Microscopic nephrocalcinosis and hypercalciuria in nephrotic syndrome

Microscopic Nephrocalcinosis and Hypercalciuria in Nephrotic Syndrome HILAL MOCAN, MD, ALl,AN YILDIRAN, MD, TEKSEN (~AMLIBEL, MD, AND GAMZE MOCAN KUZEY, MD Focal calcification is an occasional tubular abnormality seen in minimal-change nephrotic syndrome. Nephrocalcinosis was also reported in premature infants as a consequence of hypercalciuria resuiting from long-term fttrosemide therapy. We describe 4 nephrotic

children (3 minimal change, 1 diffuse proliferative glomerulonephri-

infection. We were concerned that children with nephrotic syndrome are at risk for nephrocalcinosis, and urinary calcium and pH should be monitored carefully during prolonged furosemide use, especially in children with nephrotlc syndrome with reduced initial responsiveness to corticosteroid therapy. HUM PATHOL31:1363-1367. Copyright

tis) with transient hypercalciuria and intrainminal calcifications in renal histopathological specimens without radiologic evidence of renal calcification. These children were resistant to corticosteroid therapy and were receiving furosemide therapy along with albumin for management of oedema. Two of the children also had twinary

© 2000 by W.B. Saunders Company

Furosemide-associated renal calcifications were first r e p o r t e d in 1982 by Hufnagle et aP in 10 premature infants but not in nephrotic patients. Since that time, o t h e r observations have c o n f i r m e d the association between furosemide therapy and the d e v e l o p m e n t of urolithiasis a n d nephrocalcinosis, especially in the very l o w - b i r t h weight p r e m a t u r e infants with b r o n c h o p u l m o n a r y dysplasia and hyaline m e m b r a n e diseaseY -5 Histological examination of renal tissue f r o m these patients often shows intratubular calcifications. 4,6-8 A 2-year u h r a s o n o g r a p h i c follow-up study of these patients showed that in a significant n u m b e r , discontinuation of the t r e a t m e n t with the l o o p diuretics results in resolution of the renal calcifications s with the persistence of abnormalities in some. 6 These long-term studies, however, did not provide information on the renal histology of these patients. O t h e r factors may be associated with hypercalciuria and nephrocalcinosis, such as parenteral calcium administration, alkaline urine, h y p o p h o s p h a t e m i a , fluid restriction and r e d u c e d urine output, and the absence or reduction of urinary stone inhibitors. 7 Tubular abnormalities seen in minimal-change nephrotic syndrome (MCNS) include protein droplets, lipid accumulation, focal calcification, and disruption by extruded casts. '-~ T u b u l a r abnormalities have b e e n reported to be associated with r e d u c e d initial responsiveness to corticosteroid therapy. ~° We f o u n d it noteworthy that no study has evaluated the frequency and possible risk factors for micro-

scopic nephrocalcinosis in nephrotic children. We suggest that patients with nephrotic syndrome who show r e d u c e d initial responsiveness to corticosteroid therapy and who have received m o r e furosemide t h r o u g h o u t the entire hospitalization may carry the risk of possibly furosemide-related renal calcifications, but additional risk factors should be evaluated.

From the American Hospital, Department of Paediatrics, lstanbul, Turkey; Karadeniz (Black Sea) Technical University Faculty of Medicine, Department of Paediatrics, Trabzon, Turkey; Jinemed Medical Center, istanbul, Turkey; and Hacettepe University Faculty of Medicine, Department of Pathology, Ankara, Turkey. Accepted for puhlication May 2, 2000. Address correspondence and reprint requests.to Hilal Mocan, MD, Poyraclk Sk. 43/4 Cmar Apt., Ni~anta~l, 80200, Istanbul, Turkey. Copyright © 2000 by W.B. Saunders Company 004C>8177/00/3111-0005510.00/0 doi: 10.1053/hupa.2000.19431

Key words: hypercalciurla, nephrocalcinosis, nephrotic syndrome, children, infant, childhood. Abbreviations: MCNS, minimal change nephrotic syndrome; PTH, parathyroid hormone; USG, ultrasonography.

