1998 Global Conference Proceeding
March 02 - 04, 1998
| Conference: | 1998 Global Conference |
|---|---|
| Title: | Clinical Data and Cell Biological Aspects of Mutations in Nephrogenic Diabetes Insipidus |
| Authors: | Knoers, Nine; Deen, Peter M.T. |
| Institutions: | University Medical Centre Nijmegen, University of Nijmegen |
Nephrogenic Diabetes Insipidus (NDI) is characterized by the inability of the kidney to concentrate urine in response to AVP and is inherited either as an X-linked recessive or as a recessive or dominant autosomal trait. In recent years we have studied a large group of patients with this disorder at the clinical, physiological, and molecular level. A retrospective analysis of the clinical data of thirty male NDI patients revealed that in the majority of cases NDI was diagnosed within the first 2.5 years of life. Vomiting and anorexia, failure to thrive, fever and constipation were, in order of frequency, the most reported initial symptoms. In a few older patients the disease was detected as a result of events not directly related to NDI. Long-term follow up of these patients revealed serious urological complications in four of thirty patients and growth retardation in the majority of cases. In a psychological study of 17 male patients we have found that the current prevalence of mental retardation is considerably lower than suggested in the literature.
With molecular analysis of the two known genes involved in NDI, V2R and AQP2, we have identified 41 different V2R mutations and eight different AQP2 mutations. We have shown that patients with an AQP2 mutation can be discriminated from those with a V2R mutation by means of a DDAVP-infusion test with measurement of extrarenal coagulation, fibrinolytic, and vasodilatory responses. Remarkable was the identification of V2R mutations in a few women with complete clinical manifestation of NDI, a phenomenon which may be explained by skewed X-inactivation. Several V2R mutations have been expressed in an in-vitro expression system to proof their pathogenicity. For one of these mutations, T204N, the reduced but not absent vasopressin binding capacity of the mutant receptor, as shown in the expression system, was reflected in-vivo by an increase in urinary osmolality after administration of a double dosis of DDAVP to the patient carrying this mutation. To investigate whether the identified mutations in the AQP2 gene cause NDI, missense mutants in recessive NDI were expressed in Xenopus oocytes. Most mutants were not functional (G64R, N68S, R187C, S216P), while two conferred a small increase in water permeability (T126M, A147T). The mutants with mutations in supposed transmembrane domains (A147T, S216P) were unstable compared to wild-type (wt) AQP2. Inununoblot and immunocytochemical analyses showed that all these mutants were impaired in their export from the endoplasmic reticulum (ER), which is likely to be the molecular cause of NDI in patients with these mutations. Recently, a family was identified in which NDI was inherited in a dominant fashion. Haplotype and sequence analyses revealed that in this family NDI was caused by a mutation in AQP2 gene, resulting in an E258K substitution. Expressed in oocytes, AQP2-E258K appeared to confer a reduced water permeability compared to wt AQP2. Although the E258K mutation is located close to the PKA phosphorylation site (S256), the E258K mutation did not interfere phosphorylation of this residue. Immunoblot analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not present as a high mannose glycosylated form, indicating that this mutant was not retarded in the ER. Immunocytochemistry showed that AQP2-E258K was localized just below the plasma membrane. Electron microscopy revealed that AQP2-E258K was piled up in the Golgi. AQPs appear at the plasma membrane as homotetramers, while a monomer is the functional unit. Therefore, in mixed tetramers of wt AQP2 and AQP2-E258K, intracellular retention of the AQP2E258K mutant in the Golgi offers an explanation for the dominant form of inheritance of NDI in this family.
Analysis of the data of 30 nephrogenic diabetes insipidus (NDI) patients showed:
- The majority of cases were diagnosed within the first 2.5 years of life.
- Vomiting, anorexia, failure to thrive, fever and constipation, in that order, were the most reported initial symptoms.
- A majority of the NDI patients experienced growth retardation. However, a psychological study of 17 male NDI patients showed that NDI associated mental retardation is significantly lower than the literature suggests.
NDI can be inherited either as an X-linked or as a recessive or dominant autosomal trait. If it is X-linked, the mutated gene is carried on the X chromosome and the disorder will manifest in a male and be transmitted via females. The gene involved in X-linked NDI is the vasopressin receptor gene, V2R. If NDI is autosomal, the mutant gene is carried on a non-sex chromosome and the disorder can manifest in either female or male. The gene involved in autosomal NDI is the aquaporin-2 water channel gene (AQP2). Knoers, et al., have identified 41 different mutations in VC-JR and eight different mutations in AQP2. Remarkably, several women with NDI appeared to be manifesting carriers of V2R mutations. That is, they not only carried X-linked NDI; they had the disorder.
The mutations in V2R and AQP2 were studied at the functional level to see how the mutations interfered with V2R and AQP2's ability to perform their job. It appeared that not all V2R mutations result in the same level of kidney unresponsiveness to vasopressin. For example, mutation T204N in the V2R has a reduced, but not absent, functional capacity that can be relieved somewhat by giving the NDI patient who has this mutation a synthetic form of arginine vasopressin (DDAVP).
In order to concentrate urine, the AQP2 water channel has to be synthesized in the kidney cell and needs to be transported to the side of the cell where urine passes (the plasma membrane). To find out whether and why mutations in AQP2 cause NDI, AQP2 mutated proteins were expressed in cell cultures in the laboratory. Then the researchers followed the AQP2 as it tried to travel to their job site (the plasma membrane) and tested their ability to channel water through the membrane. It appeared that all AQP2 mutants (with autosomal recessive inheritance in the patient) were impaired at an early step in their routing to the plasma membrane. They couldn't get where they needed to go. An AQP2 mutation identified in a patient with dominant NDI was also impaired in its transport to the plasma membrane, but at a later step in the routing process. Presumably, the binding of this mutant to the normal AQP2 protein hinders the further transport of the normal AQP2 protein to the plasma membrane and might explain the dominant inheritance of NDI in this case.