Primary polydipsia, but not accumulated ceramide, causes lethal renal damage in saposin D-deficient mice.

Saposin D-deficient (Sap-D(-/-)) mice develop polydipsia/polyuria and die prematurely due to renal failure with robust hydronephrosis. Such symptoms emerged when they were around 3 mo of age. To investigate the pathogenesis of their water mishandling, we attempted to limit water supply and followed sequential changes of physiological and biochemical parameters. We also analyzed renal histological changes at several time points. At 3 mo old just before water restriction challenge was started, their baseline arginine vasopressin level was comparable to the wild-type (WT) level. Twenty-four-hour water deprivation and desamino d-arginine vasopressin administration improved polydipsia and polyuria to certain degrees. However, creatinine concentrations in Sap-D(-/-) mice were significantly higher than those in WT mice, suggesting that some renal impairment already emerged in the affected mice at this age. Renal histological analyses revealed that renal tubules and collecting ducts were expanded after 3 mo old. After 6 mo old, vacuolar formation was observed, many inflammatory cells migrated around the ducts, and epithelial monolayer cells of tubular origin were replaced by plentiful cysts of various sizes. At 10∼12 mo old, severe cystic deformity appeared. On the other hand, 8-mo-long water restriction started at 4 mo old dramatically improved tubular damage and restored once-dampened amount of tubular aquaporin2 protein to the WT level. Furthermore, 10-mo-long water restriction ameliorated their renal function. Remarkably, by continuing water restriction thereafter, overall survival period became comparable with that of the WT. Together, polyuria, devastating renal tubular lesions, and renal failure were ameliorated by the mere 10-mo-long water restriction, which would trigger lethal dehydration if the disease were to be caused by any processes other than primary polydipsia. Our study demonstrates that long-term water restriction surely improved renal histopathological changes leading to prevention of premature death in Sap-D(-/-) mice.

Development and application of a system for seminolipid metabolism using mouse seminiferous tubules.

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [(35)S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [(35)S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44 degrees C for 10 min) of the isolated seminiferous tubules suppressed the (35)S-incorporation into seminolipid to 47% of heating at scrotal temperature (32 degrees C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44 degrees C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.

Effect of nutritional substrate on sulfolipids metabolic turnover in isolated renal tubules from rat.

Effects of a glycolytic (glucose) and a gluconeogenic renal nutritional substrate (glutamine) on metabolic turnover of sulfolipids, determined as [(35)S]sulfate incorporation, were compared in renal tubules prepared from well-fed rats. The results showed that the effects of glucose and glutamine, at nearly physiological serum concentration, are quite contrary to each other. Glucose increased the turnover rates of relatively long chain ganglio-series sulfoglycolipids (Gg(3)Cer II(3)-sulfate and Gg(4)Cer II(3),IV(3)-bis-sulfate) (1.7 to 2.4-fold), but not of cholesterol 3-sulfate (0.9-fold). In contrast, glutamine accelerated the turnover rates of relatively short chain sulfoglycolipids (glucosyl sulfatide, galactosyl sulfatide and lactosyl sulfatide) (1.3 to 2.7-fold), as well as cholesterol 3-sulfate (2.4-fold). The possible mechanism which causes these marked differences is also discussed.

Higher expression of renal sulfoglycolipids in marine mammals.

Patterns and contents of major acidic glycosphingolipids in the kidney of three marine mammalian species, the Steller sea lion (Pinnipedia), the rough-toothed dolphin and the broad-beaked dolphin (Odontoceti), were examined, and compared with those of terrestrial mesic mammals. The profile of major acidic glycosphingolipids was not significantly different between the terrestrial and marine mammals: predominant gangliosides were GM3 and GD3, and major sulfoglycolipids were SM4s and SM3. On the other hand, the total concentration (nmol/g wet tissue) of sulfoglycolipids was considerably higher in the marine mammals (2.3-3.0 times) than that in the terrestrial mesic mammals with comparable body weights. In contrast, there was no significant difference in the level of renal glycolipids-bound sialic acid between the marine and the terrestrial mammals. These results suggest that higher expression of renal sulfoglycolipids in marine mammals may contribute to the maintenance of osmotic balance of their body fluid against sea water.

Chemical and apoptotic properties of hydroxy-ceramides containing long-chain bases with unusual alkyl chain lengths.

