Defective renal water handling in transgenic mice over-expressing human CD39/NTPDase1

Abstract

Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders.

Fig. 1.

Water balance, urinary parameters, and aquaporin-2 (AQP2) protein abundance in the kidneys of wild-type (WT) and human CD39 (hCD39)-transgenic (TG) mice under basal conditions. WT and TG mice were allowed to feed on standard solid chow with free access to drinking water in metabolic cages. After acclimation to the housing conditions for 2 days, 24-h water consumption, urine output, and urine osmolalities were recorded. Mice were euthanized, and kidney cortical and whole medullary (outer + inner) tissue samples were processed separately for semiquantitative immunoblotting for AQP2 protein and β-actin. A–C: water intake (A), urine ouput (B), and urine osmolality (C) in the WT and TG mice (n = 10 mice/genotype). D: representative immunoblots of AQP2 and β-actin in cortical and medullary tissue samples. Total AQP2 band densities (29 kDa native + 35–50 kDa glycosylated form) were determined for each mouse and normalized to the corresponding β-actin band densities. Values are converted to %mean values in WT mice and are shown in bar graphs adjacent to the corresponding immunoblots. Statistical analysis was performed by unpaired t-test, and the P values are shown above the bars (n = 5 mice/bar).

Fig. 2.

Effect of water deprivation on urine output and urine osmolality in WT and hCD39-TG mice. The effect of 24-h water deprivation in WT and TG mice was studied as described in methods. Data collected from 2 independent experiments were pooled (n = 10 mice/genotype). A and B: urine output and urine osmolality were monitored prior to and during 24-h water deprivation. Basal, data collected prior to water deprivation; WD, data collected during water deprivation. Statistical analysis was performed by unpaired t-test, and P values are shown above the bars.

Fig. 3.

Urinary excretion of analog of vasopressin (AVP), prostaglandin E₂ (PGE₂), and metabolite and total nitrates/nitrites in WT and hCD39-TG mice under basal conditions and during water deprivation. After acclimation to housing conditions in metabolic cages, 24-h urine samples were collected from WT and TG mice under basal conditions (free access to chow and drinking water) and during the subsequent 24-h water deprivation period (free access to chow, but no drinking water). Data from 2 independent experiments were pooled (n = 10 mice/genotype). Urinary excretion of AVP (A), PGE₂ metabolite (B), and total nitrates/nitrites (C) was determined and normalized to 20 g body wt to correct for small variations in the body weights among different mice. Statistical analysis was performed by unpaired t-test, and P values are shown above the bars. NO, nitric oxide.

Fig. 4.

Effect of acute water loading in WT and hCD39-TG mice. Acute water loading was performed in WT and TG by injecting 2 ml of sterile water intraperitoneally after an overnight water deprivation to ensure a comparable state of hydration and empty bladders, as described in methods. Data from 2 independent experiments were pooled (n = 9 WT mice and 10 TG mice). Urine output, urine osmolality, and osmolar excretion were monitored over a 6-h period. A–C: urine output (A), urine osmolality (B), and osmolar excretion (C) over a 6-h period in blocks of 2 h. D–F: cumulative values over a 6-h period for urine output (D), urine osmolality (E), and osmolar excretion (F). It should be noted that although 9 or 10 mice/group were used, all mice did not void urine during the period of every time block. Therefore, the no. of responding mice in each time block is shown in parentheses over the bars in A. Statistical significance for relevant pairs of data was assessed by Mann-Whitney nonparametric method, and the P values are shown above the bars.

Fig. 5.

Water balance, urinary parameters, and AQP2 protein abundance in the medulla of WT and hCD39-TG mice under stable hydrated conditions. WT and TG mice were acclimated to metabolic cages and fed a gelled diet containing fixed proportions of nutrients and normal water content as the sole ration for 7 days. Twenty-four-hour water consumption, urine output, and urine osmolalities were monitored, and the values obtained during the last 2 days (days 6 and 7) were averaged and used in the computation of the results. Data from 3 independent experiments were pooled (n = 15 mice/genotype). A–C: water intake (A), urine ouput (B), and urine osmolality (C) in the WT and TG mice (n = 15 mice/group). Representative immunoblots of AQP2 and β-actin proteins in the medullary tissue and densitometric values are shown in A. Total AQP2 band densities (29 kDa native + 35–50 kDa glycosylated form) were determined for each mouse and normalized to the corresponding β-actin band densities. Values obtained for TG mice were converted to %mean values in WT mice and are shown (n = 5 mice/ggenotype). Statistical analysis was performed by unpaired t-test, and P values are shown above the bars.

Fig. 6.

Relative mRNA expression of P2Y and adenosine receptors in the medulla of WT and hCD39-TG mice under basal conditions (solid diet) or stable hydrated conditions (gelled diet). WT and TG mice were allowed to feed on standard solid chow, with free access to drinking water in metabolic cages, or fed a gelled diet, as described in methods. Mice were euthanized, and kidney cortical and whole medullary (outer + inner) tissue samples were processed separately for RNA extraction, followed by real-time RT-PCR for P2Y receptors and adenosine receptors, as described in methods. The mRNA expression of target genes was expressed relative to the mRNA expression of β-actin in the samples. Means ± SE in TG mice were plotted as %corresponding mean values in WT mice. Statistical analysis was performed by unpaired t-test, and the P values are shown above the bars (n = 5 mice/genotype).

Fig. 7.

Effect of water restriction in WT and hCD39-TG mice. The effect of water restriction for 3 days in WT and TG mice was performed by switching the mice from stable hydrated conditions (normal gelled diet) to a low-water-containing gelled diet, as described in methods. A–C: differences in water intake (A), urine output (B), and urine osmolality (C) in WT and TG mice prior to (Pre-) and during the 3 days of water restriction. Statistical analysis was performed by unpaired t-test, and the P values are shown above the bars (n = 5 mice/genotype).

Fig. 8.

Effect of chronic water loading in WT and hCD39-TG mice. Chronic water loading was performed in WT and TG mice by giving a high-water-containing gelled diet as the sole ration for 7 days, as described in methods. Water intake, urine output, and urine osmolality were monitored. A–C: data collected on the last 2 days were averaged for each mouse and adjusted to 20 g body wt to correct for small variations in the body weights (n = 5 mice/genotype).

Fig. 9.

Effect of subacute desamino-8-d-arginine vasopressin (dDAVP) administration. The subacute effect of dDAVP over a 4-h period was evaluated in WT and TG mice by determining the increase in urine osmolalities of spot urine samples following the administration of dDAVP compared with the osmolalities of baseline spot urine samples. Data from 2 independent experiments were pooled (n = 12 mice/genotype). Mean values of osmolalities of spot urine samples under basal conditions (baseline) and the highest values attained for osmolality within 4 h of dDAVP administration (+dDAVP) in WT and TG mice are shown. Each bar represents the mean ± SE of 10–12 spot urine samples. It should be noted that baseline osmolalities of spot urine samples collected during the daytime are generally low compared with the osmolalities of 24-h urine collections (Fig. 1). Statistical analysis was performed by Mann-Whitney nonparametric method, and P values are shown above the bars.

Fig. 10.

Effect of chronic dDAVP infusion. The chronic effect of dDAVP on urinary concentrating ability was evaluated in WT and TG mice by infusing dDAVP at a rate of 1 ng/h for 5 days. Osmolalities of urine samples were monitored daily. Mean urine osmolalities in WT and TG mice on day 0 (prior to the start of dDAVP infusion) and on days 3-5 of dDAVP infusion expressed as %changes over respective mean values on day 0 (100%; n = 5 mice/genotype) are shown.