Profiling of renal tubule Na+ transporter abundances in NHE3 and NCC null mice using targeted proteomics

The Na+–H+ exchanger NHE3 and the thiazide-sensitive Na+–Cl− cotransporter NCC are the major apical sodium transporters in the proximal convoluted tubule and the distal convoluted tubule of the kidney, respectively. We investigated the mechanism of compensation that allows maintenance of sodium balance in NHE3 knockout mice and in NCC knockout mice. We used a so-called ‘targeted proteomics’ approach, which profiles the entire renal tubule with regard to changes in Na+ transporter and aquaporin abundance in response to the gene deletions. Specific antibodies to the Na+ transporters and aquaporins expressed along the nephron were utilized to determine the relative abundance of each transporter. Semiquantitative immunoblotting was used which gives an estimate of the percentage change in abundance of each transporter in knockout compared with wild-type mice. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. This is thought to be an aldosterone-stimulated form of γ-ENaC. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. In conclusion, both mouse knockout models demonstrated successful compensation for loss of the deleted transporter. More extensive adaptation occurred in the case of the NHE3 knockout, presumably because NHE3 is responsible for much more sodium absorption in normal mice than in NCC knockout mice. The rate of sodium excretion by the kidney is the rate of sodium filtration by the glomerulus minus the net rate of sodium reabsorption along the renal tubule. The renal tubule consists of several distinct segments, each with distinct sodium transporters. The sites of expression of the major apical Na+ transporters are summarized in Fig. 1. The predominant apical Na+ transporter in the proximal tubule is NHE3, the type 3 Na+–H+ exchanger (Biemesderfer et al. 1993). In the distal convoluted tubule, the predominant apical Na+ transporter is NCC, the thiazide-sensitive Na+–Cl− cotransporter (Kaplan et al. 1996). The genes for these two transporters have been knocked out in mice as described previously (Schultheis et al. 1998a,b). Figure 1 Sodium transporter distribution along the nephron In previous studies (Schultheis et al. 1998a) the most prominent effects in homozygous NHE3 knockout mice were in the gastro-intestinal tract, with dilatation of the small bowel and sustained diarrhoea. These mice exhibited a mild metabolic acidosis and had decreased arterial blood pressure. There was an increase in their circulating aldosterone levels, along with an increase in renin mRNA expression in the kidney. The most prominent effect in homozygous NCC knockout mice was decreased arterial blood pressure when placed on a low salt diet (Schultheis et al. 1998b). In addition, these mice displayed increased renin mRNA levels in the kidney, hypomagnesemia and hypocalciuria. These two knockout models are the focus of the present study. The other apical Na+ transporters described in Fig. 1 are: (1) NaPi-2, the sodium-phosphate cotransporter expressed in proximal tubule (Biber et al. 1998); (2) NKCC-2, the bumetanide-sensitive Na+-K+-2Cl− cotransporter expressed in the thick ascending limb of Henle's loop (Kaplan et al. 1996); and (3) ENaC, the amiloride-sensitive Na+ channel of the connecting tubule and the collecting duct, consisting of α-, β- and γ-subunits (Garty & Palmer, 1997). The major basolateral sodium transporter is the same in all segments, the Na+-K+-ATPase, which in the kidney consists of a complex of α1 and β1 subunit proteins (Lingrel et al. 1994). The Na+-K+-ATPase is the primary active transporter responsible for the maintenance of low intracellular sodium concentration and a negative intracellular electrical potential in all renal tubule epithelial cells. The electrochemical driving force for apical sodium entry through the various apical Na+ transporters described above is generated by the Na+-K+-ATPase in each segment. The cDNAs encoding the apical Na+ transporters described in Fig. 1 have been cloned, allowing the production of peptide-directed rabbit polyclonal antibodies to each (Fernandez-Llama et al. 1998; Kim et al. 1998, 1999, 2000; Masilamani et al. 1999). This ensemble of antibodies has allowed the development of a so-called ‘targeted proteomics’ approach (Masilamani et al. 1999; Kim et al. 2000) to the analysis of renal adaptation. This approach profiles the entire renal tubule with regard to changes in apical Na+ transporter and aquaporin abundance in response to specific systemic physiological perturbations. Here we use this transporter-profiling approach to investigate the mechanism of compensation that allows maintenance of sodium balance in the NHE3 and NCC knockout mice. We hypothesize that the deletion of an individual sodium transporter will result in compensatory increases in abundance of one or more of the remaining Na+ transporters. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the γ-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance.