- Email: [email protected]
Lysozyme-Induced Nephropathy
NEPHROLOGY ROUNDS
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Lysozyme-Induced Nephropathy Dominick Santoriello1, Laurie May Andal2, Robert Cox3, Vivette D. D’Agati1 and Glen S. Markowitz1
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1 Department of Pathology & Cell Biology, Columbia University Medical Center, New York, New York, USA; 2Department of Medicine, Division of Nephrology, Christiana Care Health System, Newark, Delaware, USA; and 3Department of Medicine, Division of Nephrology, VA Medical Center, Wilmington, Delaware, USA
Correspondence: Dominick Santoriello, Department of Pathology, Columbia University, College of Physicians and Surgeons, Renal Pathology Laboratory, Room VC14-224, 630 W 168th Street, New York, NY 10032, USA. E-mail: [email protected]
Kidney Int Rep (2016) -, -–-; http://dx.doi.org/10.1016/j.ekir.2016.09.002 ª 2016 International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
INTRODUCTION eukemia is among the most common malignancies associated with hospital admissions for acute kidney injury (AKI).1,2 AKI develops in up to one-third of patients affected by hematologic malignancy, particularly in older patients with prior chronic kidney disease (CKD),3 and has been shown to have an adverse effect on long-term prognosis in this patient population.4 Intravascular volume depletion, tumor lysis syndrome, and drug-induced acute tubular injury are among the most common causes of leukemiaassociated AKI.4,5 Radiologic studies may be useful to exclude obstructive uropathy secondary to retroperitoneal lymphadenopathy or tumorous masses. Direct infiltration of the renal parenchyma by leukemic cells, although a common finding at autopsy,5 is only rarely associated with the development of symptomatic AKI.4 Rare cases of AKI secondary to intravascular leukostasis have also been described, particularly in patients with white blood cell counts greater than 100,000/mm3.6 Lysozyme-induced nephropathy (LyN) is a rare and underrecognized complication of chronic myelomonocytic leukemia (CMML) and other forms of monocytic leukemia in which lysozyme, a small cationic protein, is released into the circulation, filtered by the glomerulus and reabsorbed by the proximal tubule, causing toxic tubular injury.7
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CASE PRESENTATION Clinical History and Initial Laboratory Data A 69-year-old Caucasian man with a history of CKD (baseline serum creatinine, 2.0 mg/dl; estimated glomerular filtration rate, 35 ml/min/1.73 m2) and wellcontrolled HIV infection (CD4 count, 500 cells/mm3; viral load, <20 copies/ml) presented to the emergency department after 10 days of watery diarrhea. Two months prior, he had been diagnosed with CMML, but
had not commenced treatment. Maintenance antiretroviral therapy included darunavir, emtricitibine, and ritonavir. Physical examination findings were notable for a blood pressure of 119/63 mm Hg, splenomegaly, and the absence of edema. Initial laboratory evaluation (Table 1) revealed markedly elevated serum creatinine (10.9 mg/dl; estimated glomerular filtration rate, 5 ml/min/1.73 m2) associated with oliguria. Complete blood count revealed leukocytosis, anemia, and thrombocytopenia. Urinalysis revealed 3þ protein by dipstick. Urine protein:creatinine ratio was 6.7 g/g on a spot measurement. Urinary microscopy, performed on a specimen collected after Foley catheter insertion, showed 21 to 30 red blood cells and 31 to 40 white blood cells per high-power field, but no cellular casts or crystals. Serologic workup results were negative (Table 1). The patient showed no improvement in renal function with volume resuscitation. In light of the additional laboratory findings of hyperuricemia with hyperphosphatemia, hypocalcemia, and elevated lactate dehydrogenase, the patient was started on dialysis and allopurinol for suspected tumor lysis syndrome. A kidney biopsy was performed on the 10th hospital day. Kidney Biopsy Results The sampling for light microscopy included 16 glomeruli, 2 of which were globally sclerotic. The remaining 14 glomeruli appeared largely unremarkable, and no lesions of focal segmental glomerulosclerosis were identified. The predominant abnormalities involved proximal tubular epithelial cells, which exhibited widespread degenerative changes including luminal ectasia, cytoplasmic simplification and vacuolization, irregular luminal profiles, loss of brush border, enlarged nuclei with prominent nucleoli, and focal apoptotic figures (Figure 1a). The proximal tubular injury was accompanied by mild interstitial edema and
