RESEARCH AT CCSU                                            Research at Wesleyan

The first shorn rat (abbreviated shn) arose spontaneously at the Biological Sciences’ rat colony in 1994.  The most obvious physical characteristic or phenotype of shn is the lack of normal whiskers and hair over its entire body.  In addition, there are several interesting internal physical abnormalities.  The shorn rats die prematurely at around 12-14 months of age, while the typical lifespan of a rat is 24 months.  Post mortem examination of the rats reveals enlarged hearts and kidneys that are abnormal in color and shape, instead of being pink and smooth they are yellow and bumpy.  The general location of the mutation has been genetically mapped to rat chromosome 7 but the exact gene or genes that are involved remain unknown (Hall et al., 2000). 

The shorn genentic map has been refined in recent months within Dr. Thomas King’s laboratory (see Figure 1 Chrissluis et al. submitted).    Within the region of the mutation on rat chromosome 7 there lies a number of genes that are potential candidates for the shorn mutation.   These include Krt2, Hoxc, and Aqp.  The many physical characteristics of the shorn mutation make narrowing the choices of genes candidates for shorn difficult.  Krt2 is a family of genes that encodes the basic keratins, the major protein component of mammalian hair. The Hoxc gene cluster includes Hoxc13, a gene that is required for normal hair development, at least in mice (Godwin and Capecchi, 1998).   Aqp, Aquaporins, facilitate water transport and 5 of the Aquaporins figure prominently in the nephron of the kidney.   To date no role for Hoxc or Krt2 in the development or function of the mammalian kidney has been proposed leaving Aqp as the only candidate for the kidney abnormalities seen post mortem.   The other genes may be mutated as well in a complex mutation in the target area, such as with an inversion,  but the aquaporins are the most likely candidate responsible for kidney abnormalities. Using the kidneys as a guide we have decided to focus on the aquaporin genes (AQP2, AQP5, and AQP6) located on rat chromosome 7. Aquaporins are a family of water channel proteins that facilitate the transport of water across the cell membrane. To date 10 members of the aquaporin family have been identified.  This family of proteins is conserved in animals as well as in plants, bacteria, and yeast.  Aquaporins are small, hydrophobic, integral membrane proteins of approximately 30 kDa that span the membrane six times (see Figure 2).  Nowhere is the transport of water more important than in the kidney where water reabsorption depends on several of the 10 members of the aquaporin family. The role of Aquaporin 2 has been studied extensively in the kidney since mutations in this gene and its protein product figure prominently in diabetes insipidus.  Diabetes insipidus is a disease characterized by an inability to concentrate urine even under dehydrating conditions causing excessive water loss.   The water loss is attributed to; 1) Aquaporin 2 cannot be translocated into the apical membrane of the cells in the collecting duct of the kidney or 2) Aquaporin 2 is mutated so it cannot function as a water permeable protein (Marples et al. 1999).

Diagram of an aquaporin protein in a cell membrane.

APPROACH:

Kidneys from Shorn, RR (derived from Sprague-Dawley) or Charles River Hairless were isolated from unfasted rats and kidneys from Shorn and RR were isolated from fasted rats.  The kidneys were homogenized to create a lysate and immunoprecipitate with an anti-aquaporin-2 antibody.  The immunoprecipitates were resolved using SDS-PAGE and electrophoretically transferred to Immobilon-P.  The blots were probed with anti-aquaporin antibody.

Aquaporin-2 is present in the kidney cells of the collecting duct in a 29 KDa and 35 KDa (glycosylated form).  Fasting for 24 hours (no food or water) has been reported to increase the levels of aquaporin-2 expressed in the membrane of cells of the collecting duct.  Without fasting, we see a band corresponding to Aquaporin-2 in the Shorn and Charles River Hairless lysates but not in our RR rat immunoprecipitates.  After a 24 hour fast we detect a 2-3 fold increase in the expression levels of Aquaporin-2 in both the Shorn and RR rat immunoprecipitates.