—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 5 - Iatrogenic Phospholipidosis Mimicking Fabry's Disease

Charles Alpers
University of Washington Medical Center
Seattle, WA





Virtual Slides as well as Still Images are displayed below.
For the fastest viewing of virtual slides, click:



under each thumbnail image below. You must have Aperio ImageScope installed on your PC.
If you do not already have Aperio ImageScope, Windows users with administrator privileges may download and install a free version in order to view USCAP Virtual Slides. Click the icon on the right to get your free copy:  
Or, click on slide thumbnail images to view each slide
in a Web-based slide viewer, which is somewhat slower.

If you have any difficulties viewing these slides, email or call George Clay at +1.724.449.1137.



Renal Biopsy
Light microscopic examination of the paraffin-embedded sections stained with, silver methenamine counterstained with hematoxylin and eosin (one of which was provided to you) demonstrated medulla and renal cortex containing up to 4 intact glomeruli. The glomerular visceral epithelial cells were diffusely enlarged and contained abundant, finely vacuolated cytoplasm (Figure 1). The glomeruli were without inflammation or segmental sclerosis. Some of the tubular epithelial cells showed evidence of acute tubular injury, characterized by vacuolization and blebbing of the apical cytoplasm into the tubular lumina. There was mild, patchy interstitial fibrosis, associated with mild tubular atrophy. Muscular arteries demonstrated mild intimal sclerosis. Arteriolar segments showed no significant sclerosis. There was no evidence of vasculitis.


Case 5 - Slide 1
Click to view with ImageScope
Click to view with a Web-Based Viewer


Case 5 - Figure 2
Case 5 - Figure 3
Case 5 - Figure 4


Material submitted for immunofluorescence microscopy contained 2 intact glomeruli. The glomeruli, tubular basement membranes, and interstitium showed no significant staining for IgG, IgA, IgM, kappa or lambda light chains, complement, fibrinogen, or albumin. Arterial vessels were not prominent in the specimen.

Methylene blue-stained thick sections cut at 1 µm were prepared from all tissue submitted for electron microscopy and contained only one glomerulus. Light microscopic examination revealed dense intracytoplasmic granules within the glomerular visceral epithelial cells (Figure 2). Upon ultrastructural examination, the glomerular visceral epithelial cells contained prominent membrane-bound electron dense bodies with a multilamellar appearance, characteristic of myelin figures or zebra bodies (Figures 3 and 4). Similar structures were focally present within mesangial cells, glomerular endothelial cells, interstitial cells, tubular epithelial cells, and vascular endothelium. The foot processes of overlying epithelial cells showed patchy effacement. No immune type electron dense deposits were present within the glomeruli, tubular basement membranes, interstitium, or peritubular capillaries.

Interpretation and Initial Morphologic Diagnosis
Renal biopsy with features characteristic of Fabry's disease, with superimposed acute tubular injury/acute tubular necrosis and mild arteriosclerosis.

Clinical Follow-up
Based on the renal biopsy results, a measurement of the patient's leukocyte α-galactosidase A enzyme activity was immediately obtained in order to confirm the diagnosis of Fabry's disease. This test, performed on leukocytes, revealed a decreased enzyme level of 21 nmoles/hr/mg protein (normal mean 47). However, repeat testing on leukocytes and plasma obtained approximately one week later and performed in a different referral laboratory demonstrated normal enzyme activity. Since hydroxychloroquine is known to inhibit lysosomal enzymes, including the α-galactosidase A enzyme, this therapy was discontinued. Mutational analysis by denaturing high performance liquid chromatography (DHPLC) performed at the second referral laboratory showed no abnormalities in the patient's α-galactosidase A gene. The patient was placed on angiotensin converting enzyme (ACE) inhibitor therapy. At follow-up, 6 months following the renal biopsy, the patient's proteinuria had improved slightly, dropping to 2158 mg/24 hours from her previous measured high of 3384 mg/24 hours. Her serum creatinine decreased to 1.1 mg/dl (97 µmol/dl), but her reduced creatinine clearance persisted at 58 mL/min (0.97 mL/s). A repeat analysis of the patient's α-galactosidase A enzyme activity was performed 6.5 months after the initial test and was within normal limits.