PATIENTS AND METHODS Four children aged 1 to 10 years (Table 1) presented with diagnostic findings of MCNS2 Two cases (Patients 1 and 3) were in their first relapses and 1 (Patient 2) in fourth relapse who had responded to prednisone initially. Case 4 was a newly diagnosed patient. There was not consanguinity between the parents in all cases. The treatment was daily prednisone 2 mg/kg in divided doses for 4 patients. Repeated doses of furosemide 250-500 mg or 1 mg/kg daily combined with salt-free human albumin 25% in a dose of 0.5 to 1 g/kg up to 25 g and over a period of 30 to 60 minutes intravenously have been used to achieve a satisfactory diuresis and elimination of oedema. Number of diuretic therapies until renal biopsy has been shown in Table 1. Albumin infusion did not result in hypertension in any cases. Significant hypokalemia, hypernatremia, and hyperbicarbonatemia were not recorded in any cases. Renal biopsy was carried out in all because of the unresponsiveness to usual induction regimen of prednisone. Histopathologic findings were consistent with minimal change nephropathy in patients 1, 2, and 3, and diffuse proliferative glomerulonephritis in patient 4. Intratubular calcium depositions were seen in all biopsy specimens by hematoxylin-eosin (H&E), and this was confirmed by the Von Kossa stain. Following the histopathological evaluation, patients were investigated for the possible causes of nephrocalcinosis. Twenty-four-hour timed urine samples were collected 48 hours after high-dose furosemide administration in patients 1 and 2 and immediately after in patients 3 and 4. During the urine collection, patients were still in relapse with refractory oedema. Urine testing including urine calcium, creatinine, sodium, and other urinary electrolytes and were studied using ion selective electrode method and urinary oxalate excretion by colourmatic method. Urinary pH (by dipstick), microscopy, and urine culture were also performed in spot urine

1363

HUMAN PATHOLOGY Volume31, No. 11 (November 2000) TABLE 1. Clinical and Biochemical Data of Patients

Age (years) Biopsy time from onset (months) Diagnosis No. of furosemide therapy until biopsy Follow-up (years)

Case 1

Case 2

Case 3

Case 4

2.5 6 (lst relapse) MCNS 8 5

2 16 (4th relapse) MCNS 12 3

2.5 7 (1st relapse) MCNS 12 2

10 1 (nonresponder) DPGN 4 2

24.1 0.9 9.7 4.5 134 0.22 17.0 26.8

28.9 0.5 8 5.3 171 0.24 NA NA

19.2 0.9 9.1 5.2 40 0.24 Na NA

Blood Bicarbonate (mmol/L) Creatinine (mg/dL) Calcium (mg/dL) Phosphate (mg/dL) Alkaline phosphatase (t~/L; normal: 37-145) PTH (mg/mL; normal: 0.22-0.66) 25 OH vit D (ng/mL; normal 10-40 1.25 diOH vit D3 (pg/mL; normal: 24-45)

25 0.7 10.7 4.4 86 0.24 7.0 27.2

Abbreviations: DPGN, diffuse proliferative glomerulonephritis; NA, not available; MCNS, minimal change nephrofic syndrome; PTH, parathyroid hormone. samples. Hypercalciuria was defined as a 24-hour urine calcium secretion of more than 4 mg/kg or a fasting urinary calcium: creatinine ratio of more than 0.21At Blood samples were also taken and the following laboratory investigations were performed: serum bicarbonate, creatinine, urea, uric acid, calcium, phosphorus, alkaline phosphatase (Technicon RA-XT; Diagnostic Chemicals Ltd, Oxford, CT), parathyroid hormone (PTH), 25-hydroxyvitamin D (25-OH Vit D), and 1.25 diOH Vit D3 (radioimmunoassay). Blood gases were studied by electrode method, and acid loading test was performed. Renal ultrasonographic studies were normal at presentation. All 4 children had repeated renal uhrasonography at least six monthly following the detection of microscopic nephrocalcinosis.

