SULFATIDES ARE REQUIRED FOR RENAL ADAPTATION TO CHRONIC METABOLIC

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Sulfatides are required for renal adaptation to chronic metabolic acidosis

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Paula Stettnera, Soline Bourgeoisb, Christian Marschingc,d,e, Milena Traykova-Braucha, Stefan Porubskya, Viola Nordströma, Carsten Hopfd,e, Robert Köstersf, Roger Sandhoffc,d,e, Herbert Wiegandta, Carsten A. Wagnerb, Hermann-Josef Grönea,e,1, and Richard Jennemanna,1

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a Department of Cellular and Molecular Pathology, German Cancer Research Center, D-69120 Heidelberg, Germany; bInstitute of Physiology and Center for Integrative Human Physiology, University of Zurich, CH-8057 Zurich, Switzerland; cLipid Pathobiochemistry Group, Department of Cellular and Molecular Pathology, German Cancer Research Center, D-69120 Heidelberg, Germany; dInstitute of Instrumental Analytics and Bioanalytics, Department of Biotechnology, University of Applied Sciences, D-68163 Mannheim, Germany; eCenter for Applied Biomedical Mass Spectrometry; and fHopital Tenon, Unité Mixte de Recherche S 702, Institut National de la Santé et de la Recherche Médicale/University of Paris 6, 75020 Paris, France

Edited* by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved May 1, 2013 (received for review October 16, 2012)

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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. cerebroside sulfotransferase deficiency

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ow 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 corticopapillary 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 www.pnas.org/cgi/doi/10.1073/pnas.1217775110

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). 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 Pax8 promoter (20). This approach was taken Q:14 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

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Author contributions: P.S., S.B., C.M., H.W., C.A.W., H.-J.G., and R.J. designed research; Q:9; 10 P.S., S.B., C.M., M.T.-B., S.P., V.N., R.S., H.-J.G., and R.J. performed research; C.H. and R.K. contributed new reagents/analytic tools; P.S., S.B., C.M., and R.J. analyzed data; and P.S., C.A.W., H.-J.G., and R.J. wrote the paper. The authors declare no conflict of interest. Glycosphingolipids are abbreviated according to the recommendations of the UPACInternational Union of Biochemistry Joint Commission (J Mol Biol. 1999;286:963-70). *This Direct Submission article had a prearranged editor. 1

To whom correspondence may be addressed. E-mail: [email protected] or h.-j. [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1217775110/-/DCSupplemental.

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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 (47). (C and D) Neutral (C) and acidic (D) GSL extracts from one control mouse and Ugcgf/f , Cstf/f, and Ugcgf/f Cstf/f Pax8Cre mice (n = 3 each) were separated by TLC. Aliquots from extracts corresponding to 4 mg of dry weight of kidneys were loaded. Pronounced reduction of both GlcCerbased and all sulfated GSLs (SM4s, SM3, and SB1a) was found in kidneys from Ugcgf/f Cstf/f Pax8Cre mice only. *GM3 ganglioside is obviously expressed in other cell types (endothelial, stromal) than those that express the Pax8Cre recombinase and remains therefore unaltered in mutant kidney (A and D). (E– G) MALDI-MSI of renal sulfatides in control mice and mice with kidney-specific sulfatide deficiency. (E) The sum of all detected isoforms of SM4s (red) and complex SM3 (green) that overlap in the papilla. (F) SM4s isoforms with hydroxylation in the fatty acid (red) are mainly detected in the inner medulla, whereas more lipophilic isoforms without this modification (green) are mainly detected in the papillary region. (G) Complex SM3 without any modification in the ceramide anchor (green) shows a specific location to the papilla, whereas more hydrophilic isoforms with a hydration of the double bond of the sphingoid base (red) are located to the renal cortex. (Scale bar, 1 mm.) C, cortex; IM, inner medulla; OM, outer medulla; P, papilla. Each section shown is representative of n = 3 mice per genotype.

concentration in the papilla, which is needed for appropriate ammonium excretion into urine under basal conditions and during metabolic acidosis. Results Cell-Specific Depletion of GlcCer-Derived and Sulfated GSLs in Mouse Kidney. Ugcgf/f Cstf/f Pax8Cre and respective single-enzyme mu-

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tant mice were born according to Mendelian inheritance. Southern blot analysis showed cell-specific disruption of the Ugcg and Cst genes in kidney as expected from the activity of the Pax8 promoter (SI Appendix, Fig. S1 A–G). Deletion of the gene products was verified by TLC of neutral and acidic GSLs extracted from mutant mice kidneys (Fig. 1 C and D and SI Appendix, Fig. S1I). In Ugcgf/f Cstf/+ Pax8Cre kidneys, GlcCerdependent neutral and acidic GSLs were strongly decreased compared with controls as evident from bands corresponding to the globoseries (Fig. 1C) and complex sulfatides (SM3, SB1a), whereas SM4s remained unaltered (Fig. 1D and SI Appendix, Fig. S1I). In contrast, in kidneys from Ugcgf/+ Cstf/f and Ugcgf/f Cstf/f Pax8Cre mice, all sulfated GSL species could be detected in only minute amounts. Remnant traces of SM4s were possibly due to cells in which Cre recombinase was not active (Fig. 1D and SI Appendix, Fig. S1I). Single disruption of Cst did not affect the synthesis of renal neutral GSLs. The strong band at the height of monohexosylceramide detected in kidneys with Cst deficiency (Ugcgf/+ Cstf/f and Ugcgf/f Cstf/f Pax8Cre kidneys) was due to an accumulation of GalCer, as reported previously (7). In addition, Gal2Cer (at the height of dihexosylceramide) increased in kidneys with Ugcg deficiency (Ugcgf/f Cstf/+ and Ugcgf/f Cstf/f Pax8Cre kidneys) (Fig. 1C). In line, no shift to sphingomyelin was seen in any of the mutant mice kidneys (SI Appendix, Fig. S1H). The compartment-specific distribution of sulfatide species and isoforms and their deficiency in mutant mice was further demonstrated on mouse kidney sections by applying MALDI massspectrometric imaging (MSI) (Fig. 1 E–G). Both, TLC of acidic GSL extracts from cortical, medullary, and papillary tissue from wild-type mice, as well as MSI revealed that SM4s and SM3 were concentrated in the papilla (Fig. 1 E–G and SI Appendix, Fig. S1J). Lack of Renal Sulfatides Leads to More Acidic Urinary pH. Renal Ugcg/Cst and respective single-enzyme–deficient mice had a normal life span. Kidney morphology appeared regular by light microscopy (SI Appendix, Fig. S2) and transmission electron 2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1217775110

microscopy. Creatinine clearance was similar compared with control littermates (SI Appendix, Table S1). However, all mutant mouse groups had a significantly lower urinary pH (Fig. 2A). Notably, urine of renal Cst- and Ugcg/Cst-deficient mice was more acidic compared with that of renal Ugcg-deficient animals, indicating the major role of sulfatides in urinary pH regulation (Fig. 2A). Additionally, mice with Cst and Ugcg/Cst deficiency had lower ammonium excretion (Fig. 2B) as well as higher urinary potassium and urinary output (SI Appendix, Table S1). Induction of Metabolic Acidosis in Renal Ugcg/Cst-Deficient Mice.

Despite the significantly lower urinary pH and reduced ammonium excretion, blood pH and bicarbonate levels in renal Ugcg/ Cst–double-deficient mice were in the physiological range under control diet suggesting compensatory mechanisms for altered renal acid–base handling. In fact, urinary excretion of titratable acids (TAs) in mutant mice was significantly higher compared

Fig. 2. Renal deficiency of sulfatides induced a urinary pH and ammonium excretion lower than in sulfatide-expressing kidney. Urinary pH and ammonium of renal Ugcg-, Cst-, and Ugcg/Cst-deficient mice is shown compared with their respective control littermates. (A) n = 58 in controls, n = 26 in Ugcgf/f Pax8Cre, n = 14 in Cstf/f Pax8Cre, and n = 19 in Ugcgf/f Cstf/f Pax8Cre mice. (B) n = 19 in controls, n = 7 in Ugcgf/f Pax8Cre, n = 8 in Cstf/f Pax8Cre, and n = 9 in Ugcgf/f Cstf/f Pax8Cre mice. Means ± SEM are shown (**P < 0.01; ***P < 0.001).

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with controls (SI Appendix, Table S1). Foremost renal Ugcg/Cst double-enzyme–deleted mice with no ability to compensate for the lack of sulfatides by de novo synthesis of Ugcg-derived anionic GSLs were chosen to study further the influence of sulfatide deficiency on renal handling of an acid challenge. In addition, single mutant mice were exposed to a chronic acid load. Mutant mice and respective control littermates were exposed to an acute (2 d) and chronic (9 d) hydrochloric acid (HCl) diet. The acute HCl load induced a significant decrease in blood pH and bicarbonate levels and an increase in blood chloride levels in both groups (Ugcg/Cst–double-deficient mice vs. controls). Control mice adapted well by a strong increase in ammonium excretion and partially recovered by day 9 their blood pH and bicarbonate (Fig. 3 A–C and E). Ugcg/Cst-deficient mice, however, showed a reduced capacity (−30%) to increase their ammonuria resulting in less net acid excretion (NAE) than controls (Fig. 3 E and F). Urinary pH in control mice declined significantly upon acute acid loading followed by a recovery during chronic acid challenge, whereas renal Ugcg/Cst-deficient mice maintained their low pH throughout the 9-d experimental period (Fig. 3D). The urinary phenotype in Ugcg/Cst-deficient mice was paralleled by chronic hyperchloremic metabolic acidosis as indicated by more acidic blood pH, lower blood bicarbonate, and decreased body weight compared with control mice (Fig. 3 A– C, SI Appendix, Fig. S3, and SI Appendix, Table S2). To exclude the possibility of involvement of neutral and other acidic glycosphingolipids, blood pH, bicarbonate, and chloride as well as urinary pH and ammonuria was measured in parallel in single- and double-mutant mice after chronic acid loading. Consistent with significantly reduced ammonium excretion, only mice lacking renal Cst expression showed significantly increased metabolic acidosis compared with controls (SI Appendix, Fig. S4 A–E). Reduced Papillary Ammonium Accumulation in Renal Cst- and Ugcg/ Cst-Deficient Mice. Medullary TAL ammonium reabsorption and

the associated cortico-papillary ammonium concentration gradient are enhanced during chronic acidosis (23, 24). To examine whether Ugcg/Cst-deficient mice are able to create such a gradient, we dissected cortical, medullary, and papillary parenchyma from kidneys of chronic acid-loaded mice. In the papilla, ammonium content was reduced by 30% in renal Ugcg/Cst-deficient

Expression and Activity of Ammoniagenic Enzymes and Ammonium Transporters in Renal Ugcg/Cst-Deficient Mice. Renal mRNA and

protein levels, respectively, of the proximal tubular glutamine transporter sodium-coupled neutral amino acid transporter (SNAT3) and of ammoniagenic enzymes such as kidney-specific glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) slightly increased in mutant vs. controls (Fig. 5 A–C, G, and H). The phosphate-dependent renal glutaminase activity was not significantly changed (SI Appendix, Fig. S4F). In addition, ammoniagenesis in isolated cortical proximal tubules from double-mutant mice was similar to controls (SI Appendix, Fig. S4G). Ammonium produced is secreted at least in part via the brush border membrane-localized Na+-H+/NH4+ exchanger-3 (NHE-3) into the lumen (25). NHE-3 mRNA levels in kidneys of renal Ugcg/Cst-deficient and control mice were similar (SI Appendix, Fig. S5A). In line, total NHE activity in isolated brush border membrane vesicles of Ugcg/Cst-deficient kidneys was unaffected, as revealed by using the acridine orange quenching method (SI Appendix, Fig. S5B). mRNA and protein expression, respectively, of transporters involved in TAL ammonium reabsorption such as the apical Na+-K+/NH4+-2Cl− cotransporter 2 (NKCC2), basolateral Na+/NH4+-H+ exchanger 4 (NHE-4), as well as electroneutral Na+-bicarbonate cotransporter 1 (NBCn1) were significantly increased or similar to controls in Ugcg/Cstdeficient kidneys (Fig. 5 D–H). mRNA expression of major acid–base transporters in the collecting duct (CD) was not affected in renal Ugcg/Cst-deficient mice (SI Appendix, Fig. S6 A–F). The cellular activity of some of those transporters can be also regulated by their subcellular localization (26, 27). However, the cellular distribution of the V-ATPase subunits V1B1 (SI Appendix, Fig. S6 G–J), V0a4, and the major ammonium transporter Rhcg (SI Appendix, Fig. S6 K– Q:16 N) was not changed.

Fig. 3. Mice with lack of renal sulfatides show impaired acid stress handling. Renal Ugcg/Cst-deficient mice (n = 6–12) and control littermates (n = 8–14) were exposed to an oral acid challenge with hydrochloric acid (HCl). (A) Blood pH; (B) blood bicarbonate; (C) blood chloride; (D) urinary pH. (E and F ) Reduced ammonuria (E ) in response to acid loading in renal Ugcg/Cst-deficient mice resulted in reduced net acid excretion (F) compared with control mice. Excretion of titratable acidity (TA) (F) was not reduced in mutant mice. Means ± SEM are shown (*P < 0.05; **P < 0.01; ***P < 0.001).

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kidneys. Medullary and cortical ammonium content did not significantly differ, although medullary ammonium content tended to be reduced. Renal Ugcg/Cst-deficient mice thus exhibited a significantly reduced cortico-papillary ammonium gradient compared with control littermates (Fig. 4A). In contrast, in mice with renal Ugcg deficiency, papillary ammonium content was not significantly changed compared with controls (Fig. 4B).

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Fig. 4. Ammonium content in papilla of renal sulfatide-deficient mice was significantly reduced compared with controls. (A) Ammonium content in dissected cortical, medullary, and papillary kidney tissue from 9-d acidloaded control and renal sulfatide-deficient mice was measured. n = 8 in controls and n = 12 in mutant mice. (B) Comparison of cortical and papillary ammonium content in Ugcgf/f-deficient, Cstf/f-deficient, and Ugcgf/f Cstf/f– double-deficient mouse kidneys (n = 5–7 per group). Mean ± SEM is shown (*P < 0.05).

Unaltered Transepithelial NH3 Transport in CDs from Renal Ugcg/CstDeficient Mice. The lack of sulfatides could possibly have influ-

enced transporter-mediated or diffusive epithelial ammonium permeability or proton transport in the CD (4). We assessed this question by two different approaches. First, we measured apical NH3, NH4+, and H+ excretion by the NH4Cl prepulse technique in in vitro-microperfused outer medullary CDs (OMCDs). The OMCD was chosen because it contains mostly acid-secretory type A intercalated cells but only very few bicarbonate-secretory type B intercalated cells. Second, we examined total transepithelial NH3 permeability in the in vitro microperfused cortical CDs (CCDs) by imposing a bath-to-lumen NH3 gradient in the nominal absence of an NH4+ gradient. Both approaches demonstrated that the capacity to transport NH3 and protons was not altered by the renal lack of Ugcg and Cst (Fig. 6). Q:17

The strongly lowered pH in Cst- and Cst/Ugcg-deficient mice was accompanied by lower ammonium excretion compared with control littermates. Lower urinary pH may be caused by a reduced capacity to buffer protons. Apparently, increased excretion of titratable acidity did not fully compensate for the lack of ammonium buffer in Ugcg/Cst-deficient mice. The lower amount of ammonium excretion is remarkable because it occurs in the presence of a steeper pH gradient. Nevertheless, acidic urine can increase the driving force for ammonium excretion, thereby partially compensating the defect in medullary ammonium accumulation and thereby partially rescuing the defect in ammonium excretion. By this mechanism, mice lacking the NHE-4 exchanger in the medullary TAL fully compensated deficient ammonium excretion at basic conditions (29). Lower urinary pH in combination with higher NAE, but impaired use of ammonium buffer, has been described as a characteristic for patients with uric acid nephrolithiasis and was also found in patients with metabolic syndrome and type 2 diabetes (30, 31). The mechanisms underlying the dysfunctional renal ammonium excretion in humans with either of these diseases have not been explored so far (30). In Zucker diabetic fatty rats, ammoniagenesis and ammonium excretion by the PT may be affected (32). Under standard diet, the reduced ammonium excretion (by about 4–5 mM·mM−1 creatinine) did not affect systemic acid– Q:18 base homeostasis in renal Cst- and Ugcg/Cst-deficient mice. However, during an oral acid load, increased excretion of TA could not substitute for the ∼30% reduction in urinary ammonium excretion. Renal Cst- and Ugcg/Cst-deficient mice developed hyperchloremic metabolic acidosis compared with controls; this phenomenon did not occur in renal Ugcg-deficient

