Original article

Urinary N-acetyl-β-D-glucosaminidase activityin rabbits with experimental hypercalciuria

Mehmet Turkmen1, Salih Kavukcu1, Huray Islekel2, Sulen Sarioglu3, Hulya Akhunlar 2, Neriman Gokden3,and Ataman Gure4

1 Pediatrics,2 Biochemistry,3 Pathology, and4 Physiology, Dokuz Eylu¨l University, Faculty of Medicine, Inciraltı-Izmir, Turkey

Received February 12, 1996; received in revised form November 25, 1996; accepted November 27, 1996

Abstract. Routinely used renal function tests remain nor-mal in uncomplicated hypercalciuria. The aim of this studywas to assess the value ofN-acetyl-β-D-glucosaminidase(NAG), a sensitive marker of renal proximal tubular dam-age, in experimental hypercalciuria. Oral calcium providing75 mg/kg per day elementary calcium and 20,000 IU/dayvitamin D3 was administered for 15 days to 7 rabbits(Orytolagus cuniculus-New Zealand white) and 7 rabbitswere given placebo as a control group. Serum calcium,phosphorus, and alkaline phosphatase, daily urinary cal-cium excretion and NAG/creatinine ratio were measuredbefore and after drug administration. Kidneys were ex-amined macroscopically and microscopically following thestudy period. Serum calcium, phosphorus and urinary cal-cium excretion increased, while alkaline phosphatase de-creased significantly in response to drug treatment[10.8+1.5 vs. 12.2+1.3 mg/dl, 4.6+0.6 vs. 6.7+0.7 mg/dl, 22.3+8.3 vs. 46.8+22.5 mg/kg per day, and138.0+57.1 vs. 70.1+33.1 IU/l, respectively (P 50.05)].The NAG/creatinine ratio prior to the study (0.5+0.1 mU/mg) was significantly different from that after the study(5.4+1.5 mU/mg,P50.01). In the control group, changesin serum and urinary parameters were not significant(P 40.05). The relationship between the urinary NAG/creatinine ratio and the daily urinary calcium excretion wasstatistically significant (r = 0.67, P 50.05). In the studygroup, nephrocalcinosis was present in all rabbits except 1(85.7%), whereas none of the control group rabbits hadnephrocalcinosis. In conclusion, in rabbits urinary NAGexcretion increases significantly in nephrocalcinosis in-duced by hypercalciuria.

Key words: Hypercalciuria – Nephrocalcinosis – Rabbit –N-Acetyl-β-D glucosaminidase

Introduction

Nephrocalcinosis and nephrolithiasis are the most impor-tant complications of hypercalciuria. Nephrocalcinosismost commonly involves the corticomedullary junction andto a lesser extent the medulla [1, 2]. The degree of renaldysfunction varies with the extent of nephrocalcinosis.According to some in vivo studies, proximal tubuli remainintact [3]. N-Acetyl-β-D-glucosaminidase (NAG) is a ly-sosomal enzyme indicative of proximal renal tubular le-sions [4, 5]. NAG might be useful in monitoring patientswith clinical hypercalciuria. The aim of this study was toassess the value of NAG as a sensitive marker of renalproximal tubular damage in experimental hypercalciuria.

Materials and methods

Fourteen 1-year-old healthy New Zealand white rabbits (Orytolaguscuniculus) were included in the study. They were weighed before andafter the study period. Seven rabbits (group A) received vitamin D3