We analysed four types of free ceramides (Cer 1, Cer 2, Cer 3 and Cer 4) from equine kidneys by electrospray ionization mass spectrometry. Cer 1 was composed of dihydroxy long-chain bases (dLCBs) of (4E)-sphingenine (d18:1), sphinganine and non-hydroxy fatty acids (NFAs); Cer 2 was composed of trihydroxy LCBs (tLCBs) of 4-hydroxysphinganine, t16:0, t18:0, t19:0 and t20:0, and NFAs; Cer 3 was composed of dLCBs, d16:1, d17:1, d18:1, d19:1 and d20:1, and hydroxy FAs (HFAs); and Cer 4 was composed of tLCBs, t16:0, t17:0, t18:0, t19:0 and t20:0, and HFAs. The results indicate all ceramide species containing LCBs with non-octadeca lengths (NOD-LCBs) can be classified into hydroxy-ceramides since these species always consist of tLCBs, and/or HFAs. Furthermore, such species tend to contain FAs with longer acyl chains but contain neither palmitate (C16:0) nor its hydroxylated form (C16:0h). The apoptosis-inducing activities of these hydroxyl-ceramides towards tumour cell lines were compared with that of non-hydroxy-ceramides, dLCB-NFA (Cer 1). Monohydroxy-ceramides, tLCB-NFA (Cer 2) and dLCB-HFA (Cer 3), exhibited stronger activities, whereas dihydroxy-ceramides, tLCB-HFA (Cer 4), exhibited similar or weaker activity than dLCB-NFA (Cer 1), depending on cell lines.

De-N-acetyllactotriaosylceramide as a novel cationic glycosphingolipid of bovine brain white matter: isolation and characterization.

A novel cationic lipid was separated from bovine brain white matter by a series of chromatographies on carboxymethyl-Sephadex and silica gel in chloroform and methanol. Its structure was identified unambiguously as de-N-acetyllactotriaosylceramide (deNAcLc(3)Cer) by mass spectrometry and (1)H NMR. The natural occurrence of this glycolipid in white matter extract was detected by immunostaining of thin-layer chromatography with monoclonal antibody 5F5, which is directed to deNAcLc(3)Cer and recognizes the terminal beta-glucosaminyl (GlcNH(2)) residue, having a free NH(2) group. A de-N-acetylase capable of hydrolyzing the N-acetyl group of Lc(3)Cer was detected in bovine brain extract using N-[(14)C]acetyl-labeled Lc(3)Cer as a substrate. The biogenesis and possible functional significance of deNAcLc(3)Cer are discussed.

Metabolism of sulfolipids in isolated renal tubules from rat.

Proximal-rich tubules were prepared from rat kidneys by using collagenase treatment. The isolated rat renal tubules were compared with the intact kidney on the following characteristics. (1) Composition of the sulfoglycolipid. (2) Sulfoglycolipid metabolism based on incorporation of [35S]sulfate or some properties of sulfoglycolipid metabolism, including the activities of anabolic and catabolic enzymes. The results indicated following characteristics of the isolated renal tubules in comparison to the kidney in vivo. (1) The sulfoglycolipid compositions are qualitatively similar, except that the content of glucosyl sulfatide, Gg3Cer II3-sulfate, and GM4 was slightly higher in the isolated tubules. (2) The apparent half-lives (15-55 min) of sulfoglycolipids in the isolated tubules could indicate the existence of a rapid turnover pool of these lipids. (3) The sulfotransferase and sulfatase activities related to sulfoamphiphiles in the renal tubule were similar to those reported for the whole kidney. Based on the above criteria, we conclude that the isolated rat renal tubule should be a useful metabolic system for clarification of the short-term physiological events, up to 90 min, of proximal tubular sulfoglycolipids. By using the present system, we showed that biosynthesis of the renal total sulfoglycolipid was significantly elevated in rats deprived of water for 24 h.

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse.

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Evaluation of counterions for electrospray ionization mass spectral analysis of a highly sulfated carbohydrate, sucrose octasulfate.