Decreased AQP2 Protein Levels in Renal Ugcg/Cst-Deficient Mice.

Renal Ugcg/Cst-deficient mice showed higher urinary output and polydipsia under baseline and upon HCl loading (SI Appendix, Fig. S7 A and B). As renal sulfatides have been implicated in the urinary concentration process (10), we tested the ability of renal Ugcg/Cst-deficient mice to produce concentrated urine. Mice were exposed to an osmotic stress by water restriction for 24 h. Again, urine was less concentrated and urinary osmolality was lower in mutant mice (SI Appendix, Fig. S8 A and B). Plasma osmolality and renal osmotic gradient, as examined by measurement of tissue osmolality in separated cortical, outer medullary, and inner medullary tissue, were unaltered (SI Appendix, Fig. S8 C and D). In contrast, the expression of the AQP2 water channel was significantly decreased compared with controls indicating reduced water reabsorption in CDs of renal Ugcg/ Cst-deficient mice (SI Appendix, Fig. S8 E and F). Discussion Our study demonstrates that sulfatides influence urinary acidification; they are needed for acid–base homeostasis during acid loading. Cell-specific genetic deletion of sulfatide synthesis was achieved by disruption of GSL-specific sulfotransferase while the possible compensatory synthesis of other negatively charged GSLs was inhibited by deletion of glucosylceramide synthase (Ugcg) activity. Renal Ugcg-, Cst-, and Ugcg/Cst-deficient mice displayed lower urinary pH. By far more acidic urinary pH in renal Cst- and Ugcg/ Cst-deficient compared with single Ugcg-deficient mice per se indicated the crucial role of sulfatides, in particular SM4s. In support of this, Gb3 synthase−/− mice with exclusive deficiency for neutral globo-series GSLs, another major component of renal GSLs, showed regular urinary pH (28). 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1217775110

Fig. 5. Expression of enzymes and transporters relevant for ammonium production, TAL reabsorption, and delivery to the medullary interstitium. Q:23 (A–F) Total mRNA was isolated from kidneys of 9-d acid-loaded control and renal Ugcg/Cst-deficient mice (n = 5 or 6 per genotype) and analyzed by realtime RT-PCR. (A) The sodium-coupled neutral amino acid transporter (SNAT3) and (B) the mitochondrial glutaminase (GA) involved in production of NH4+, as well as (C) the cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) mediating the generation of HCO3− and (D) the apical Na+-K+/NH4+-2Cl− cotransporter 2 (NKCC2) mediating net NH4+ reabsorption in the TAL were significantly increased in renal Ugcg/Cst-deficient kidneys. (E) The basolateral Na+/NH4+-H+ exchanger 4 (NHE-4) and (F) the electroneutral Na+-bicarbonate cotransporter 1 (NBCn1) were elevated too, however not significantly. Means ± SEM are shown (*P < 0.05; **P < 0.01). (G and H) Western blot analysis of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH), both involved in the production of NH4+, as well as the NKCC2 cotransporter in chronic acid-loaded mice. The cytoplasmic and membrane protein samples shown are representative of n = 3 in controls and n = 5 in mutant mice. Intensities of bands were densitometrically evaluated. Data are expressed as mean ± SEM (*P < 0.05).

