Sulfatides are required for renal adaptation to chronic metabolic acidosis

Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH₃ gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na⁺-K⁺/NH₄⁺-2Cl⁻ cotransporter 2 were higher, and transepithelial NH₃ transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid–base homeostasis.Low blood pH, as it occurs in metabolic acidosis, affects cellular functions and can lead to increased morbidity and mortality (1). The mammalian kidney plays a central role in the regulation of extracellular osmolality and fluid volume as well as the maintenance of blood pH in a narrow range of pH 7.35–7.45 (2). Excess protons are buffered by bicarbonate synthesized during renal ammoniagenesis and excreted into urine mainly bound to NH₄⁺ (ammonium) and titratable acids. During metabolic acidosis, enhanced ammonium excretion accounts for more than 80% of the increase in urinary net acid excretion in humans and rodents (3, 4). Ammonium (NH₃ and NH₄⁺) is produced in proximal tubular (PT) epithelia, secreted into the PT lumen, and is largely reabsorbed in the thick ascending limb of Henle’s loop (TAL). This process results in high interstitial ammonium concentrations in the medulla and the papilla and thereby, in parallel to the gradient of hypertonicity, in a cortico-papillary ammonium gradient facilitating secretion of ammonium into medullary collecting ducts (4). Several transport proteins have been shown to mediate medullary TAL NH₄⁺ reabsorption (5). However, the mechanisms that underlie the maintenance of high interstitial NH₄⁺ concentrations in the medulla and papilla, thereby avoiding backflux into the systemic circulation, have remained unexplored.Sulfatides are a subclass of anionic glycosphingolipids (GSLs), which consist of ceramide and carbohydrate residues to which one or several sulfate esters are bound via enzymatic catalysis by cerebroside sulfotransferases (CST; Cst). In mammals, sulfatides accumulate in the kidney with particularly high concentrations in the distal nephron segments and the renal medulla (6). The major renal sulfatide in humans and rodents is the galactosylceramide (GalCer)-derived SM4s. Other sulfated GSL species such as the glucosylceramide (GlcCer)-derived SM3 (sulfated lactosylceramide) in humans and mice and SB1a (gangliotetraosylceramide-bis-sulfate) in mice are even more polar than SM4s (7) (Fig. 1A).Fig. 1.Pax8-driven deletion of renal neutral and sulfated GSLs. (A) Ugcg- and Cst-dependent synthesis of neutral and acidic GSLs in the mouse kidney. (B) Cloning strategy for the disruption of the Cst gene. Disruption of the Ugcg gene has been described previously ...Various kidney diseases such as renal cell carcinoma and polycystic kidney disease are associated with disturbances in renal sulfatide metabolism (8, 9). However, the basic physiological function of renal sulfatides is not known. They are mainly found in the outer part of the plasma membrane. Apart from mediating cellular interactions with various external ligands, e.g., l-selectin, the anionic charge carried by sulfatides on the membrane surface may point to a modulatory role in membrane ion fluxes and/or binding of cationic extracellular substrates (7, 10, 11). Sulfatides at the cell membrane were surmised to function as ion barriers to extracellular osmolality oscillation (12, 13). Furthermore, they have been discussed to act as cofactors of basolateral Na⁺-K⁺-ATPase activity by binding K⁺ or by facilitating the membrane relocalization of the enzyme (14–16). Zalc et al. (17) have proposed a role of sulfatides rather in passive sodium chloride diffusion in the TAL.However, renal abnormalities have not been reported either in mice with systemic disruption of UDP-galactose:ceramide galactosyltransferase (Cgt; CGT) lacking SM4s or in Cst^−/− mice with deficiency of all sulfatides most probably due to the dominant and finally lethal central nervous system phenotypes (18, 19).The aim of this study was to assess in vivo the hypothesis that sulfatides are involved in ion transport processes in the kidney by a combined molecular genetic and physiological approach. Therefore, we have generated mice with disruption of the Cst gene and UDP-glucose:ceramide glucosyltransferase (Ugcg) gene and combinations of both in a tubular epithelial cell-specific manner under control of the paired box gene 8 (Pax8) promoter (20). This approach was taken to reduce the possibility for compensatory synthesis of charged GSLs as well as to circumvent systemic effects (18, 21, 22). Here, we show that sulfatides, most probably by their anionic extracellular charge, are required to maintain high interstitial ammonium concentration in the papilla, which is needed for appropriate ammonium excretion into urine under basal conditions and during metabolic acidosis.