20,000 U/kg per day and calcium glucolactobionate, providing a doseof 75 mg/kg per day of elementary calcium orally for 14 days. Sevenrabbits (group B) were given placebo to serve as the control group.Blood samples were obtained from ear veins of the rabbits. Twenty-four-hour urine collections were obtained by insertion of a 6-Fr Foleycatheter into the bladder of the rabbits. The rabbits were fed accordingto their daily requirements. Blood urea nitrogen (BUN), creatinine,calcium, phosphorus, and alkaline phosphatase (ALP) were measuredusing routine spectrophotometric methods (DACOS XL otoanalyzer).Control sera (Coulter, level1, level2) were included in each run. BUNwas measured using the modified Talke and Schubert [6] ureasemethod at 340 nm, inorganic phosphorus as phosphomolybdate com-plex using ammonium molybdate at 340 nm [7], creatinine as picricacid complex in alkaline media at 492 nm [8], ALP using modifiedBowers and Mc Comb’s [9]p-nitrophenyl phosphate method at 420 nm,and calcium using methylthymol as the indicator at 612 nm [10].Creatinine, calcium, inorganic phosphorus, and NAG were also mea-sured in 24-h urine samples. The urine samples were centrifuged at500 rpm for 5 min. Following the removal of unclear supernatant dueto hypercalciuria, creatinine and inorganic phosphorus were measuredwith a similar method as used in the blood samples. Urinary proteinwas assessed using the Biuret system (DACOS XL otoanalyzer). Be-fore measurement of urine calcium, uncentrifuged urine samples were

Correspondence to:S. Kavukcu, Mithatpas˛a Cad.,665/4, TR-35280 Izmir, Turkey

Pediatr Nephrol (1997) 11: 481–484 IPNA 1997

treated with 0.1 N hydrochloric acid to obtain a pH of 3–4 and thenassessed with the same technique used for the measurement of serumcalcium. Urinary NAG was measured at 580 nm as 3-cresol sul-fonphthalein released from 3-cresol sulfonphthaleinylβ-D-glucosami-nide [11].

Endogenous creatinine clearance and tubular phosphorus reabsorp-tion were measured for each rabbit. Nephrocalcinosis and metastaticcalcification were assessed by total body X-ray films following thetermination of the drugs. Following cervical dislocation, the kidneyswere examined histopathologically by pathologists blinded to thestudy. Hematoxylin eosin and von Kossa’s stains for calcium depositswere used. The amount of glomerular, proximal and distal tubular, andvascular calcium deposits were assessed.

Statistical analysis.Variables before and after the termination of themedications were compared using the Wilcoxon signed rank test. Therelationship between the parameters was assessed by the simple linearregression test using the Stat View statistical package program.

Results

At the end of the study, the decrease in weight of bothgroups was insignificant and there was no statistical sig-nificance between group A and group B at the beginning

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Table 1. Comparison of the weights serum biochemical parameters and endogenous creatinine clearance of the study group (group A) and controlgroup (group B) at the beginning and at the end of the study

Group Aat the beginning of studyMean+ SD(range)

Group Aat the end of studyMean+ SD(range)

Group Bat the beginning of studyMean+ SD(range)

Group Bat the end of studyMean+ SD(range)

Weight (g) 1,750+175(1,550–1,970)

1,680 +140(1,580–1,840)

1,690+150(1,530–1,900)

1,730+190(1,590–1,930)

BUN 19.1+3.4 16.9+2.3 17.7+3.1 17.1+3.0(mg/dl) (14.0–23.0) (14.0–21.0) (13.0–21.0) (14.0–22.0)Creatinine 1.3+0.2 1.4+0.3 1.2+0.3 1.3+0.2(mg/dl) (1.0–0.2) (1.1–1.9) (1.0–1.8) (1.1–1.6)Protein 6.6+0.7 6.3+0.4 6.4+0.5 6.3+0.5(g/dl) (7.5–5.6) (5.7–7.0) (5.5–7.2) (5.6–6.8)Calcium 10.8+1.5* 12.2+1.3*** 10.6+1.2 10.4+1.1(mg/dl) (8.1–13.0) (10.0–14.0) (8.0–12.7) (8.3–11.9)Phosphorus 4.6+0.6** 6.7+0.7*** 4.9+0.8 4.3+0.7(mg/dl) (3.9–5.8) (5.7–8.0) (3.8–5.4) (3.9–5.9)Alkaline 138.8+57.1** 70.1+33.2*** 140.7+49.3 146.6+50.3phosphatase(U/l)

