Prasugrel suppresses development of lithium-induced nephrogenic diabetes insipidus in mice

Abstract

Previously, we localized ADP-activated P2Y₁₂ receptor (R) in rodent kidney and showed that its blockade by clopidogrel bisulfate (CLPD) attenuates lithium (Li)-induced nephrogenic diabetes insipidus (NDI). Here, we evaluated the effect of prasugrel (PRSG) administration on Li-induced NDI in mice. Both CLPD and PRSG belong to the thienopyridine class of ADP receptor antagonists. Groups of age-matched adult male B6D2 mice (N = 5/group) were fed either regular rodent chow (CNT), or with added LiCl (40 mmol/kg chow) or PRSG in drinking water (10 mg/kg bw/day) or a combination of LiCl and PRSG for 14 days and then euthanized. Water intake and urine output were determined and blood and kidney tissues were collected and analyzed. PRSG administration completely suppressed Li-induced polydipsia and polyuria and significantly prevented Li-induced decreases in AQP2 protein abundance in renal cortex and medulla. However, PRSG either alone or in combination with Li did not have a significant effect on the protein abundances of NKCC2 or NCC in the cortex and/or medulla. Immunofluorescence microscopy revealed that PRSG administration prevented Li-induced alterations in cellular disposition of AQP2 protein in medullary collecting ducts. Serum Li, Na, and osmolality were not affected by the administration of PRSG. Similar to CLPD, PRSG administration had no effect on Li-induced increase in urinary Na excretion. However, unlike CLPD, PRSG did not augment Li-induced increase in urinary arginine vasopressin (AVP) excretion. Taken together, these data suggest that the pharmacological inhibition of P2Y₁₂-R by the thienopyridine group of drugs may potentially offer therapeutic benefits in Li-induced NDI.

Keywords: Arginine vasopressin; Diabetes insipidus; Extracellular nucleotides; Nephrogenic; Polyuria; Purinergic receptors.

Fig. 1

Effect of prasugrel on lithium-induced polydipsia and polyuria. Groups of mice were fed regular diet (CNT) or lithium-added diet (LI) or prasugrel in drinking water (PRSG) or a combination of Li in the diet and PRSG in drinking water (LI + PRSG) for 14 days and euthanized. Twenty-four-hour water intake and urine output were determined, prior to the experimental period (day 0), on days 7 and 8, and prior to euthanasia (days 13 and 14; terminal). Mean values for days 7 and 8 and 13 and 14 were used in computing the data. a, b show water intake during the three time points or terminally, respectively. c, d show urine output during the three time points or terminally, respectively. e, f show urine osmolality during the three time points or terminally, respectively. Values shown are mean ± SE (N = 5 mice/per group). *significantly different from other groups at the same time point by ANOVA (P < 0.05 or better). **significantly different from all other groups by Tukey-Kramer multiple comparison test followed by ANOVA (P < 0.05 of better)

Fig. 2

Effect of prasugrel on lithium-induced decreases in AQP2 protein abundances in the kidney. Groups of mice were fed regular diet (CNT) or lithium-added diet (LI) or prasugrel in drinking water (PRSG) or a combination of Li in the diet and PRSG in drinking water (LI + PRSG) for 14 days and euthanized. Whole tissue homogenates of renal medulla or cortex were processed for semiquantitative immunoblotting for abundances of AQP2 water channel and β-actin proteins. a shows immunoblot profiles for AQP2 protein in the medulla, where each lane represents sample for one mouse. AQP2 protein band densities were normalized by corresponding β-actin band densities and are shown in b (mean ± SE). *significantly different from the CNT and PRSG groups by Tukey-Kramer multiple comparison test following ANOVA (P < 0.01); **significantly different from the LI group by Bonferroni Test following ANOVA (P < 0.05). c shows immunoblot profiles for AQP2 protein in the cortex, where each lane represents sample from one mouse. AQP2 protein band densities were normalized by corresponding β-actin band densities and are shown in d (mean ± SE) for the corresponding immunoblots of medulla and cortex. *significantly different from all other groups by Tukey-Kramer multiple comparison test following ANOVA (P < 0.05 or better)

Fig. 3

Effect of prasugrel alone or in combination with lithium on the protein abundances of NKCC2 and NCC in the renal cortex and/or medulla. Groups of mice were fed regular diet (CNT) or lithium-added diet (LI) or prasugrel in drinking water (PRSG) or a combination of Li in the diet and PRSG in drinking water (LI + PRSG) for 14 days and euthanized. Whole-tissue homogenates of renal medulla or cortex were processed for semiquantitative immunoblotting for abundances of sodium transporters, NKCC2 or NCC, and β-actin. a, b show immunoblot profiles for NKCC2 protein bands in the medulla and cortex, respectively, where each lane represents sample from one mouse. Bar graphs on the right side of each panel show corresponding densitometric values (mean ± SE) for the protein bands. *significantly different from the CNT group by Tukey-Kramer multiple comparison test following by ANOVA (P < 0.05). c immunoblot profile for NCC protein bands in the cortex, where each lane represents sample from one mouse. Bar graph on the right side of shows corresponding densitometric values (mean ± SE) for the immunoblot. *significantly different from the CNT and PRSG groups by Tukey-Kramer multiple comparison test following ANOVA (P < 0.03)

Fig. 4

Immunofluorescence (IF) imaging of cellular expression and disposition of APQ2 (green) and P2Y₁₂ receptor (red) proteins in the inner medullary collecting ducts of mice treated with PRSG and/or lithium in comparison with control mice. Representative low magnification profiles show IF labeling in mice treated with no drug (a), LI (b), prasugrel (PRSG) (c), or a combination of LI and PRSG (d). Insets show corresponding higher magnification profiles. Fluorescence imaging settings were the same in all groups. Bar is 20 μm

Fig. 5

Morphological appearance of medullary collecting ducts in mice treated with PRSG and/or lithium. Formalin-fixed and paraffin-embedded kidney samples were sectioned and stained with hematoxylin-eosin. Representative profiles of medullary collecting duct in a control mouse (a) or a mouse fed lithium (b) or treated with prasugral (c) or a combination of lithium and prasugrel (d). Bar is 20 μm

Fig. 6

Effect of prasugrel and/or lithium on blood parameters. a Terminal serum osmolalities in all the groups. b Terminal serum sodium levels in all the groups. c Terminal serum lithium levels in lithium-treated groups. Values shown are mean ± se (N = 5 mice/group)

Fig. 7

Effect of prasugrel and/or lithium on urinary parameters. a Urinary sodium excretion. *significantly different from CNT group by Tukey-Kramer multiple comparison test following ANOVA (P < 0.01). b Urinary AVP excretion. *significantly different from CNT group by Tukey-Kramer multiple comparison test following ANOVA (P < 0.05). All data are mean ± se, N = 5 mice/group