New Insights into the Pivotal Roles of Claudins in Proximal Tubule Electrolyte Reabsorption

• proximal tubule
• claudins

The extraordinarily high GFR of the kidneys (160–180 L of plasma ultrafiltrate per day in humans) is greatly in excess of what is required to maintain electrolyte and fluid balance. Thus, extraordinarily high energy production and oxygen consumption are needed to reabsorb >99% of the filtered Na⁺. The proximal tubule is the site where 60%–70% of the filtered Na⁺ is reabsorbed by active transcellular and passive paracellular pathways.^1,2 The latter is important for allowing overall Na⁺ reabsorption to occur at less energy cost, and with less oxygen consumption than predicted by the 3 Na⁺ per ATP stoichiometry of the basolateral membrane Na,K-ATPase that mediates primary active Na⁺ transport.³

In the early proximal tubule, Na⁺ reabsorption is predominantly transcellular and takes place in part by electrogenic Na⁺-solute cotransporters, such as Na⁺-glucose cotransporter SGLT2, which generate a small lumen negative potential difference that drives paracellular Cl⁻ reabsorption in parallel. A much larger quantity of transcellular Na⁺ reabsorption occurs by Na⁺-H⁺ exchanger NHE3 that has the effect of reabsorbing NaHCO₃. Reabsorption of solutes in the proximal tubule drives nearly isosmotic water reabsorption, due to very high constitutive water permeability, principally mediated by aquaporin-1 water channels. The nearly isosmotic reabsorbate in the early proximal tubule is enriched in NaHCO₃ relative to NaCl, so the luminal fluid left behind has a progressively falling HCO₃ concentration and rising Cl⁻ concentration. Consequently, a lumen to plasma Cl⁻ concentration difference and an equal and opposite HCO3⁻ concentration difference are generated. Because the paracellular permeability to Cl⁻ is much higher than that of HCO₃⁻, passive Cl⁻ reabsorption is favored over HCO₃⁻ backflux, and a lumen-positive potential difference is generated. This, in turn, provides a driving force for passive paracellular Na⁺ reabsorption despite the absence of a transtubular Na⁺ concentration gradient. The result is a brisk rate of passive paracellular NaCl reabsorption, representing as much as 50%–60% of overall proximal tubule Na⁺ reabsorption.

This model for passive paracellular NaCl reabsorption requires appropriate Na⁺ and Cl⁻ permeabilities of the tight junctions of the proximal tubule. Claudins that form paracellular ion channels are major determinants of the ion selectivity of the tight junction.³ Previous studies have demonstrated the pivotal role of claudin-2 in mediating paracellular Na⁺ permeability, thereby facilitating NaCl reabsorption in the proximal tubule.^4,5 But which claudin is responsible for the selective Cl⁻ over HCO₃⁻ anion permeability that is required for passive paracellular NaCl reabsorption in this nephron segment? And what are the functional consequences of the loss of this selective Cl⁻ permeability? These important questions have been answered by the elegant and comprehensive studies by Breiderhoff and colleagues reported in this issue of JASN.⁶

Claudin-10a and claudin-10b are splice isoforms of claudin-10 that differ in ion selectivity and sites of expression along the nephron. Whereas claudin-10a is anion selective and expressed in the proximal tubule, claudin-10b is cation selective and expressed in the thick ascending limb.⁶⇓^–8 In their new study, Breiderhoff et al. generated knockout mice specifically deficient for expression of the claudin-10a isoform.⁶ They found that proximal tubules from claudin-10a null mice have a large decrement in transtubular Cl⁻ permeability and complete loss of selectivity for Cl⁻ over HCO₃⁻. Thus, they established that claudin-10a is responsible for the selective Cl⁻ over HCO3⁻ permeability that is a crucial requirement for passive paracellular NaCl reabsorption to take place in the proximal tubule.

