455 Original Paper Cell Physiol Biochem 2007;20:455-464 Accepted: March 29, 2007 Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology Cellular Physiology and Biochemistr and Biochemistr and Biochemistr and Biochemistr and Biochemistry Copyright © 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com © 2007 S. Karger AG, Basel 1015-8987/07/0205-0455$23.50/0 Accessible online at: www.karger.com/cpb Absence of ClC5 in Knockout Mice Leads to Glycosuria, Impaired Renal Glucose Handling and Low Proximal Tubule GLUT2 Protein Expression 1,2 Jackson Souza-Menezes BSc, 2 Marcelo M. Morales PhD, 1 Deepali N. Tukaye MBBS, 1 Sandra E. Guggino PhD and 1 William B. Guggino PhD 1 Department of Physiology, The Johns Hopkins University, School of Medicine, Baltimore and, 2 Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro Marcelo Marcos Morales Instituto de Biofísica Carlos Chagas Filho-UFRJ CCS- Bloco G, 21949-900, Rio de Janeiro, RJ (Brasil) Tel. +55 21 2562-6572, Fax +55 21 2290-8193 E-Mail mmorales@biof.ufrj.br Key Words ClC-5  Glucose homeostasis  Megalin  Proximal tubule  Glucose transporters and insulin Abstract Glycosuria is one of the well-documented characteristics in ClC-5 knockout (KO) mice and patients with Dent’s disease. However, the underlying pathophysiology of its occurrence is unknown. In this study, we have compared ClC-5 KO mice with age and gender matched wild-type (WT) control mice to investigate if the underlying cause of manifested glycosuria is an impairment of glucose homeostasis and/or an alteration in expression levels of proximal tubule (PT) glucose transporters. We observed that, the blood glucose concentration (n=12, p<0.01) and the fractional excretion of glucose and insulin (n=6, p<0.05) were higher in KO mice. In contrast, the fasting blood glucose levels (n=7) were not significantly different in the two groups. Plasma glucose increased to a greater extent in KO mice (n=7, p<0.05) when challenged by an intraperitoneal injection of glucose. However, no peripheral tissue insulin resistance was observed following an intraperitoneal injection of insulin (n=9) in the KO mice. ELISA analysis demonstrated low plasma insulin concentrations after a 12 hour fasting period and also following glucose injection in KO mice. The total insulin released during a 2 hour period following glucose challenge was significantly lower in KO mice (n=6, p<0.05). By western blot, we observed a significant decrease in GLUT2 protein expression levels in isolated PT ((n=10, p<0.01)) of KO mice. This decrease in protein levels was corroborated by a significant decrease in GLUT2 mRNA levels estimated semi quantitatively by RT-PCR in isolated PT (n=10, p<0.01). No significant changes in mRNA expression levels of SGLT2, SGLT1 and GLUT1, as analyzed by RT-PCR, could be detected in the isolated PT (n=10). Also, we have shown by western blot analysis that expression of megalin is lower in the renal cortex of KO mice when compared to WT mice (n=3, p<0.05). Our results suggest that low plasma insulin concentration together with renal function changes observed in KO mice significantly contribute towards the glucose intolerance and documented glycosuria observed in this animal.