RESULTS

Laboratory Investigations The peripheral blood c o u n t and serum electrolytes were normal. Blood gas analysis showed normal p H values; PCO z values between 26-40 m m o l / L and bicarbonate levels between 19.2-28.9 m m o l / L . The serum creatinine concentration was elevated in 3 of the 4 children, and urea and uric acid levels were normal. Serum total calcium levels were between 8-10.7 m g / d L with serum p h o s p h o r u s 4.4-5.3 m g / d L and alkaline phosphatase 40-134 /z/L (normal values 37-145). Ser u m p h o s p h o r u s concentration was increased in patients 3 and 4. Intact PTH; serum 25-OH Vit D; 1.25d i O H Vit D 3 performed; serum calcitonin levels were within normal ranges in patients 1 and 2 (not performed in patients 3 and 4) (Table 1). Thyroid horm o n e s were normal in all patients. Urinary o u t p u t and urine osmolatities were normal in all patients. Urine microscopy showed basiluria in patient 2 and some erythrocytes in patient 4. Urine culture in patient 2 showed Proteus (>105 c f u / m l ) and Klebsiella (>105 cfu/ml) in patient 3. Fractional excretion of sodium was 0.7% in patient 1; 1.4% in patient 2; 4% in patients 3, and 2% in patient 4. Hypercalciuria and elevated calcium/creatinine ratios (Ca/Cr) were

recorded in all patients. Oxalate excretion was normal in patients 1, 2 (Table 2).

Imaging O n renal ultrasonography (USG), both kidneys were normal shaped and displayed grade II echo increase without any evidence of nephrocalcinosis in all patients. Cystography was unremarkable. Intravenous urography showed normal pyelocaliceal systems. Radiographic examination of the wrist showed no abnormality.

Renal Biopsy Histopathological findings of patients were as follows: Patient 1: Normal glomeruli. Slight tubular atrophy and interstitial inflammation was seen. Presence of intratubular calcium depositions in the majority of the tubules, and distrophic calcification on tubular walls have been de-

TABLE 2

Urinary Data of Patients

Case 1 Daily volume (mL) pH Microscopy Culture Fe Na(%) Ca (mg/kg/day) Ca/Cr Oxalate (/zmol/24 h; normal: 140420)

Case 2

Case 3

Case 4

600 7.5 Negative Negative 0.7

700 6.5 Basiluria Proteus 1.4

500 6.5 Negative Klebsiella 4

1200 6.5 Eryhrocytes Negative 2

6 0.3

6.5 0.6

40 5

35 1.6

190

190

NA

NA

NA, not available

NEPHROCALCINOSIS IN NEPHROTIC SYNDROME (Mocan et al)

tected. Amyloid reaction was negative. Von Kossa staining for calcium was positive. Patient 2: Normal glomeruli but extensive pathological changes in tubules with slight dilatation, epithelial degeneration, cellular casts, and partial exfoliation of tubular epithelium. Extensive intraluminal calcification. Interstitium was normal. Amyloid reaction was negative. Von Kossa staining for calcium in tubular lumina was positive (Fig 1). Patient 3: Normal glomeruli. Intraluminal calcification in the majority of tubules. Interstitium was normal. Amyloid reaction was negative. Von Kossa staining for calcium was positive. Patient 4: Acute diffuse proliferative glometrulonephritis. Narrowed Bowman spaces. Neutrophilic infiltration in glomeruli. Interstitial inflammation was present. Dystrophic calcification in some tubules. Amyloid reaction was negative. Von Kossa staining for calcium was positive. Although H&E staining is the optimal m e t h o d to detect intratubular calcium, Von Kossa staining was also performed to indicate that no artifacts, but calcium-salts were present in the renal tubules (Fig 1). Three patients responded well to high-dose methylprednisolone (30 m g / k g / d a y ) with subsequent alternate day prednisolone. Patient 4 was unresponsive to several combinations including cyclosporin and had persistent proteinuria. Patients 1 and 2 had 4 more relapses during follow-up. Patient 3 is still in remission during 2-year follow-up. Urinary calcium and urinary C a / C r returned to normal during follow-up including patient 4. Follow-up USG findings in patients remained normal. Urinary pH values varied between 5-7.5 during follow-up.