Final Clinicopathological Diagnosis
The renal biopsy morphologic findings were re-evaluated in light of the patient's α-galactosidase A enzyme activity tests results, the lack of a detectable mutation in the α-galactosidase A gene, and the patient's medication history. A final diagnosis of iatrogenic phospholipidosis due to hydroxychloroquine was made.

Discussion
Fabry's disease is one of several well-characterized lipidoses with a renal phenotype. This X-linked disease is due to the inactivation of the lysosomal enzyme, α-galactosidase A, which can result in the intracellular accumulation of globotriaosylceramide within a variety of renal cells, including podocytes, glomerular endothelial cells, mesangial cells, tubular epithelium, interstitial foam cells, vascular myocytes, and vascular endothelium [1, 2]. First described approximately 50 years ago, the renal biopsy features of classic Fabry's disease are well characterized [1, 2, 3]. Light microscopic examination of glomeruli characteristically shows enlarged podocytes with abundant, finely-vacuolated cytoplasm, due to accumulated globotriaosylceramide. Although podocytes are predominantly affected, accumulation of this storage product can occur in a variety of renal cells, including parietal epithelial cells, mesangial cells and glomerular endothelial cells, distal and proximal tubular epithelium, interstitial foam cells, vascular endothelium and vascular myocytes. As the disease progresses, mesangial expansion, segmental glomerulosclerosis, and global glomerulosclerosis can occur. Advanced disease is marked by progressive tubular atrophy and interstitial fibrosis [3]. Immunofluorescence microscopic studies are generally non-contributory. Electron microscopy reveals enlarged lysosomes filled with characteristic electron dense lamellated membrane structures (Zebra bodies, myelin figures). Progressive proteinuria is often associated with increased podocyte foot process effacement.

The differential diagnosis of intrarenal lipid accumulation includes various lysosomal storage diseases. The most useful feature in distinguishing Fabry's disease from other forms of renal lipidosis is the ultrastructural appearance of accumulated lipid as electron dense, multi-lamellated myelin figures [3, 4]. All other primary renal lipidoses have morphologic features distinct from Fabry's disease, which reflects differences either in the cellular distribution of accumulated lipid material or in the appearance of the storage material at the electron microscopic level (See Table 1 in Ref 4). Lipid accumulation primarily affecting glomerular podocytes, resulting in abundant foamy cytoplasm similar to that seen in Fabry's disease, is commonly found in infantile nephrosialidosis, GM-1 gangliosidosis, I-Cell disease (mucolipidosis, type 2), Hurler's syndrome, Niemann-Pick disease, and Farber's disease [4, 5, 6, 7, 8, 9, 10]. Accumulation of lipid with predominant involvement of glomerular mesangial and endothelial cells is seen in Gaucher's disease, and lecithin-cholesterol acyltransferase (LCAT) deficiency [5, 11, 12]. Disorders such as Refsum's disease and Sandhoff's disease (GM-2 gangliosidosis) primarily involve the tubular epithelium and are unlikely to be confused with Fabry's disease [5, 7, 8, 13, 14]. None of the above lipidoses demonstrate prominent and widespread whorled myelin figures upon ultrastructural examination, although such figures are occasionally encountered in Niemann-Pick disease.

This biopsy illustrated classic renal pathologic features of Fabry's disease, including prominent vacuolization of visceral epithelial cells by light microscopy and demonstration of characteristic intracellular myelin figures by electron microscopy. Many pathologists (including me) would regard these features as diagnostic of Fabry's disease until proven otherwise, and this was conveyed to the referring nephrologist as soon as the electron microscopic findings were obtained. However, both we and the referring nephrologist had concerns about this surprising and unsuspected diagnosis, given the lack of other symptoms or family history of Fabry's disease. We sought confirmation of this interpretation. Measurement of the patient's α-galactosidase A enzyme activity demonstrated depressed enzymatic activity, a finding widely considered as definitive proof of the carrier state in a female suspected of having Fabry's Disease, seemingly confirming the diagnosis. We then scored this as a triumph for the superiority of diagnostic nephropathology, and its servant, the nephropathologist, over other diagnostic modalities with their lack of such penetrating insight into the basis for a patient's disease.

