Defective Aquaporin-2 Trafficking in Nephrogenic Diabetes Insipidus and Correction by Chemical Chaperones

Title: Defective Aquaporin-2 Trafficking in Nephrogenic Diabetes Insipidus and Correction by Chemical Chaperones
Authors: Tamarappoo, B.K.; Verkman, Alan S.
Publisher: Journal of Clinical Investigation
Date Published: May 01, 1998
Reference Number: 161
Five single-point aquaporin-2 (AQP2) mutations that cause non-X-linked nephrogenic diabetes insipidus (NDI) were characterized to establish the cellular defect and to develop therapeutic strategies. In Xenopus oocytes expressing AQP2 cRNAs, single-channel water permeabilities of mutants L22V, T126M, and A147T were similar to that of wild-type AQP2, whereas R187C and C181W were nonfunctional. In [35S] methionine pulse-chase experiments in transiently transfected CHO cells, half-times for AQP2 degradation were ~ 4 h for wild-type AQP2 and L22V, and mildly decreased for T126M (2.7 h), C181W (2.4 h), R187C (2.0 h), and A147T (1.8 h). Immunofluorescence showed three distinct AQP2-staining patterns: plasma membrane and endosomal staining (wild-type, L22V), endoplasmic reticulum (ER) staining (T126M > A147T ~ R187C), or a mixed pattern of reticular and perinuclear vesicular staining. Immunoblot of fractionated vesicles confirmed primary ER localization of T126M, R187C, and A147T. To determine if the AQP2-trafficking defect is correctable, cells were incubated with the "chemical chaperone" glycerol for 48 h. Immunoblot showed that glycerol produced a nearly complete redistribution of AQP2 (T126M, A147T, and R187C) from ER to membrane/endosome fractions. Immunofluorescence confirmed the cellular redistribution. Redistribution of AQP2 mutants was also demonstrated in transfected MDCK cells, and using the chaperones TMAO and DMSO in place of glycerol in CHO cells. Water permeability measurements indicated that functional correction was achieved. These results indicate defective mammalian cell processing of mutant AQP2 water channels in NDI, and provide evidence for pharmacological correction of the processing defect by chemical chaperones.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

In order to concentrate urine, the principal cell membranes of the kidney collecting duct must become more water permeable than usual. The signal that sets off the increase in cell membrane permeability comes from the antidiuretic hormone, arginine vasopressin (AVP). If this increase does not occur, the result is nephrogenic diabetes insipidus (NDI), a disorder marked by the kidney's inability to concentrate urine.

One cause of NDI is mutations of the aquaporin-2 gene (AQP2). AQP2 is the water-transporting protein that, when signaled by AVP, inserts itself into the kidney collecting-duct principal cells to allow more water than usual to flow through them. Mutated AQP2 genes can produce defective AQP2s that fail to do their job, resulting in NDI.

There are several reasons why mutant AQP2s could fail to do their job: they could be absent, intrinsically nonfunctional, improperly processed and therefore subject to rapid degradation, or not able to travel from their holding place (the endoplasmic reticulum) inside the cell. Tamarappoo and Verkman investigated to discover and define the mechanism(s) by which five AQP2 mutants failed to do their job.

The authors studied these five mutant AQP2s, each of which has been known to cause NDI, in laboratory cell cultures. Although some of the AQP2 mutants showed mild differences in their structural stability and their ability to increase water permeability, none could reach the cell membrane. Had they been able to, they would have inserted themselves in order to increase membrane permeability. Instead, these AQP2s were detained in the endoplasmic reticulum (ER).

Newly synthesized proteins like AQP2 are further modified in the ER. The ER then exerts a quality control function, and if the protein is improperly folded or not fully assembled, then the ER will not let the protein out and targets it for degradation.

The accumulation of the mutant AQP2s in the ER suggested they were misfolded or assembled incorrectly. The authors thought that chemical agents that promote protein-folding might help the mutant AQP2s travel from the ER to the cell membrane. They took one such chemical chaperone (a chemical that can guide a protein), glycerol, and added it to the cell cultures with the mutant AQP2s for 40 hours. They then analyzed the cell culture and found that four of the mutant strains of AQP2 were able to make it out of the ER to the cell membrane. The authors tested two other chemical chaperones, TMAO and DMSO, and found they also could correct the ER-retention of AQP2 mutants. The success of the chemical chaperone in laboratory cell cultures suggests that they may have similar success in living beings. The authors feel the next step is to test this hypothesis in mice afflicted with NDI.