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Fig. 6. Similar proton and ammonia transport activity in CDs segments from renal sulfatide-deficient mice. (A–C) In vitro microperfusion experiments of single isolated CCDs and OMCDs. (A and B) Transepithelial NH3 and NH4+ inward (A) as well as proton outward movements (B) showed similar rates in intracellular alkalinization and pH recovery, suggesting unaltered luminal NH3 entry and proton secretion by Ugcg/Cst-deficient OMCD intercalated cells. Means ± SEM are shown (n = 4 mice per genotype and n = 1–2 OMCDs per mouse). (C) Direct measurements of transepithelial NH3 fluxes show unimpaired NH3 permeability of Ugcg/Cst-deficient CCD epithelium. Means ± SEM are shown (n = 7 mice per genotype; n = 1 CCD per mouse).

mice with unaffected sulfatide SM4s expression. Consistent with impaired ammoniuria, acidotic mutant mice with complete sulfatide deficiency had significantly lower ammonium concentration selectively in the papilla compared with control mice. Notably, the phenotype of reduced ammonuria as well as deficient papillary ammonium content upon chronic acid loading was slightly less pronounced in single Cst-deficient mice compared with Ugcg/Cst-deficient mice. This could possibly be explained by the synthesis of an Ugcg-derived, sialylated GSL in single Cst-deficient mice (below the expected band of SB1a) (Fig. 1D), which may have exerted compensatory function for the lack of anionic sulfatides. In contrast to other ions, ammonium is produced by the kidney itself and renal ammonium handling at baseline and during acidosis involves several nephron segments. Although the molecular mechanisms of renal ammonium transport are not completely elucidated, numerous proteins mediating NH3 or NH4+ transport along the renal tubule have been identified so far (4). Despite the pronounced reduced urinary ammonium excretion, acidosis-induced renal Ugcg/Cst-deficient mice showed slightly enhanced expression of ammoniagenic enzymes and glutaminase activity to control mice. In addition, increased expression of NKCC2 was detected. Both changes in the PT and the TAL were apparently compensatory and very likely exclude the possibility of reduced ammonium delivery to the TAL and medullary interstitium. Similar to characteristics of distal renal tubular acidosis (dRTA), acidotic renal Ugcg/Cst-deficient mice exhibited impaired NAE. However, classic dRTA is manifested by a high urinary pH even in the presence of systemic acidosis (33, 34). Furthermore, net NH3 and NH4+ fluxes as well as H+-ATPase– mediated proton secretion across the apical membrane in isolated OMCDs or transepithelial ammonia fluxes in CCDs of acidotic mutant mice were not reduced. Renal Ugcg/Cst-deficient mice had higher urinary K+ at baseline and upon acid loading. Sulfatides have been reported to modulate basolateral Na+-K+-ATPase activity in osmoregulatory organs of vertebrates and in the kidney of rodents and pig through different mechanisms (15, 17, 35, 36). Moreover, they have been shown to interact with H+-K+-ATPase and influence its activity in the rabbit gastric mucosa (16). However, the slight changes in blood K+ concentrations at baseline and upon acid loading (SI Appendix, Tables S1 and S2) were unlikely to affect renal acid–base handling in mutant mice (37, 38). Concomitantly, ammoniagenesis in isolated proximal tubular segments of double-mutant cortices was similar to controls. Stettner et al.