Only a week later, clouds appeared on the horizon. A second study of enzyme activity in plasma and in leukocytes performed in a different laboratory on specimens obtained only 1 week later was within normal limits, as was a third study performed 6 and ˝ months after the initial test. This prompted a reconsideration of this patient's previous diagnosis and medication history. The predominant alternate diagnostic consideration to Fabry's disease was iatrogenic phospholipidosis of the kidney, which has morphologic features identical to Fabry's disease. Iatrogenic phospholipidosis has been reported in association with several drugs, the most common being chloroquine and amiodarone [15, 16, 17]. Chloroquine is a weak base that becomes concentrated within lysosomes and inhibits key lysosomal enzymes, including α-galactosidase A [15, 18, 19]. Due to the similarities in chemical structure, it is reasonable to assume that hydroxychloroquine causes iatrogenic phospholipidosis in a manner similar to chloroquine.

Given our patient's long-term treatment with hydroxychloroquine, lack of family history, and lack of additional symptoms of Fabry's disease, we favored a diagnosis of iatrogenic phospholipidosis due to reduced circulating α-galactosidase activity resulting from inhibition by hydroxychloroquine. This interpretation was confirmed with the results of the DNA mutational analysis test, which demonstrated a normal α-galactosidase A gene sequence in this patient, and excluded the possibility that the patient was a female carrier of a mutant gene.

Most reports of iatrogenic phospholipidosis have described morphologic features identical to those of classic Fabry's disease. Due to the inability to distinguish the genetic and iatrogenic conditions based on morphologic grounds alone, as this case illustrates, a diagnosis of Fabry's disease must be confirmed by the demonstration of depressed enzymatic activity and/or by mutational analysis of the α-galactosidase A gene. Genetic analysis is necessary to ensure detection of female carriers, who may demonstrate normal enzymatic activity due to unequal X-chromosome inactivation. Any history of exposure to implicated drugs should prompt the pathologist to consider the possibility of iatrogenic phospholipidosis. Several case reports of patients with iatrogenic phospholipidosis indicate that removal of the offending agent may lead to complete recovery of renal function and resolution of proteinuria [15, 16]. Although these reports are encouraging, our patient has not demonstrated similar recovery. Six months after discontinuation of hydroxychloroquine, our patient continues to excrete significant urinary protein (2158 mg/24 hours) and shows a decreased creatinine clearance of 58 mL/min (0.97 mL/s). This may reflect the fact that our patient was treated with hydroxychloroquine intermittently for 10 years, while the patients reported by Albay et al. [16] and Muller-Hocker et al. [15] were exposed to chloroquine for much shorter time periods (18 months and 17 months, respectively).

It is noteworthy that the studies which established the efficacy of enzyme replacement therapy leading to its approval for treatment of patients with Fabry's disease in the United States was based on removal of globotriaosylceramide from endothelial cells in biopsy samples obtained from treated patient and controls [20]. Similar efficacy in removal of lipid from podocytes was not achieved. It is not clear why this therapy is less effective for podocyte accumulation. One possibility is that the exogenously administered enzyme is less able to gain entrance to podocytes compared to endothelial cells. In our patient, the predominant lipid accumulation was within podocytes, while the endothelium was much less affected. It is possible that our patient's failure to demonstrate significant improvement in clinical renal parameters may reflect a similar inability to achieve adequate α-galactosidase A activity within podocytes. The level of α-galactosidase A expression in normal podocytes and the degree of lipid removal from podocytes required for disease abatement is not known. If the endogenous enzyme activity in podocytes is normally low or absent, the patient may still require exogenous enzyme to remove the lipid. However, if the process of enzyme entry into the podocyte is normally slow or nonexistent, some patients may recover slowly or incompletely when the offending drug is removed and normal measurable circulating enzyme activity is restored and/or replacement therapy is instituted.

In summary, we present a case of iatrogenic phospholipidosis with renal biopsy features morphologically identical to classic Fabry's disease. In this case, an atypical presentation with lack of additional Fabry's symptoms and a history of exposure to hydroxychloroquine led to further laboratory testing which confirmed the proper diagnosis. Removal of the offending agent may not be sufficient to restore renal function in patients with acquired phospholipidosis.

