Laboratory Investigation

Kidney International (1981) 20, 181–187; doi:10.1038/ki.1981.120

Phosphatase activity along the nephron of mice with hypophosphatemic vitamin-D-resistant rickets

Michele G Brunette1, Meanthan Chan1 and Manon Lebrun1

1Department of Pediatrics, Maisonnenve-Rosemont Hospital, School of Medicine, University of Montreal, Montreal, Canada

Correspondence: Dr M G Brunette, Maisonneuve-Rosemont Hospital, Research Center, 5415 l'Assomption Blvd., Montreal H1T 2M4 P.Q. Canada

Received 5 February 1980; Revised 5 November 1980.

Top

Abstract

Phosphatase activity along the nephron of mice with hypophosphatemic vitamin-D-resistant rickets. It has recently been suggested that alkaline phosphatase may be involved in the renal transport of inorganic phosphorus (Pi). Because a specific defect of Pi reabsorption in the proximal tubule is detected in genetic hypophosphatemic vitamin-D-resistant rachitic mice, we measured the alkaline phosphatase activity along the nephron of this mutant strain, using an in vitro microassay of alkaline phosphatase on single tubules. In a series of preliminary experiments performed in normal mice, enzyme activity was detected in glomeruli (1.28 plusminus [SEM] 0.08 pmoles of hydrolyzed substrate per millimeter of glomerulus per 30 min of incubation), in the initial part of the proximal tubule (S1) (in pmoles/mm/30 min: 20.3 plusminus 1.04), and to a lesser extent in the middle part of this same segment (S2) (13.8 plusminus 1.1). No enzyme activity was detected in the late pars recta (S3) or in the loop of Henle, the distal tubule, and the collecting tubule. In 24 experiments performed in rachitic mice paired with normal mice, alkaline phosphatase activity was 14.8 plusminus 0.8vs 19.7 plusminus 1.2 in S1; 13.3 plusminus 1.3 vs. 13.8 plusminus 1.0 in S2, and 0.1 plusminus 0.5 vs. 0.7 plusminus 0.5 in S3, in the two series of animals, respectively. Thus, in rachitic mice, the enzyme activity is significantly lower in S1 compared with the corresponding normal value (P < 0.005). This difference is not due to variations in nephron diameters because it remains significant when enzyme activities are expressed as picomoles of dephosphorylated substrate per microgram of tubular protein (P < 0.02). It is not due to variations in ages between the animals because phosphatase activity per microgram of tubular protein does not significantly vary within the age range of our mice. Because of the renal leak of Pi in the rachitic mice, these animals are chronically Pi depleted. To compare the alkaline phosphatase activity in hypophosphatemic mice with the activity which should normally result from a comparable Pi depletion, that enzyme was measured in a separate series of 14 rachitic mice paired with genetically normal animals depleted of Pi for 7 to 14 days. Alkaline phosphatase activity in S1 was 24.6 plusminus 1.2 vs. 14.4 plusminus 0.9 in Pi-depleted and rachitic mice, respectively (P < 0.001). Pi depletion in normal mice increased rather than decreased the enzyme activity in S1, therefore accentuating the abnormality observed in the rachitic mice.

L'activité de phosphatase alcaline le long du néphron de souris chez rachitisme vitamino-sistant avec hypophosphatémie Plusieurs travaux ont récemment suggéré que la phosphatase alcaline joue un rôle dans le transport tubulaire rénal du phosphore inorganique. Par ailleurs, un défaut spécifique de ce transport fut mis en évidence au niveau du tube proximal de la souche mutante de souris présentant un rachitisme vitamino-résistant avec hypophosphatémie. Nous avons donc mesuré l'activité phosphatasique le long du néphron de cette souche mutante, à l'aide d'une microtechnique enzymatique in vitro sur tubule isolé. Dans une série d'expériences préliminaires chez des souris normales, une activité enzymatique fut trouvée dans les glomérules: 1.28 plusminus (SEM) 0.08 pmoles de substrat hydrolyse par glomérule par 30 min d'incubation; dans la partie initiale du tube proximal ou S1 (pmoles/mm/30 min): 20.3 plusminus 1.04; et à un degré moindre, dans la partie moyenne de ce segment ou S2: 13.8 plusminus 1.1. Aucune activité enzymatique ne fut détectée dans la portion terminale de la pars recta ou S3, ni dans l'anse de Henle, le tube distal ou le tube collecteur. Dans 24 expériences effectuées chez des souris rachitiques pairées avec des souris normales l'activité de la phosphatase alcaline était de 14.8 plusminus 0.8 vs. 19.7 plusminus 1.2 dans S1, de 13.3 plusminus 1.3 vs. 13.8 plusminus 1.0 dans S2, et 0.1 plusminus 0.5 vs. 0.7 plusminus 0.5 dans S3, chez les deux séries d'animaux, respectivement. Chez la souris rachitique. l'activité enzymatique est donc significativement abaissée au niveau de S1 en comparaison de la valeur correspondante chez l'animal normal (P < 0.005). Cette différence n'est pas due à des variations de diamètre des néphrons puisqu'elle persiste lorsque les activités enzymatiques sont exprimées en pmoles de substrat déphosphorylé par mug de protéines tubulaires (P < 0.02). Elle n'est pas due non plus à des variations d'age des animaux puisque l'activité phosphatasique par mug de protéines tubulaires ne varie pas de façon significative en dedans des limites d'age de nos souris. A cause de la fuite rénale de phosphore que présentent les souris rachitiques, ces animaux en sont chroniquement déplétés. Afin de comparer l'activité phosphatasique des souris rachitiques avec celle que devrait entraîner normalement une déplétion en phosphore analogue, l'enzyme fut dosée chez une nouvelle série de 14 souris rachitiques pairées avec des souris génétiquement normales déplétées en phosphore durant 7 à 14 jours. L'activité de la phosphatase alcaline au niveau de S1 fut de 24.6 plusminus 1.2 vs. 14.4 plusminus 0.9 chez les souris normales déplétées en phosphore et chez les souris rachitiques respectivement (P < 0.01). La déplétion en phosphore augmente plutôt qu'elle ne baisse l'activité phosphatasique en S1, ce qui accentue l'anomalie observée chez la souris rachitique.