Renal Cst- and Ugcg/Cst-deficient mice had less concentrated urine at baseline and upon HCl loading. Lower ammonium excretion as well as polyuria may be a consequence of a general dysfunction of the countercurrent system. This was excluded as water-deprived renal Ugcg/Cst-deficient mice exhibited a normal gradient of tissue osmolality. We suggest that polyuria in sulfatide-deficient mice is caused by reduced water reabsorption due to the reactively decreased protein levels of the luminal water channel AQP2 in the CD. Renal tubular acidosis has been associated with polyuria (33). Increased water intake and diuresis have been suggested as a compensatory regulatory process during metabolic acidosis (39). Decreased water reabsorption by the late distal tubule and CD may reduce NH3 backflux from lumen to blood by several effects (24). At baseline and during water deprivation, Ugcg/Cstdeficient mice had normal systemic acid–base parameters. We surmise that, in sulfatide-deficient mice, AQP2 decreased in a compensatory mode at baseline and during the acid load. Based on our data, we hypothesize that renal sulfatidedeficient mice have an impaired electrostatic ability to keep high interstitial ammonium concentrations in the papilla. Titze et al. (40) have previously demonstrated that increased sodium storage in skin correlates with increased negative charge density provided by sulfated polyanionic glycosaminoglycans in the extracellular matrix of the skin. In analogy, kidney sulfatides may act as counterions of interstitial NH4+ neutralizing its positive charge and thereby facilitating its accumulation. We could demonstrate that sulfatation of GalCer is essential for the binding of NH4+ (SI Appendix, Fig. S9). Renal sulfatide deficiency therefore would result in decreased medullary/papillary interstitial ammonium retention and consequent backrelease into the systemic circulation through the renal vein. Consistent with this interpretation, renal Ugcg/Cstdeficient mice showed elevated levels of plasma urea, an ammonium metabolite of the liver (41). We did not observe an adaptive increase in renal sulfatide levels in wild-type mice upon acute as well as chronic acid loading. High concentrations of sulfatide in the inner medulla and papilla apparently provide sufficient capacity to cope with the bulk of renal ammonium in response to acidosis. In summary, we report a renal dysfunction caused by a renal Q:19 epithelia-specific genetic disruption of sulfatide synthesis. Our data suggest that renal sulfatides may be required to bind ammonium and maintain its high interstitial concentrations in the papilla needed for appropriate urinary ammonium excretion. The impact of sulfatides on renal ammonium excretion is highlighted by the impaired acid stress handling and resulting acidosis in sulfatide-deficient mice. Materials and Methods Generation of Ugcgf/f, Cstf/f, and Ugcgf/f Cstf/f Pax8Cre Mice. Mice with disruption of the Cst and/or Ugcg gene specifically in the kidney were generated by crossing Cst-, Ugcg-, and Ugcg/Cst-floxed mice, respectively, with mice expressing the Cre-recombinase under control of the Pax8 promoter (20) as described in SI Appendix. Generation of mutant mice and all experiments were approved and performed in accordance with federal laws (Regierungspräsidium Karlsruhe, Karlsruhe, Germany). Acid Loading. Acid loading, blood as well as urine pH, blood gases and electrolytes, titratable acids, bicarbonate, and glutaminase activity were measured as described in SI Appendix. Ammoniagenesis. Ammoniagenesis on isolated proximal tubule segments was measured as described in SI Appendix. GSLs Extraction and Detection. GSLs were extracted according to Sandhoff with modifications (42) and detected by TLC as described in SI Appendix. MALDI Imaging Mass Spectrometry. Mass-spectrometric measurements were performed using Autoflex III MALDI-TOF/TOF instruments (Bruker Daltonics) as described previously (43). Measurements of Tissue Ammonium and Osmolality. Ammonium content in kidney tissue was measured as previously described (29). Data were initially

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calculated as micromoles of ammonium per milligram of protein. Data of two independent experiments were pooled. Renal tissue osmolality measurements were performed as described (44).

measurements on isolated OMCDs and transepithelial NH3 permeability measurements on isolated CCDs from acid-loaded mice were performed as described previously (46).

Real-Time RT-PCR. Specific mRNA was analyzed using the LightCycler 2.0 System and the LightCyler-FastStart DNA MasterSYBR Green I kit (Roche Diagnostics). Primer sequences are described in SI Appendix.

Statistics. Datasets were compared by performing the Mann–Whitney test. Unless otherwise stated, data are expressed as means ± SEM. Differences were considered significant if P < 0.05.

Western Blotting. Proteins were isolated, blotted, and detected as described in SI Appendix.

ACKNOWLEDGMENTS. We thank S. Kaden, S. Meldner, U. Rothermel, C. Schmidt, T. Sijmonsma, S. Wang, and M. Volz for excellent technical assistance. We thank N. Curthoys and D. Weiner for kindly providing antibodies. H.-J.G. and S.P. were supported by German Research Foundation Grant Sonderforschungsbereich 938, and C.A.W. was supported by Swiss National Science Foundation Grant 31003A_138143. C.H., R.S., and H.-J.G. were supported by a joint grant (Zentrum für Angewandte Forschung–Applied Biomedical Mass Spectrometry) from the Landesstiftung Baden-Württemberg and the Europäischer Fonds für Regionale Entwicklung. Q:20