DISCUSSION

Corticosteroids and cytotoxic medications have dramatically improved the outcome of children with the nephrotic syndrome. Unfortunately, the nephrotic syndrome in some children does not respond either to corticosteroids or cytotoxic agents, while in others resistance to therapy may develop during the course of the disease. 12 The m a n a g e m e n t of the oedema in these children is particularly difficult. Vigorous diuretic therapy in these patients may result in electrolyte disturbances, vascular collapse, acute renal failure, and vascular thrombosis, a3 Intravenous infusion of albumin in combination with diuretics has been advocated as an effective m e t h o d of treating oedema owing to nephrotic syndrome. 14 Infusion of albumin may increase intravascular volume 15 and improve diuretic delivery to sites of secretion into the urine. 16 The most common complications observed in patients treated with albumin and diuretic are acute hypertension, transient reduction in weight, electrolyte disturbances (ie, hypokalemia, hypernatremia), and cardiopulmonary complications. 17 Critical analysis of complications of this therapy, however, is not available sufficiently in the literature. When we reviewed the hospital records of our cases, we found no hypokalemia, hypernatremia, or hypercarbonatemia related to the diuretic treatment. Hypertension was not recorded in any of the cases during the treatment course. Nephrocalcinosis is microscopic calcification in the tubules, tubular epithelium, or intestinal tissue of the kidneys. ~s The formation of renal calcification is a complex process dependent on the interaction of multiple ions, promoters and inhibitors of crystallization, urine volume, and pH. v-~

FIGURE I.

Von Kossastaining for calcium in tubular iumina is positive for patient 2. (Original magnification ×40, von Kossa).

1365

HUMAN PATHOLOGY

Volume 31, No, 11 (November 2000)

Nephrocalcinosis with hypercalciuria can be seen in childhood in mainly 6 different groups. 2° Of these, we have evaluated hyperparathyroidism and hyper- or hypothyroidism, malignancy, sarcoidosis and other granulomatous diseases, Williams syndrome, and milkalkali syndrome la in our patients which could be causes for hypercalciuria and nephrocalcinosis. Furthermore, the immobilization hypothesis was not applicable in our patients. Vitamin D levels were normal in 2 patients. Although we did not check the vitamin D levels in patients 3 and 4, the clinical picture was not consistent with hypervitaminosis D. Long-term furosemide therapy for chronic lung disease in the preterm infant is the most c o m m o n recognized cause of hypercalciuria and nephrocalcinosis in infants. 1 Short and Cooke 5 noted that no difference at the time of nephrocalcinosis was detected, in the mean total dose of furosemide given to infants with or without nephrocalcinosis. Hence, other factors may be associated with hypercalciuria and nephrocalcinosis, such as parenteral calcium administration, alkaline urine, hypophosphatemia, fluid restriction, and reduced urine output and the absence or reduction of urinary stone inhibitors. 21,22 The association of renal calcification with furosemide therapy was considered to be confined to preterm infants until M o n e t al 2s reported nephrocalcinosis and nephrolithiasis in non-very low birth weight infants treated with chronic furosemide for congestive heart failure. In their 4 patients, renal calcification was detected by USG and in 1 by autopsy. Discontinuation of the furosemide in 3 children resulted in resolution of the calcifications in 2 patients. Residual renal morbidity included reduced creatinine clearance, microscopic hematuria, and hypercalciuria. Katz et al24 reported microscopic nephrocalcinosis in 35 of 38 kidney specimens from autopsies of patients with cystic fibrosis, including 2 neonates and 1 stillborn infant. They found hypercalciuria in 5 of 14 cystic fibrosis patients and attributed the nephrocalcinosis to a primary abnormality of calcium metabolism in the kidneys of patients with cystic fibrosis. Bentur et a125 found no nephrocalcinosis by ultrasound in 17 patients with cystic fibrosis and only 4 of 34 with hypercalciuria. They found only sparse microscopic nephrocalcinosis at autopsy in 5 patients with cystic fibrosis while 6 of 12 patients with other chronic illness and similar preterminal events had nephrocalcinosis also. Distal renal tubular acidosis characterized by the inability of the distal n e p h r o n to acidify urine below pH 5.5 and the presence of hypercalciuria (26) was seen. Urine citrate levels are often low or undetectable, so these patients lack an important inhibitor of renal calcification. Alkaline pH favors calcium-phosphate crystallization. The combination of high urine pH, hypercalciuria, and low urine citrate makes nephrocalcinosis a common finding in distal renal tubular acidosis. We have excluded distal renal tubular acidosis in our cases. Bartter's syndrome is a renal tubular disorder characterized by hypokalemic metabolic alkalosis, hyper-reninemia, hyperaldosteronism, normal blood