Top

References

  1. Eicher EM, Southard JL, Scriver CR, Glorieux FH: Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D resistant) rickets. Proc Natl Acad Sci USA 73:4667–4671, 1976 | Article | PubMed | ChemPort |
  2. Giasson SD, Brunette MG, Danan G, Vigneault N, Carriere S: Micropuncture study of renal phosphorus transport in hypophosphatemic vitamin D-resistant rickets mice. Pfluegers Arch 371:33–38, 1977
  3. Tenenhouse HS, Scriver CR: The defect in transcellular transport of phosphate in the nephron is located in brush border membrane in X-linked hypophosphatemia (Hyp mouse model). Can J Biochem 56:640–646, 1978 | PubMed | ChemPort |
  4. Tenenhouse HS, Scriver CR, McInnes RR, Glorieux F: Renal handling of phosphate in vivo and in vitro by the X linked hypophosphatemic male mouse: evidence for a defect in the brush border membrane. Kidney Int 14:236–244, 1978 | PubMed | ChemPort |
  5. Horiuchi T, Horiuchi S, Mizuno D: A possible negative feedback phenomenon controlling formation of alkaline phosphomono-esterase in E. coli. Nature 183:1529, 1959 | PubMed | ISI | ChemPort |
  6. Torriani A: Influence of inorganic phosphate in the formation of phosphatase by Escherichia coli. Biochem Biophys Acta 38:460, 1960
  7. Garen A, Levinthal C: A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. Biochem Biophys Acta 38:470–483, 1960
  8. Melani F, Farnararo M, Chiarugi VP: Molecular aspects of the regulation of rat kidney alkaline phosphatase. Biochem J 121:33–40, 1971
  9. Kempson SA, Dousa TD: Phosphate transport across renal cortical brush border membrane vesicles from rats stabilized on a normal, high or low phosphate diet. Life Sci 24:881–888, 1979 | PubMed |
  10. Kempson SA, Kim JK, Northrup TE, Knox FG, Dousa TP: Alkaline phosphatase in adaptation to low dietary phosphate intake. Am J Physiol 237(5):E465–E473, 1979 | ChemPort |
  11. Plante GE, Erian R, Nawar T, Petitclerc C: Renal transport of phosphate: Role of alkaline phosphatase. VII Int Congr Nephrol, Montreal, 1978, abst E13
  12. Imbert M, Chabardes D, Montegut M, Clique A, Morel F: Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments. Pfluegers Arch 354:213–228, 1975
  13. Morel F, Chabardes D, Imbert M: Functional segmentation of the rabbit distal tubule by microdetermination of hormone dependent adenylate cyclase activity. Kidney Int 9:264–277, 1976 | PubMed | ISI | ChemPort |
  14. Brunette MG, Chabardes D, Imbert-Teboul M, Clique A, Montegut M, Morel F: Hormone sensitive adenylate cyclase along the nephron of genetically hypophosphatemic mice. Kidney Int 15:356–369, 1979
  15. Butcher EC, Lowry OH: Measurement of nanogram quantities of protein by hydrolysis followed by reaction with orthophtalaldehyde or determination of glutamate. Analyt Chem 76:502–503, 1976
  16. Brunette MG, Chan M, Ferriere C, Roberts KD: Site of 1,25(OH)2 vitamin D3 synthesis in the kidney. Nature 276:287–289, 1978 | Article | PubMed | ISI | ChemPort |
  17. Cornish CJ, Neale FC, Posen S: An automated fluorometric alkaline phosphatase microassay with 4 methyl umbelliferyl phosphate as a substrate. Am J Clin Pathol 53:68–76, 1970
  18. Kelly MH, Hamilton JR: A microtechnique of the assay of intestinal alkaline phosphatase. Clin Biochem 3:33–43, 1970 | PubMed |
  19. Longley JB, Fisher ER: A histochemical basis for changes in renal tubular function in young mice. Quart J Micro Sci 97:187–195, 1956
  20. Hardonk MJ, Koudstaal J: Localization of 5'; Nucleotidase and Alkaline phosphatase isoenzymes in the kidney of young and adult mice. Histochemie 15:290–299, 1968
