Cell Tissue Res (2005) 319: 299–313 DOI 10.1007/s00441-004-0999-y REGULAR ARTICLE Tatjana Haenggi . Marcus C. Schaub . Jean-Marc Fritschy Molecular heterogeneity of the dystrophin-associated protein complex in the mouse kidney nephron: differential alterations in the absence of utrophin and dystrophin Received: 14 April 2004 / Accepted: 8 September 2004 / Published online: 25 November 2004 # Springer-Verlag 2004 Abstract The dystrophin-associated protein complex Keywords Dp71 . Dystrophin-associated protein (DPC) consisting of syntrophin, dystrobrevin, and dystro- complex . Kidney epithelial cells . Mouse (mdx3Cv; glycan isoforms is associated either with dystrophin or its utrophin-knockout) homolog utrophin. It is present not only in muscle cells, but also in numerous tissues, including kidney, liver, and Abbreviations AChR: Acetylcholine receptor . AQP1/2: brain. Using high-resolution immunofluorescence imaging Aquaporin 1/2 . ATL: Ascending thin limbs . BV: Blood and Western blotting, we have investigated the effects of vessels . CNT: Connecting tubules . CCT: Cortical utrophin and dystrophin gene deletion on the formation collecting ducts . CTAL: Cortical thick ascending limbs . and membrane anchoring of the DPC in kidney epithelial DPC: Dystrophin-associated protein complex . DTL: cells, which co-express utrophin and low levels of the C- Descending thin limbs . IR: Immunoreactivity . MCT: terminal dystrophin isoform Dp71. We show that multiple, Medullar collecting ducts . MRP1/2: Multidrug resistant- molecularly distinct DPCs co-exist in the nephron; these associated protein 1/2 . MTAL: Medullar thick ascending DPCs have a segment-specific distribution and are only limbs . NKCC2: Na+/K+/Cl− cotransporter 2 . NMJ: partially associated with utrophin in the basal membrane Neuromuscular junction . PT: Proximal tubules of tubular epithelial cells. In utrophin-deficient mice, a selective reduction of β2-syntrophin has been observed in medullary tubular segments, whereas α1-syntrophin and Introduction β1-syntrophin are retained, concomintant with an upre- gulation of β-dystroglycan, β-dystrobrevin, and Dp71. The dystrophin-associated protein complex (DPC) is a These findings suggest that β2-syntrophin is dependent on multimeric membrane-spanning complex linked either to utrophin for association with the DPC, and that loss of dystrophin, a large X-linked cytoskeletal protein, or to its utrophin is partially compensated by Dp71, allowing the autosomal homolog utrophin (Matsumura et al. 1992). In preservation of the DPC in kidney epithelial cells. This skeletal muscle cells, the DPC is composed of α- hypothesis is confirmed by the almost complete loss of all dystroglycan (an extracellular protein bound to laminin), DPC proteins examined in mice lacking full-length β-dystroglycan and sarcoglycans (spanning the mem- utrophin and all C-terminal dystrophin isoforms brane), and dystrobrevins and syntrophins (located intra- (utrophin0/0/mdx3Cv). The DPC thus critically depends on cellularly). Utrophin and dystrophin anchor this complex these proteins for assembly and/or membrane localization to the cytoskeleton by binding to the intracellular tail of β- in kidney epithelial cells. dystroglycan and to actin filaments (Way et al. 1992; Winder et al. 1995). Utrophin is selectively localized at the neuromuscular junction (NMJ), whereas dystrophin is present in the NMJ as well as along the inner face of the This project was supported by the Swiss National Science surface membrane, the sarcolemma. The DPC seems to Foundation (no. 31-63901.00 to J.M.F.). contribute to the aggregation of acetylcholine receptors T. Haenggi . M. C. Schaub (*) . J.-M. Fritschy (AChRs) at the NMJ and to enhance the structural stability Institute of Pharmacology and Toxicology, University of of the sarcolemma (Grady et al. 2000). Association of Zurich, signaling molecules, such as nitric oxide synthase, α5β1 Winterthurerstrasse 190, integrin, and focal adhesion kinase, with the DPC also 8057 Zurich, Switzerland e-mail: [email protected] suggests a role in signal transduction (Bredt 1999; Hillier Tel.: +41-1-6355919 et al. 1999; Winder 2001). Disruption of the DPC by Fax: +41-1-6356874 300 mutations in its main components results in severe muscle proteins are not readily detected in morphologically well- dystrophies. preserved tissue, and the molecular composition of the Dystrophin is expressed mainly in skeletal and cardiac DPC may be cell-type specific (Loh et al. 2000). muscle and in the brain (Mehler 2000; Blake et al. 2002). The aim of the present study has been to investigate the Short C-terminal dystrophin isoforms, ranging in size from role of utrophin and the short dystrophin isoform Dp71 for 71 kDa to 260 kDa and lacking the actin-binding