The investigators measured Li⁺ clearance and magnitude of diuresis in response to furosemide or hydrochlorothiazide as measures of NaCl delivery out of the proximal tubule. Using these indirect methods for assessing proximal tubule function, no net change in proximal tubule Na⁺ reabsorption was detected in claudin-10a null mice. However, there were several additional observations suggesting there is a decrement in passive paracellular NaCl reabsorption in the proximal tubule of claudin-10a null mice that results in compensatory responses within the proximal tubule itself and in downstream nephron segments. In microdissected proximal tubules of claudin-10a null mice, RNA sequencing analyses revealed an increased expression of genes involved in energy metabolism, and respirometry measurements demonstrated increased oxygen consumption. These findings suggested enhanced transcellular Na⁺ reabsorption in the proximal tubule as an adaptation to loss of claudin-10a. Moreover, immunoblotting and immunofluorescence microscopy demonstrated increased expression of NKCC2 and NCC, and the activated, phosphorylated form of NCC in kidneys of claudin-10a null mice. These findings suggested increased Na⁺ reabsorption in the thick ascending limb and distal convoluted tubule as additional adaptations to the loss of claudin-10a expression in the proximal tubule.

How might enhanced transcellular NaCl reabsorption take place as an adaptation to decreased paracellular NaCl reabsorption resulting from deletion of claudin-10a? Previous studies had demonstrated that the proximal tubule is capable of mediating transcellular NaCl reabsorption by Cl⁻–base (i.e., Cl^––OH⁻, Cl^––HCO₃⁻, or Cl^––formate) exchange functionally coupled to apical membrane Na⁺-H⁺ exchange, and by apical membrane Cl⁻-oxalate exchange functionally coupled to oxalate-sulfate exchange and Na⁺-sulfate cotransport.^1,2 Anion transporter SLC26A6 expressed on the apical membrane of proximal tubule cells is capable of operating in the multiple modes of Cl⁻-anion exchange described in the proximal tubule. Although studies of SLC26A6 null mice did not detect a defect in proximal tubule NaCl reabsorption under in vivo conditions when passive paracellular NaCl reabsorption is operating normally,⁹ it is possible that SLC26A6 contributes more significantly to transtubular NaCl reabsorption as part of the adaptation to deletion of claudin-10a when there is decreased paracellular NaCl reabsorption.

Breiderhoff and colleagues observed important additional features of the phenotype of claudin-10a null mice that were due not to loss of anion permeability, but rather to enhanced paracellular cation permeability resulting from redistribution of claudin-2 into the tight junctions.⁶ Claudin-2 redistribution in the claudin-10a knockout mice was associated with increased paracellular Ca⁺⁺ and Mg⁺⁺ permeability that, in turn, correlated with reduced fractional excretion of Ca⁺⁺ and Mg⁺⁺ and mild elevation of serum Mg⁺⁺. These findings are important because they suggest the proximal tubule may contribute to the abnormalities in electrolyte handling found in some patients with HELIX syndrome. HELIX syndrome, due to biallelic loss of function mutations in claudin-10, has a clinical phenotype of hypohydrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia.¹⁰ The abnormal electrolyte phenotype includes salt wasting with secondary aldosteronism and hypokalemia, and hypocalciuria and hypermagnesemia.¹⁰ Mutations causing HELIX syndrome either disrupt function of claudin-10b or disrupt function of both claudin-10b and claudin-10a. A similar phenotype of electrolyte abnormalities had been described in mice with selective deletion of claudin-10b expression in the thick ascending limb, which led to decreased paracellular Na⁺ permeability, resulting in reduced Na reabsorption and enhanced reabsorption of Ca⁺⁺ and Mg⁺⁺.⁸ Accordingly, the electrolyte abnormalities in HELIX syndrome have been attributed to the loss of claudin-10b expression in the thick ascending limb.¹⁰ The new findings of Breiderhoff et al. that deletion of claudin-10a in the proximal tubule leads to enhanced Ca⁺⁺ and Mg⁺⁺ reabsorption suggest altered proximal tubule transport may contribute to the electrolyte abnormalities in patients with HELIX syndrome who have mutations affecting both claudin-10a and claudin-10b.

Taken together, the findings in this new study by Breiderhoff et al. have greatly advanced our understanding of the physiologic and pathophysiologic roles of claudins in the proximal tubule.⁶ But new questions now arise. What are the mechanisms by which transcellular NaCl reabsorption in the proximal tubule takes place and is enhanced when paracellular NaCl reabsorption is reduced? What are the mechanisms leading to increased expression of Na⁺ transporters in downstream nephron segments when paracellular NaCl reabsorption in the proximal tubule is reduced? What causes claudin-2 to redistribute into the tight junctions of the proximal tubule when claudin-10a is absent? Is there a detectable clinical phenotype in patients with genetic variants causing selective loss of expression of claudin-10a? Clearly, many more secrets of proximal tubule physiology and pathophysiology remain to be revealed by future studies.

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