  21. Jacobsen NO, Jorgensen FL, Thomsen AC: On the localizion of some phosphatases in three different segment of the proximal tubules in rat kidney. J Histochem Cytoehem 15:456, 1967
  22. Gomori G: The distribution of phosphatase in normal organ and tissues. J Cell Comp Physiol 17:71, 1941 | Article | ChemPort |
  23. Wachstein M: Histochemical staining reactions of the normally functioning and abnormal kidney. J Histochem Cytochem 3:246, 1955
  24. Bonting SL, Tsoodle AD, de Bruin H, Mayron BR: Quantitative histochemistry of the nephron: VII. Various phosphatases in the healthy human kidney. Arch Biochem 91:130, 1960
  25. Bonting SL, Pollak VR, Muehrcke RC, Kark RM: Quantitative histochemistry of the nephron: II. Alkaline phosphatase activity in man and other species. J Clin Invest 39:1372, 1960 | PubMed | ISI | ChemPort |
  26. Wachstein M, Bradshaw M: Histochemical localization of enzyme activity in the kidney of three mammalian species during their post natal development. J Histochem Cytochem 13:44, 1965
  27. Mizutani A, Barrnett RJ: Fine structural demonstration of phosphatase activity at pH 9. Nature 206:1001, 1965 | Article |
  28. Scherberich JE, Gaul C, Mondorf W: Biochemical, immunological, and ultra structural studies on brush border membranes of human kidney. Curr Probl Clin Biochem 8:85–95, 1978
  29. Ramadoss CS, Selvam R, Shanmugasundaram KR, Shanmugasundaram ERB: Alkaline phosphatase from rabbit kidney: Studies on the catalytic properties of its multiforms. J Biochem 81:1813–1823, 1977
  30. Murer H, Stoll R, Storelli C, Kinne R, Bonjour JP, Fleish H: Transport of phosphate by rat proximal tubular brush border vesicle: Effect of dietary phosphate, parathyroid hormone, 1,25(H)2 vitamin D3, diphosphonatates (EHDP) and inhibitors of alkaline phosphatase (abst). Min Electr Metab 2:272, 1979
  31. Evers C, Murer H, Kinne R: Effect of parathyrin on the transport properties of isolated renal brush-border vesicles. Biochem J 172:49–56, 1978
  32. Tenenhouse HS, Scriver CR: Renal brush border membrane adaptation to phosphorus deprivation in the Hyp/Y mouse. Nature 281:225–227, 1979 | Article |
  33. Arnaud C, Glorieux F, Scriver C: Serum parathyroid hormone in X linked hypophosphatemia. Science 173:845–847, 1971 | PubMed | ISI | ChemPort |
  34. Meyer RA, Gray RW, Meyer MH: Paradoxical normal levels of vitamin D metabolites in the plasma of X linked hypophosphatemic mouse. 60th Annu Meeting Am Endoc Soc, Miami, 1978, abst 816, p. 486
  35. Drezner MK, Haussler MR, Lyles KW, Harrelson JM: Abnormal homeostatic regulation of 1-25 di HCC biosynthesis: A factor in the genesis of X linked hypophosphatemic rickets. 4th Workshop on Vitamin D, Berlin 1979, abst. 299
  36. Stoll R, Klnne R, Murer H, Fleish H, Bonjour JP: Phosphate transport by rat renal brush border membrane vesicles: Influence of dietary phosphate, thyroparathyroidectomy and 1,25-di hydroxy vitamin D3. Pfluegers Arch 380:47–52, 1979
  37. Walling MW, Levine BJ, Brautbar N, Lee DBN, Coburn JW: Renal cortical alkaline phosphatase activity and renal P retention during P depletion in vitamin D deficient rats. 11th Annu Meeting Am Soc Nephrol, abst 9A, 1978
  38. Shah SV, Kempson SA, Northrup TE, Dousa TP: Renal adaptation to a low phosphate diet in rats blockade by actinomycin D. J Clin Invest 64:955–966, 1979 | PubMed | ISI | ChemPort |
  39. Tenenhouse HS, Scriver CR: Membrane alkaline phosphatase activity and phosphate transport in mouse renal brush border membrane vesicles. Am Soc Nephrol Annu Meeting, Boston, 1979, abst 70A
  40. Cowgill L, Goldfarb S, Goldberg M, Slatopowsky E, Agus ZS: Nature of the renal defect in familial hypophosphatemic rickets (abst). Clin Res April:505A, 1977

Extra navigation

.
ADVERTISEMENT