(60.0–235.0) (48.0–120.0) (68.0–227.0) (74.0–2310)

Endogenous 2.9+1.2 3.7+0.7 3.2+1.0 3.6+0.8creatinineclearance(ml/min)

(1.6–4.7) (3.0–4.6) (1.7–4.5) (2.4–4.6)

BUN, Blood urea nitrogen* In group A, beginning of study versus end of study,P 50.025** In group A, beginning of study versus end of study,P 50.05*** At the end of study, group A versus group B,P 50.05

Table 2.Comparison of urinary biochemical parameters and tubular reabsorption of phosphorus (TRP) in group A and group B at the beginning andat the end of the study

Group Aat the beginning of studyMean+ SD(range)

Group Aat the end of studyMean+ SD(range)

Group Bat the beginning of studyMean+ SD(range)

Group Bat the end of studyMean+ SD(range)

Urinary creatinine 45.5+4.4 51.2+6.6 50.8+5.2 49.0+6.8(mg/kg per day) (38.3–51.1) (43.6–64.7) (45.6–61.7) (38.4–61.1)Urinary protein 0.26+0.13 0.25+0.11 0.27+0.13 0.26+0.12(mg/kg per day) (0.15–0.52) (0.14–0.43) (0.14–0.47) (0.12–0.48)NAG 1.1+0.4* 10.5+2.5*** 1.7+0.6 1.5+0.5(U/l) (0.1–3.4) (2.8–21.5) (0.3–3.6) (0.2–3.2)NAG/creatinine 0.5+0.1* 5.4+1.5*** 0.4+0.1 0.5+0.1(mU/mg) (0.07–1.19) (1.2–12.0) (0.1–1.1) (0.1–0.9)TRP 86.9+6.9 76.7+11.7 87.2+7.4 85.4+9.5(%) (76.0–93.4) (60.0–88.0) (72.0–95.6) (70.4–97.6)Calciuria 22.3+8.3** 46.8+22.5*** 18.4+9.6 21.4+7.6(mg/kg per day) (7.0–28.7) (17.9–88.6) (5.4–26.5) (8.3–27.6)

NAG, N-Acetyl-β-D-glucosaminidase* In group A, beginning of study versus end of study,P 50.01** In group A, beginning of study versus end of study,P 50.05*** At the end of study, group A versus group B,P 50.05

and end of the study (P 40.05) (Table 1). Differences inBUN, serum creatinine, endogenous creatinine clearance,and protein levels in group A were insignificant whencompared with levels at the end of the study (Table 1).Serum calcium and inorganic phosphorus were signifi-cantly elevated following the study (P 50.05), while ALPlevels decreased significantly after the study in group A(P 50.05) (Table 1). There was no statistical difference inall parameters of group B at the end of study (P 40.05)(Table 1)

Urinary levels of creatinine and protein were not sig-nificantly different before and after the study in groups Aand B (Table 2). Calciuria and urinary NAG levels weresignificantly elevated following drug administration(P 50.05, P 50.01). The NAG/creatinine ratio was sig-nificantly elevated (P 50.01) following the study ingroup A. The alteration in tubular phosphorus reabsorptionwas not statistically significant in group A. In the controlgroup no significant changes were found in levels of ur-inary creatinine, protein, NAG, NAG/creatinine ratio, tub-ular reabsorption of phosphorus, and calciuria at the end ofthe study period. Calciuria, urinary NAG, and NAG/crea-tinine ratio in group A were significantly higher than ingroup B at the end of the study (Table 2). The relationshipbetween calciuria and urinary NAG/creatinine ratio wasstatistically significant (r = 0.67,P 50.01) (Fig. 1).

None of the rabbits manifested nephrocalcinosis ormetastatic calcification using conventional radiologicalmethods. Six rabbits had calcium deposits in their glo-meruli Bowman’s capsules, proximal and distal tubuli, andvascular walls. These were more prominent in the cortex(Fig. 2). One of the rabbits given vitamin D3 and calciumdid not show any calcium deposition within the kidneys.The same was true for the control rabbits who were givenonly placebo. None of the rabbits showed calcium depositsin the liver.

