renal and Metabolism BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg. Johan S€allstr€om, PhD, researcher at the Department of Medical Cell Biology, Uppsala University and clinical assessor at the Swedish Medical Products Agency. ORCID Krister Bamberg http://orcid.org/0000-0002-0538-6083 Lena William-Olsson http://orcid.org/0000-0002-2604-2183 Anders Arner http://orcid.org/0000-0003-1386-090X UPSALA JOURNAL OF MEDICAL SCIENCES 279 References 1. Bouby N, Trinh-Trang-Tan MM, Bankir L. Stimulation of tubular reabsorption of magnesium and calcium by antidiuretic hormone in conscious rats. Study in Brattleboro rats with hereditary hypo- thalamic diabetes insipidus. Pflugers Arch. 1984;402:458–64. 2. Hanouna G, Haymann J, Baud L, Letavernier E. Vasopressin regu- lates renal calcium excretion in humans. Physiol Rep. 2015;3: e12562. 3. Maack T. 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Hager H, Kwon TH, Vinnikova AK, Masilamani S, Brooks HL, Frøkiaer J, et al. Immunocytochemical and immunoelectron micro- scopic localization of alpha-, beta-, and gamma-ENaC in rat kid- ney. Am J Physiol Renal Physiol. 2001;280:F1093–106. 280 K. BAMBERG ET AL. https://doi.org/10.3389/fphar.2019.00800 https://doi.org/10.3389/fimmu.2019.02036 https://doi.org/10.3389/fimmu.2019.02036 Abstract Introduction Materials and methods Perfusion system Pressure control system Experimental series Calculations and statistics Results Functional optimisation of the isolated perfused kidney Effect of desmopressin (DDAVP) on calcium reabsorption Effect of epithelial sodium channel (ENaC) inhibition Discussion Conclusion Disclosure statement References