From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters

Abstract

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

Figure 1

The expression and role of cotransporters of the SLC12 family in the kidney. KCC3 and KCC4 are expressed in the basolateral membrane. NKCC2 is present in the apical membrane driving the salt reabsorption and KCC4 in the basolateral membrane and participate in the KCl efflux. NCC is the salt reabsorption pathway. In the basolateral membrane heterodimers of Kir4.1 and Kir5.1 form the K⁺ efflux pathway. In the α intercalated cells KCC4 provides an efflux pathway for Cl⁻ allowing the continuous function of the Cl-HCO3 antiporter for the efflux of HCO3. In the B and non-A, non-B cells, KCC3 is present in the apical membrane functionally coupled with pendrin. In all cases the driven force for lumen to interstitum transport is due to the Na-K-ATPase located at the basolateral membrane, but it was not depicted to show mainly the role of the SLC12 transporters. CaSR, calcium sensing receptor; CNT/CD, connecting tubule and collecting duct. DCT, distal convoluted tubule; NCC, Na-Cl; NKCC2, Na-K-2Cl; PCT, proximal convoluted tubule; ROMK, renal outer medulla potassium channel; TAL, thick ascending limb.

Figure 2

Water living species that were key to identify the NCC and NKCC2 encoding genes. (A) Winter flounder (Psuedopleuronectes americanus). (B) African frog (Xenopus laevis). (C)+ Dogfish shark (Squalus acanthias).

Figure 3

The Hebert's laboratory approach for cloning the Na⁺-coupled chloride cotransporters. mRNA was extracted from Winter flounder's urinary bladder was used for functional expression cloning of NCC in the heterologous expression system of Xenopus laevis oocytes. Then, flounder's NCC probe was used to clone both, NCC and NKCC2 from rat and mouse cDNA libraries. Finally, taking advantage that transmembrane 1 and ten segments of NCC and NKCC2 exhibited almost 100% identity, degenerate primers from these sequences were used for PCR to clone NKCC1 from an inner medullary collecting duct cell line.

Figure 4

The Forbush's laboratory approach for cloning NKCC1 and NKCC2. Shark rectal gland proteins were subjected to separation of putative Na-K-2Cl cotransporter by [3H]-4-benzoyl-5-sulfamoy1-3-(3- theny1oxy)benzoic acid binding (a bumetanide-like photolabeling molecule). The isolated protein was then used to construct monoclonal antibodies that were used to screen a shark rectal gland cDNA library, to identify the NKCC1 cDNA. The, NKCC1 probes were used to clone NKCC2 from a rabbit renal cDNA library.

Figure 5

Phylogenetic tree of the SLC12A family of electroneutral cation-coupled chloride cotransporters. The name of the genes and transporters, the associated disease and the phenotype of knockout mice are depicted. Gamba G. Membranes. 2022;12:911.