A systematic approach was used to evaluate the electrospray ionization mass spectral (ESI-MS) analysis of sucrose octasulfate (SOS), an important pharmaceutical agent. SOS represents a model for other suffated carbohydrates, such as heparin and glycosaminoglycan-derived oligosaccharides that also are highly sulfated and pose difficult analytical problems. A survey of ammonium counterions showed that 1 degree, 2 degrees, and 3 degrees ammonium salts of SOS gave substantial fragmentation as a result of sulfate loss. In contrast, quaternary ammonium and phosphonium salts gave excellent ESI spectra, particularly in the positive ion mode. This represents the first report of the ESI-MS analysis of sulfated carbohydrates in the positive ion mode.

Internal residue loss produced by rearrangement of a novel cationic glycosphingolipid, glyceroplasmalopsychosine, in collision-induced dissociation.

A novel plasmal conjugate of galactosylsphingosine (psychosine), Gro1(3)-O-plasmal-O-6Galbeta-sphingosine (glyceroplasmalopsychosine), was analyzed by electrospray ionization and liquid secondary ion mass spectrometry with low- or high-energy collision-induced dissociation (CID). In the product ion spectra of the [M + H](+) ions, [M + H - glycerol](+) ions arising from the loss of a glycerol were predominant. Unexpectedly, CID of the [M + H - glycerol](+) ion produced an outstanding ion, [(M + H - glycerol) - Hex](+), which required the loss of the galactose from inside the molecule. This ion was greatly reduced in the spectra of N,N-dimethyl derivatives, indicating that the [(M + H - glycerol) - Hex](+) ion is formed from an intramolecular rearrangement with migration of the plasmal residue to the free amino group of sphingosine. It would be expected that the rearrangement occurs simultaneously with the elimination of glycerol or a rearranged [M + H](+) ion leads to the elimination of glycerol, to form a Schiff base-type [M + H - glycerol](+) ion, from which the terminal galactose could be removed by the normal mechanism of glycosidic cleavage. On the other hand, the [M + Na - glycerol](+) ion derived from the sodiated molecule did not produce an ion corresponding to the rearrangement reaction, possibly owing to a higher stability of the sodiated ions against conformational changes.

Seminolipid and its precursor/degradative product, galactosylalkylacylglycerol, in the testis of saposin A- and prosaposin-deficient mice.

Sphingolipid activator proteins (saposins A, B, C, and D) are derived from a common precursor protein (prosaposin) and specifically activate in vivo degradation of glycolipids with short carbohydrate chains. A mouse model of prosaposin deficiency (prosaposin-/-) closely mimics the human disease with an elevation of multiple glycolipids. The recently developed saposin A-/- mice showed a chronic form of globoid cell leukodystrophy, establishing the essential in vivo role of saposin A as an activator for galactosylceramidase to degrade galactosylceramide. Seminolipid, the principal glycolipid in spermatozoa, and its precursor/degradative product, galactosylalkylacylglycerol (GalEAG), were analyzed in the testis of the two mouse mutants by electrospray ionization mass spectrometry. Saposin A-/- mice showed the normal seminolipid level, while that of prosaposin-/- mice was approximately 150% of the normal level at the terminal stage. In contrast, GalEAG increased up to 10 times in saposin A-/- mice, whereas it decreased with age in the wild-type as well as in prosaposin-/- mice. These analytical findings on the two saposin mutants may shed some light on the physiological function of seminolipid and GalEAG.

Cationic glycosphingolipids in neuronal tissues and their possible biological significance.

During the course of studies on natural occurrence of sphingosine base in brain, cationic glycosphingolipids bound to carboxymethyl-Sephadex and eluted with triethylamine in organic solvents were isolated and characterized. Four classes of compounds were identified: (i) plasmalopsychosine-A and -B; (ii) glyceroplasmalopsychosine; (iii) glycosphingolipids having de-N-acetyl-hexosamine, e.g., de-N-acetyl-Lc3Cer; (iv) glycosylsphingosine, i.e., lysoglycosphingolipid. Only two kinds, galactosylsphingosine (psychosine) and lactosylsphingosine, were found to occur naturally in brain. All these compounds were isolated from extract of brain white matter. Their occurrence, quantity, and distribution pattern differ from one species to another. Their quantity is much lower than that of regular acidic and neutral glycosphingolipids. They may interact with regular glycosphingolipids in glycosphingolipid-enriched microdomains to elicit signal transduction, to modify cellular phenotype, although studies along this line are highly limited at this time.