domain, the assembly and subcellular localization of the DPC in have a more widespread distribution (Ahn and Kunkel the various kidney nephron segments. Dp71 has been 1993; Austin et al. 1995; Sadoulet-Puccio and Kunkel reported to represent the main dystrophin isoform in 1996; Durbeej et al. 1997; Culligan et al. 1998; Lidov and kidney (Lumeng et al. 1999; Loh et al. 2000, 2001). The Kunkel 1998; Blake et al. 1999, 2002; Lumeng et al. 1999; distribution of DPC proteins was analyzed by high- Chavez et al. 2000). Mice lacking dystrophin and all its C- resolution immunofluorescence staining in wild-type and terminal isoforms including Dp71 (mdx3Cv) are viable, mutant mice (utrophin0/0 and utrophin0/0/mdx3Cv). Chang- allowing the role of these proteins in non-muscle tissues to es in their expression levels have been assessed by be investigated (Cox et al. 1993). Utrophin has a Western blotting experiments. The results reveal a distinct ubiquitous expression pattern, with high levels in lung, molecular heterogeneity of the DPC in the different kidney, liver, nervous system, and blood vessels (Love et segments of the nephron, identified with specific markers. al. 1993; Dixon et al. 1997; Grady et al. 1997a; Tinsley et The DPC exhibits a polarized distribution in the basal al. 1998; Rafael et al. 1999; Knuesel et al. 2000; Loh et al. membrane of tubular epithelial cells in partial dependence 2000; Raats et al. 2000; Regele et al. 2000; Zuellig et al. on utrophin expression. However, in the absence of 2000; Jimenez-Mallebrera et al. 2003; Haenggi et al. utrophin and all dystrophin isoforms in utrophin0/0/mdx3Cv 2004). DPC proteins are molecularly heterogeneous, with mice, the DPC is completely disrupted throughout the the isoforms of each protein being encoded by distinct nephron, demonstrating that the assembly and/or mem- genes or being generated by alternative splicing or brane localization of the complex critically depends on differential promoter usage. In particular, dystrobrevins, utrophin and Dp71. which are structurally homologous to dystrophin and utrophin, are encoded by two separate genes, α-dystro- brevin and β-dystrobrevin, the former being alternatively Materials and methods spliced into five distinct isoforms (Blake et al. 1996; Sadoulet-Puccio et al. 1996; Ambrose et al. 1997; Peters et Animals al. 1997b; Nawrotzki et al. 1998; Newey et al. 2001). The syntrophin family likewise comprises several Utrophin0/0 mice were generated by crossing dystrophin– isoforms in muscle encoded by separate genes (α1, β1, utrophin double-mutants (mdx/utrophin0/0, generated on a β2) and two brain-specific genes (γ1 and γ2; Piluso et al. mixed 129-C57Bl/6 background; Grady et al. 1997a), with 2000; Blake et al. 2002). Whereas sarcoglycans are wild-type C57Bl/6 mice (Institute of Animal Science, muscle-specific (Blake et al. 2002), the dystrobrevin and University of Zurich) giving an F1 generation heterozy- syntrophin isoforms exhibit a differential tissue distribu- gous for utrophin (utrophin+/0; males) or utrophin and tion and are selectively associated with either dystrophin dystrophin (mdx: utrophin+/0; females). Utrophin0/0 mice or utrophin, suggesting the existence of several molecu- were obtained by the crossbreeding of heterozygous or larly and functionally distinct DPCs in non-muscle cells. homozygous mice. Utrophin0/0/mdx3Cv double-knockout Analysis of mice with targeted mutations has revealed mice were generated by crossing utrophin0/0 mice with the interdependence of the DPC with either utrophin or mdx3Cv mice (Jackson Laboratories, Bar Harbor, Me., dystrophin for assembly in muscle. For instance, mice USA) giving an F1 generation heterozygous for utrophin with dystroglycan-deficient skeletal muscles and mice (utrophin+/0; females) and hemizygous for dystrophin lacking α-syntrophin show severely reduced levels of (mdx3Cv; males). Utrophin0/0/mdx3Cv double-knockout utrophin at the NMJ (Cote et al. 1999; Adams et al. 2000). mice were obtained by the crossbreeding of heterozygous Conversely, α-dystrobrevins and the syntrophins disap- mice. For genotyping, DNA from tail biopsies was pear from the muscle sarcolemma in the absence of analyzed by the polymerase chain reaction (PCR). The dystrophin (mdx mice) but are retained at the NMJ because double-knockout mice lived only 3–4 weeks postpartum. of their association with utrophin (Deconinck et al. 1997; They displayed severe muscle impairment and were Grady et al. 1997a; Peters et al. 1997b). The phenotype of smaller by one third than wild-type mice. All animal utrophin0/0 mice has not been investigated in detail. These experiments were approved by the Cantonal Veterinary mice are fertile and exhibit normal behavior and life span. Office of Zurich. Kidney tissue of