Immunofluorescence. Staining of formalin-fixed kidneys was performed on 3-μm paraffin sections as described in SI Appendix. Physiological in Vitro Studies. Brush border membrane vesicles were assessed by the acridine orange technique as described in ref. 45. Intracellular pH

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25. Bobulescu IA, Moe OW (2009) Luminal Na+/H+ exchange in the proximal tubule. Pflugers Arch 458(1):5–21. 26. Seshadri RM, et al. (2006) Changes in subcellular distribution of the ammonia transporter, Rhcg, in response to chronic metabolic acidosis. Am J Physiol Renal Physiol 290(6):F1443–F1452. 27. Stehberger PA, et al. (2003) Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis. J Am Soc Nephrol 14(12):3027–3038. 28. Porubsky S, et al. (2012) Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells. J Immunol 189(6):3007–3017. 29. Bourgeois S, et al. (2010) NHE4 is critical for the renal handling of ammonia in rodents. J Clin Invest 120(6):1895–1904. 30. Maalouf NM, Cameron MA, Moe OW, Sakhaee K (2010) Metabolic basis for low urine pH in type 2 diabetes. Clin J Am Soc Nephrol 5(7):1277–1281. 31. Sakhaee K (2009) Recent advances in the pathophysiology of nephrolithiasis. Kidney Int 75(6):585–595. 32. Bobulescu IA, Dubree M, Zhang J, McLeroy P, Moe OW (2009) Reduction of renal triglyceride accumulation: Effects on proximal tubule Na+/H+ exchange and urinary acidification. Am J Physiol Renal Physiol 297(5):F1419–F1426. 33. Pereira PC, Miranda DM, Oliveira EA, Silva AC (2009) Molecular pathophysiology of renal tubular acidosis. Curr Genomics 10(1):51–59. 34. Karet FE (2002) Inherited distal renal tubular acidosis. J Am Soc Nephrol 13(8): 2178–2184. 35. Jedlicki A, Zambrano F (1985) Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase. Arch Biochem Biophys 238(2):558–564. 36. Lingwood D, Harauz G, Ballantyne JS (2005) Regulation of fish gill Na+-K+-ATPase by selective sulfatide-enriched raft partitioning during seawater adaptation. J Biol Chem 280(44):36545–36550. 37. DuBose TD, Jr., Good DW (1991) Effects of chronic hyperkalemia on renal production and proximal tubule transport of ammonium in rats. Am J Physiol 260(5 Pt 2): F680–F687. 38. Good DW (1987) Effects of potassium on ammonia transport by medullary thick ascending limb of the rat. J Clin Invest 80(5):1358–1365. 39. Nowik M, Kampik NB, Mihailova M, Eladari D, Wagner CA (2010) Induction of metabolic acidosis with ammonium chloride (NH4Cl) in mice and rats—species differences and technical considerations. Cell Physiol Biochem 26(6):1059–1072. 40. Titze J, et al. (2004) Glycosaminoglycan polymerization may enable osmotically inactive Na+ storage in the skin. Am J Physiol Heart Circ Physiol 287(1):H203–H208. 41. Häussinger D, Lamers WH, Moorman AF (1992) Hepatocyte heterogeneity in the metabolism of amino acids and ammonia. Enzyme 46(1–3):72–93. 42. Sandhoff R, et al. (2002) Kidney sulfatides in mouse models of inherited glycosphingolipid disorders: Determination by nano-electrospray ionization tandem mass spectrometry. J Biol Chem 277(23):20386–20398. 43. Marsching C, Eckhardt M, Gröne HJ, Sandhoff R, Hopf C (2011) Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry. Anal Bioanal Chem 401(1):53–64. 44. Herrera M, Garvin JL (2005) A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1. Am J Physiol Renal Physiol 288(1):F58–F64. 45. Honegger KJ, et al. (2006) Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC). Proc Natl Acad Sci USA 103(3):803–808. 46. Biver S, et al. (2008) A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility. Nature 456(7220):339–343. 47. Jennemann R, et al. (2005) Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth. Proc Natl Acad Sci USA 102(35): 12459–12464.

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