Acknowledgment
Erika Bracamonte, MD contributed extensively to the evaluation of this patient during her tenure as a renal pathology fellow at the University of Washington and authored a case report (ref 4) from which much of this presentation was obtained.

References
  1. Sessa A, Meroni M, Battini G, et al: Renal pathological changes in Fabry disease. J Inherit Metab Dis 24:66-70, 2001

  2. Zarate YA, Hopkin RJ: Fabry's disease. Lancet 372:1427-1435, 2008

  3. Alroy J, Sabnis S, Kopp JB: Renal pathology in Fabry's disease. J Am Soc Nephrol 13:S134-138, 2002

  4. Bracamonte ER, Kowalewska J, Starr J, Gitomer J, Alpers CE: Iatrogenic phospholipidosis mimicking Fabry Disease. Am J Kidney Dis 48:844-850, 2006.

  5. Renwick N, Nasr SH, Chung WK, Garvin J, Markowitz GS, Marboe C et al: Foamy podocytes. Am J Kid Dis 41:891-896, 2003

  6. Kashtan CE, Nevins TE, Posalaky Z, Vernier RL, Fish AJ: Proteinuria in a child with sialidosis: case report and histological studies. Pediatr Nephrol 3:166-174, 1989

  7. Bernstein J, Churg J: Heritable metabolic diseases, in Jennette JC, Olson JL, Schwartz MM, Silva FG (ed): Heptinstall's Pathology of the Kidney (5th ed), chap 30. Philadelphia , PA, Lippincott-Raven Publishers, 1998, pp 1287-1320

  8. Faraggiana T, Churg J: Renal lipidoses: a review. Hum Pathol 18:661-679, 1987

  9. Brady RO: Sphingomyelin Lipidoses: Niemann-Pick Disease, in Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS (ed): The Metabolic Basis of Inherited Disease (5th ed), chap 41. McGraw-Hill, Inc, 1983, pp 831-841

  10. D'Agati VD, Jennette JC, Silva FG: Hereditary Nephropathies, in D'Agati VD, Jennette JC, Silva FG (ed): AFIP Atlas of Nontumor Pathology Non-Neoplastic Kidney Diseases, chap 4. Silver Spring, MD, ARP Press, 2005, pp 75-104

  11. Chander PN, Nurse HM, Pirani CL: Renal involvement in adult Gaucher's disease after splenectomy. Arch Pathol Lab Med 103:440-445, 1979

  12. Lager DJ, Rosenberg BF, Shapiro H, Bernstein J: Lecithin cholesterol acyltransferase deficiency: ultrastructural examination of sequential renal biopsies. Mod Pathol 4:331-335, 1991

  13. Pabico RC, Gruebel BJ, McKenna BA, Griggs RC, Hollander J, Nusbacher J et al: Renal involvement in Refsum's disease. Am J Med 70:1136-1143, 1981

  14. Tatematsu M, Imaida K, Nobuyuki I, Togari H, Suzuki Y, Ogiu T: Sandhoff disease. Acta Pathol Jpn 31:503-512, 1981

  15. Muller-Hocker J, Schmid H, Weiss M, Dendorfer U, Braun GS: Chloroquine-induced phospholipidosis of the kidney mimicking Fabry's disease: case report and review of the literature. Hum Pathol 34:285-289, 2003

  16. Albay D, Adler SG, Philipose J, Calescibetta CC, Romansky SG, Cohen AH: Chloroquine-induced lipidosis mimicking Fabry's disease. Mod Pathol 18:733-738, 2005

  17. Kaplan LJ, Cappaert WE: Amiodarone-induced corneal deposits. Ann Opthalmol 16:762-766, 1984

  18. Hostetler KY, Richman DD: Studies on mechanism of phospholipids storage induced by amantadine and chloroquine in Madin Darby canine kidney cells. Biochem Pharmacol 3:3795-3799, 1982

  19. Fredman P, Klinghardt GW, Svennerholm L: Effect of chloroquine on the activity of some lysosomal enzymes involved in ganglioside degredation. Biochem Biophys Acta 917:1-8, 1987

  20. Desnick RJ, Brady R, Barranger J, et al: Fabry's disease, an under-recognized multisystemic disorder: expert recommendations for diagnosis, management, and enzyme therapy. Ann Intern Med 138:338-346, 2003