New autosomal recessive mutations in aquaporin-2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes

Abstract

Aquaporin-2 (AQP2), located at the luminal side of the collecting duct principal cells, is a water channel responsible for the final concentration of urine. Lack of function, often occurring through mistargeting of mutated proteins, induces nephrogenic diabetes insipidus (NDI), a condition characterized by large urinary volumes. In the present study, two new mutations (K228E and V24A) identified in NDI-affected individuals from distinct families along with the already reported R187C were analysed in comparison to the wild-type protein (AQP2-wt) using Xenopus laevis oocytes and a mouse collecting duct cell-line (mIMCD-3). Initial data in oocytes showed that all mutations were adequately expressed at reduced levels when compared to AQP2-wt. K228E and V24A were found to be properly targeted at the plasma membrane and exhibited adequate functionality similar to AQP2-wt, as opposed to R187C which was retained in internal stores and was thus inactive. In coexpression studies using oocytes, R187C impeded the functionality of all other AQP2 variants while combinations with K228E, V24A and AQP2-wt only showed additive functionalities. When expressed in mIMCD-3 cells, forskolin treatment efficiently promoted the targeting of AQP2-wt at the plasma membrane (>90%) while K228E only weakly responded to the same treatment (approximately 20%) and both V24A and R187C remained completely insensitive to the treatment. We concluded that both V24A and K228E are intrinsically functional water channels that lack a proper response to vasopressin, which leads to NDI as found in both compound mutations studied (K228E + R187C and V24A + R187C). The discrepancies in plasma membrane targeting response found in both expression systems stress the need to evaluate such data using mammalian cell systems.

Figure 7. Functional analysis of mutant AQP2 in coexpression studies

cRNA (5 ng) for either K228E (A) or V24A (B) was injected along with increasing concentrations of R187C cRNA (0–10 ng) and water permeability was evaluated using a 20 mosmol l⁻¹ hyposmotic shock. P_f values are given in 10⁻⁴ cm s⁻¹ and represent 7–8 determinations for each condition. Asterisks indicate statistical significance (P < 0.05) in comparison to pure K228E or V24A values while # indicates non-significance compared to controls (P < 0.05). Data are representative of three individual assays.

Figure 2. Expression of AQP2 variants in X. laevis oocytes

A, Western blot of oocytes injected with increasing amounts of cRNA for wild-type (0.5, 1 and 2 ng) and mutated forms (1, 2 and 5 ng) of AQP2. Each lane represents total membranes from 4 oocytes. B, densitometry evaluation of AQP2 bands (combination of both 29 and 31 kDa bands) extracted from A correlated against cRNA quantities. Data represent mean ±s.d. from triplicates and fitted to monoexponential equation.

Figure 5. Expression of AQP2 in relation to family genotypes

Oocytes were injected with AQP2 cRNA combinations in accordance with Fig. 1 reproducing genotypes presented for family 1 (A) and family 2 (B). With regard to translation efficiency, cRNAs were injected at 1 ng (HA-AQP2-wt) or 5 ng (mutant) with the exception of double mutant conditions (K228E + R187C and V24A + R187C) where both mutants were injected at 5 ng each. Functionality was evaluated using a mild hyposmotic shock (−20 mosmol l⁻¹). P_f values are expressed as percentage activity of pure AQP2-wt and represent 7–8 determinations for each condition. Western blots of purified plasma membrane were first probed using α-AQP2 for total AQP2 staining then stripped and probed again using α-HA to specifically identify wt-AQP2. Data are representative of three individual assays.

Figure 1. Segregation of AQP2 alleles in two families

Genotypes presenting segregation of both wild-type (open symbols) and mutated (filled symbols) forms of AQP2 in the two families tested. Clinical evaluation indicates that only individuals with compound mutations are affected with NDI. n indicates normal alleles.

Figure 3. Expression of AQP2 variants in X. laevis oocytes

A, functional evaluation of all AQP2 variants. Oocytes were injected with cRNA coding for either wild-type (WT, 1 ng) or mutated (V24A, R187C and K228E, 5 ng) AQP2 along with controls (Ctrl) and incubated for 72 h prior to testing for water permeability capacities (see Methods). P_f values are in 10⁻⁴ cm s⁻¹ and represent 7–8 determinations per condition. Data are representative of 5–7 individual assays. The same oocytes were tested in Western blot using either total membranes (B, 1 oocyte per lane) or purified plasma membranes (C, 40 oocytes per lane) fractions. PDI detection was performed on the same material to confirm the quality of the purification procedure for plasma membrane in C. D, immunofluorescence labelling of all AQP2 in the same samples showing retention of R187C within intracellular stores as opposed to AQP2-wt, V24A and K228E.

Figure 4. Immunofluorescence analysis of AQP2 in transfected IMCD-3 cells

mIMCD-3 cells were transfected with pcDNA6-AQP2-wt, -V24A, -R187C or -K228E, incubated for 16–24 h and treated (+forskolin) or not (–forskolin) with forskolin (50 μm, 45 min) prior to fixation. PDI was used as an ER marker (red) and DAPI was used as a nuclear stain (blue). Plasma membrane staining is found for both AQP2-wt and -K228E (arrows) but not for R187C or V24A. Magnification ×60. Scale bar, 10 μm.

Figure 8. Effects of mutant's coexpression on AQP2-wt abundance and plasma membrane targeting

Oocytes injected with water (Ctrl), HA-AQP2-wt alone (WT, 1 ng) or with untagged mutants (+, 5 ng) were probed for overall AQP2 abundances in total membranes using α-AQP2 (A) or specifically for AQP2-wt in purified plasma membranes using α-HA (B).

Figure 6. Functionality of AQP2-wt in coexpression studies

A standard 1 ng AQP2-wt cRNA was injected along with increasing concentrations (0–10 ng) of cRNA for K228E (A), V24A (B) or R187C (C) and water permeability was evaluated using a 20 mosmol l⁻¹ hyposmotic shock. P_f values are given in 10⁻⁴ cm s⁻¹ and represent 7–8 determinations for each condition. Asterisks indicate statistical significance (P < 0.05) in comparison to pure 1 ng AQP2-wt value while # indicates non-significance compared to controls (P < 0.05). Data are representative of three individual assays.

Figure 9. Schematic representation of AQP2 protein

Depiction of the human AQP2 protein showing the positions of V24A, R187C and K228E mutations. Brackets indicate conserved NPA motifs.