pressure, and hypertrophy of the juxtaglomerular apparatus. 27 Hypercalciuria and nephrocalcinosis were present in more than 50% of the cases. We have also excluded Bartter's syndrome with the absence of the characteristic diagnostic criteria in our cases. None of the traditional aetiologies of nephrocalcinosis could be identified, and nephrocalcinosis was not evident by repeated ultrasonographic studies in our patients. Dietary story of high calcium intake, a high protein, and sodium chloride intake, which predispose to hypercalciuria, were also not present. Hypercalciuria in our cases was transient and disappeared with remission. Patients with nephrotic syndrome, in general, have reduced calcium excretion and have decreased gastrointestinal absorption of calcium. The transient hypercalciuria most likely was the result of furosemide in these patients, although chronic prednisolone and pyelenephritis might be also contributing. We do not know whether a vulnerable period may occur during which the transient aggregation of several risk factors could lead to nephrocalcinosis in nephrotic children with prolonged furosemide use. We have not seen nephrocalcinosis in other nephrotic patients who had furosemide within the first 3 days of diuretic therapy either in steroid responsive or nonsteroid responsive nephrotic syndrome who underwent renal biopsy. 28 Because we were not aware of those possible effects of furosemide, we did not check the urinary calcium levels routinely in our early practice. The long-term prognosis of furosemide-associated nephrocalcinosis in the infant is still unclear. Although discontinuation of the diuretic often results in radiological resolution of the calcifications, functional abnormalities may persist. 6 The natural history of the renal histopathology of these patients is yet unknown. Alon et a129 investigated the histological long-term outcome of fnrosemide-induced nephrocalcinosis in the young rat. They concluded that most of the renal calcifications induced by furosemide occur during the early days of treatment and that up to 12 weeks after discontinuation of the diuretic, the resolution of the calcifications was only partial. Although hypercalciuria was transient in our patients, we do not know the importance of the present renal histopathology of our cases. We also do not know whether microscopic nephrocalcinosis persists below the threshold of ultrasonographic detection. During the 2- to 5-year follow-up period we could not record any radiologically evidence of nephrocalcinosis by serially performed renal ultrasonography. Although we are aware of the importance, we could not study all of the promoters and inhibitors simultaneously for occurrence of renal calcification partly owing to technical problems. However, we suggest that microscopic nephrocalcinosis in nephrotic children possibly associated with furosemide treatment is an important observation because, to our knowledge, such data were not available in the literature in nephrotic patients. We are concerned that furosemide therapy in nephrotic patients may cause hypercalciuria, alkaline urine, and subsequent nephrocalcinosis, especially in

1366

NEPHROCALCINOSIS IN NEPHROTICSYNDROME (Mocan et al)

prolonged use. It is advisable to follow urinary pH and calcium excretion during the furosemide therapy for the risk of nephrocalcinosis. Further observations are required to determine the importance of this condition on clinical course and final outcome for kidney function.