Discussion

Hypercalciuria is a clinical entity resulting from either in-creased intestinal absorption of calcium increased mobili-zation from the bones or from renal tubular lesions [12].Vitamin D3 and calcium were given to rabbits to inducehypercalciuria. Hypercalciuria itself was indicative of suf-ficient drug administration in the rabbits of the study group.Nephrocalcinosis, which may be defined as an increasedrenal tissue calcium content, may not be demonstratedusing conventional radiological methods. None of therabbits showed calcification in X-ray examination.

The administration of parathormone and vitamin D3

have been shown to induce degeneration and necrosis dueto calcium deposits, mainly in the ascending limb of Henle,distal convoluted tubules, and collecting tubules, sparingthe proximal loop. Nephrocalcinosis mainly involves themedullary or corticomedullary junction and rarely thecortex [3]. Caulfield and Shrag [13] reported that parat-hormone resulted in calcium deposition in the distal prox-imal tubule, whereas calcium gluconate administration re-sulted in calcium deposition in the convoluted portion ofthe proximal tubule in the rat kidney. It has been speculatedthat the region and type of calcium deposition varies withthe etiology of hypercalcemia [1, 2]. Calcium phosphatedeposition was shown in the proximal tubules, glomeruli,and vascular region of the cortex using von Kossa’s stain.However, the animal species may influence the depositionof calcium in different sites of the kidney in hypercalciuria.

Renal lesions have been assessed using more than 50enzymes, NAG and alanine aminopeptidase being the mostcommonly used [14]. Such enzyme levels are increasedbefore any conventional renal function tests are abnormal.Moreover, the enzymatic activity varies in parallel with thedisease activity [14]. Urinary excretion is independent ofserum levels, because NAG is not subject to glomerularfiltration. The increase in serum creatinine and decrease inBUN together with the slightly increased endogenouscreatinine clearance lead us to propose an anorexigeniceffect of vitamin D3 which may result in lower food intakeand increased catabolism, as suggested by lower bodyweights at the end of the study. Increased endogenous

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Fig. 1. Relationship between urinaryN-acetyl-β-D-glucosaminidase(NAG)/creatinine ratio and calciuria (r = 0.67,P 50.05).*, At thebeginning of study urinary NAG/creatinine (mU/mg);*, at the end ofstudy urinary NAG/creatinine (mU/mg)

Fig. 2. Calcium deposition in the glomerulus, Bowman’s capsule,tubule, and vascular tissues (von Kossa,×100)

creatinine clearance secondary to elevated serum creatininemay be explained by polyuria due to hypercalcemia. Theurinary NAG/creatinine ratio is increased due to hy-percalciuria. Minimally decreased tubular phosphorus re-absorption suggests that NAG increases very early in theprocess of renal tubular damage. Decreased tubular phos-phorus reabsorption together with increased serum phos-phorus may be explained by increased intestinal phos-phorus absorption due to vitamin D3 administration.

In conclusion, NAG increases significantly before anyother renal function parameters in rabbits with hy-percalciuria-induced nephrocalcinosis which remains un-defined by radiological techniques. By definition an in-crease in NAG implies renal damage.

References

1. Stapleton RB, Roys S, Noe HN, Jerkins G (1984) Hypercalciuriain children with hematuria. N Engl J Med 310: 1345–1348

2. Hill GS (1992) Calcium and the kidney. Nephrolithiasis and hy-dronephrosis. In: Heptinstall RH (ed) Pathology of kidney,volume 3, 4th edn. Little Brown, Boston, pp 1563–1579

3. Massry SG, Pitts TO (1989) Effects of electrolyte disturbances onthe kidney. In: Massry SG, Glassock RJ (eds) Textbook of ne-phrology, vol 1, 2nd edn. Williams and Wilkins, Baltimore,pp 419–420