REFERENCES 1. Hufnagle KG, Kahn SN, Penn D, et al: Renal calcifications: A complication of long-term furosemide therapy in preterm infants. Pediatrics 70:360-363, 1982 2. Gilsanz V, Fernal W, Reid BS, et al: Nephrolithiasis in premature infants. Radiology. 154:107-110, 1985 3. Jacinto JS, Modanlou HD, Crade M, et al: Renal calcification incidence in very low birth weight infants. Pediatrics 81:31-35, 1988 4. Adams ND, RoweJC: Nephrocalcinosis. Clin Perinatol 19:179195, 1992 5. Short A, Cooke RWI: The incidence of renal calcification in preterm infants. Arch Dis Child 66:412-417, 1991 6. Ezzedeen F, Adelman RD, Ahlfors CE: Renal calcification in preterm infants: Pathophysiology and long-term sequelae. J Pediatr 113:532-539, 1988 7. Karlowicz MG, Katz ME, Adelman RD, et al: Nephrocalcinosis in very low birth weight neonates: Family history of kidney stones and ethnici D' as independent risk factors. J Pediatr 122:635-638, 1993 8. Downing GJ, EgelhoffJC, Daily DK, et al: Furosemide related renal calcifications in the premature infant. A longitudinal ultrasonographic study. Pediatr Radiol 21:563-565, 199l 9. Nash MA, Edelmann CM, Jr, Burnstein J, et ah Minimal change nephrotic syndrome, ditfuse mesengial hypercellularity and focal glomerulosclerosis, in Edelmann CM (ed): Pediatric Kidney Disease. Boston, MA, Little Brown, 1992, pp 1272-1290 10. International Study of Kidney Disease in Children: Primary nephrotic syndrome in children: Clinical significance of histopathologic variants of minimal change and of diffuse mesangial hypercellularity. Kidney Int 20:765-771, 1981 11. Milliner DS, Stickler GB: Hypercalcemia, hypercalciuria and renal disease, in Edehnann CM (ed): Pediatric Kidney Disease. Boston, MA, Little Brown, 1992, pp. 1675-1685 12. Trainin EB, Boichis H, Spitzer A, et al: Late nonresponsiveness to steroids in children with the nephrotic syndrome. J Pediatr 87:519-523, 1975

13. Arnold WC: Efficacy of metazolone and furosemide in children with furosemide-resistant oedema. Pediatrics 74:872-875, 1984 14. Davison AM, Lambie AT, Vert AH, et al: Salt-poor human albumin in management of nephrotic syndrome. BMJ 1:481-484, 1974 15. Garnett ES, Land MB, Webber CE: Changes in blood volume produced by treatment in the nephrotic syndrome. Lancet 2:798-799, 1961 16. MetcoffJ, Janeway CA: Studies on the pathogenesis of nephrotic oedema. J Pediatr 58:640-685, 1961 17. Haws RM, Baum M: Efficacy of albumin and diuretic therapy in children with nephrotic syndrome. Pediatrics 91:1142-1146, 1993 18. Karlowicz MG, Adelman RD: Renal calcification in the first year of life. Pediatr Clin North Am 42:139%1413, 1995 19. Coe FL, Parks JH, Asplin JR: The pathogenesis and treatment of kidney stones. N Engl J Med 327:1141-1152, 1992 20. GearhartJP, Herzeberg GZ,Jeffs RD: Childhood urolithiasis: Experiences and advances. Pediatrics 87:445-450, 1991 21. Kruse K, Kracht W, Kruse U: Reference values for urinary calcium excretion and screening for hypercalciuria in children and adolescents. E u r J Pediatr 143:25-31, 1984 22. Reusz GS, Dobos M, Byrd D, et al: Urinary calcium and oxalate excretion in children. Pediatr Nephrol 9:39-44, 1995 23. Alon US, Scagliotti D, Garola RE: Nephrocalcinosis and nephrolithiasis in infants with cognitive heart failure treated with furosemide. J Pediatr 125:149-151, 1994 24. Katz SM, Krueger LJ, Falkner B: Microscopic nephrocalcinosis in cystic fibrosis. N EnglJ Med 319:263-266, 1988 25. Bentur L, Kerem E, Couper R, et al: Renal calcium handling in cystic fibrosis: Lack of evidence for a primary renal defect. J Pediatr 116:556-560, 1990 26. Norman ME, Feldman NI, Cohn RM, et ah Urinary citrate excretion in the diagnosis of distal renal tubular acidosis. J Pediatr 92:394400, 1978 27. Bartter FC, Pronove P, Gill JR, et al: Hyperplasia of the juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. Am J Med 33:811, 1962 28. Mocan H, Aksoy A, Uydu HA, et al: Oxidative damage of erythrocyte membrane in nephrotic syndrome. Pediatr Nephrol 13: 326-332, 1999 29. Alon US, Kaplan RA, Gratny LL, et al: Histological long-term outcome of furosemide-induced nephrocalcinosis in the young rat. Pediatr Nephrol 10:191-194, 1996

1367