4. Ring E, Zobel G, Erwa W, Haim-Kuttnig M (1992) Urinary ex-cretion of N-acetyl-β-D glucosaminidase in proteinuric states.Child Nephrol Urol 12: 15–18

5. Flynn FV (1990) Assessment of renal function: selected devel-opments. Clin Biochem 23: 49–54

6. Talke H, Schubert GE (1965) Enzymatische Harnstoffbestimmungin blut-und serumoptischen Test nach Warburg. Klin Wochenschr43: 174

7. Daly JA, Ertingshausen G (1972) Direct method for determininginorganic phosphate in serum with the “CentrifiChem“. Clin Chem18: 263–265

8. Houot O (1985) In: Siest G, Henry J, Schiele F (eds) Interpretationof clinical laboratory tests. Young DS Biomedical Publications,London, pp 220–234

9. Bowers GN, Mc Comb RB (1975) Measurement of total alkalinephosphatase activity in human serum. Clin Chem 21: 1988

10. Yendt ER, Gagne RJ (1968) Detection of primary hyperparathyr-oidism with special reference to its occurence in hypercalciuricfemales with “normal“ to borderline serum calcium. Can MedAssoc J 98: 331–336

11. Yakata M, Sugita O, Sakai T, Uchiyama K, Wada K (1983) Ur-inary enzyme determination and its clinical significance. C. En-zyme derived from the kidney tubular epithelium-N-acetyl-beta-D-glucosaminidase. 4. Preclinical evaluation of the urinary NAGactivity and changes in renal diseases. Rinsho Byori 56: 90–101

12. Barratt MT (1994) Urinary calculi. In: Holliday MA, Barratt MT,Vernier RL (eds) Pediatric nephrology, 3rd edn. Williams andWilkins, Baltimore, pp 1070–1079

13. Caulfield JB, Shrag PE (1964) Electron microscopic study of renalcalcification. Am J Pathol 44: 365

14. Price RG (1982) Urinary enzymes, nephrotoxicity and renal dis-ease. Toxicology 23: 99–134

Literature abstractsJ Clin Endocrinol Metab (1996) 81: 4496–4499

Association of a mutation in thiazide-sensitive Na-Cl cotransporterwith familial Gitelman’s syndrome

K. Takeuchi, S. Kure, T. Kato, Y. Taniyama, N. Takahashi, Y. Ikeda, T. Abe, K. Narisawa, Y. Muramatsu, and K. Abe

Gitelman’s syndrome is a variant of Bartter’s syndrome, characterizedby hypokalemia, hypomagnesemia, hypocalciuria, and hypovolemia.We have observed familial cases of Gitelman’s syndrome, and a pos-sible in thiazide-sensitive Na-Cl cotransporter was investigated in thiskindred. The proband was a 47-year-old Japanese female, and hermother was also affected. Her parents and maternal grandparents areconsanguineous. By using PCR-amplification and direct sequencing,we identified a novel non-conservative missense mutation at 623amino acid position, which substitutes proline for leucine (L623P), and

also creates anNci I restriction site in the exon 15. The mutation wasnot detected in normal healthy subjects (n = 102). Nci I digestion ofPCR-amplified exon 15 DNA fragments from individuals in the familyindicated the autosomal recessive inheritance of the disorder. In con-clusion, the L623P mutation in the thiazide-sensitive Na-Cl co-transporter gene is suggested to impair the transporter activity, and tounderlie this familial Gitelman’s syndrome; Gitelman’s syndromeobserved in this kindred has been inherited in an autosomal recessivefashion.

Arch Dis Child (1997) 76: 65–67

Growth hormone treatment without a needle using the Preci-Jet 50 transjector

P. Bareille, M. MacSwiney, A. Albanese, C. De Ville, and R. Stanhope

A new delivery system (Preci-Jet 50) which administers growth hor-mone through the skin using high pressure and without a needle wasevaluated. This device was inconvenient and painful compared with a

pen injection system. The conclusion is that the Preci-Jet is not thepanacea for solving the problem of compliance with subcutaneousgrowth hormone injections.

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