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103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156
D Santoriello et al.: Lysozyme-Induced Nephropathy
Table 1. Initial laboratory findings Parameter SCr (mg/dl) eGFR (ml/min/1.73 m2)
Value (reference range) 10.9 (0.6–1.2) 5 (>90)
Serum urea nitrogen (mg/dl)
72 (8–20)
Serum potassium (mmol/l)
4.4 (3.5–5.5)
Serum uric acid (mg/dl)
25 (3.8–8.0)
Serum phosphorus (mg/dl)
9.7 (2.4–4.1)
Serum calcium (mg/dl)
7.1 (8.5–10.2)
Serum albumin (g/dl)
2.5 (3.5–5.1)
LDH (IU/l) Hemoglobin (g/dl) WBC count ( 103/ml)
1100 (140–280) 9 (12.0–16.0) 67.4 (4.5–13.5)
Differential blood count (%) Neutrophils
48 (40–70)
Monocytes
39 (0–10)
Lymphocytes
9 (20–50)
Eosinophils
0 (0–6)
Urine dipstick protein Urine RBC (/hpf) Urine WBC (/hpf) Spot urine PCR (g/g)
31–40 (0–2)a 6.7 (<0.3)
Urine culture
No growth
C3 (mg/dl)
124 (88–165)
C4 (mg/dl)
37 (14–44)
ANA Q7
3þ 21–30 (none)a
Neg (neg)
MPO-ANCA
<1:20 (<1:20)
PR3-ANCA
<1:20 (<1:20)
Hepatitis C antibody
Neg (neg)
Anti-GBM antibody
Neg (neg)
Hepatitis B core antigen
Neg (neg)
Serum cryoglobulins
Neg (neg)
SPEP
No M-spike
ANA, antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; anti-GBM, antiglomerular basement membrane; eGFR, estimated glomerular filtration rate; hpf, high-power field; LDH, lactate dehydrogenase; Neg, negative; PCR, protein:creatinine ratio; RBC, red blood cell; SCr, serum creatinine; SPEP, serum protein electrophoresis WBC, white blood cell. Conversion factors for units: SCr in mg/dl to mmol/l, 88.4; SUN in mg/dl to mmol/l, 0.357. a Results obtained from Foley cathetercollected urine.
mild interstitial inflammation composed of lymphocytes and monocytes. Many proximal tubular cells had hypereosinophilic granular cytoplasm owing to the presence of abundant intracytoplasmic, PAS-positive granulules (Figure 1b). Scattered larger rounded, eosinophilic inclusions that were moderately periodic acid–Schiff positive and nonargyrophilic with the Jones methenamine silver stain were also seen (Figure 1h). Immunohistochemical staining for lysozyme (Lysozyme EP134; RabMAb, Rocklin, CA) revealed strong positivity in the distribution of the proximal tubular cell cytoplasm (Figure 1c, d). No atypical casts or intracytoplasmic crystalline-type inclusions were seen. Mild tubular atrophy and interstitial fibrosis involved 15% of the cortex sampled. Immunofluorescence staining for IgG, IgM, IgA, C3, C1, fibrinogen, albumin, and kappa (k) and lambda (l) light chains revealed no significant positivity. Immunofluorescence staining for k and l light chains 2
repeated on pronase-digested paraffin tissue sections again was negative, providing evidence against light chain proximal tubuloapthy. On ultrastructural evaluation, glomeruli exhibited no significant abnormalities. Specifically, there was only 10% foot process effacement, and no electron-dense deposits were seen. Proximal tubules displayed diffuse degenerative changes including loss of apical brush border, cytoplasmic simplification, dilatation of the endoplasmic reticulum, and intraluminal cellular debris. The most distinctive finding was abundant membranebound cytoplasmic vacuoles containing clumped, electron-dense, degenerating cellular organellar debris, consistent with autophagolysosomes (Figure 1e, f). The autophagolysosomes were diffusely distributed in proximal tubular epithelia and in some instances formed larger, membrane-bound aggregates that approached the size of the nucleus (Figure 1g). No intracellular crystals or dysmorphic mitochondria were identified. Diagnosis A diagnosis of lysozyme-induced nephropathy (LyN) was made. Clinical Follow-up Additional testing revealed markedly elevated serum lysozyme levels (101 mg/ml; nl range 2.7–9.4) and lysozymuria (>11 mg/mL; nl <3), supporting a diagnosis of lysozyme-induced nephropathy. The patient was started on chemotherapy with azacitidine, resulting in normalization of his white blood cell count. Serum lysozyme levels remained elevated 4 months after initiation of chemotherapy, but ultimately decreased to within reference range. The patient remained dialysis dependent from the time of kidney biopsy, and expired 18 months later in the setting of relapsing leukemia with blast crisis. DISCUSSION CMML is a rare, aggressive, malignant, hematopoietic neoplasm that most commonly affects patients over the age of 65 years and is characterized by peripheral blood monocytosis with myelodysplastic features. Lysozyme, also known as muramidase, is a lytic enzyme with bactericidal properties that is synthesized by monocytes and can be produced in large amounts by neoplastic cells of monocyte lineage.7 Increased serum and urine lysozyme levels (lysozymuria) were first described in patients with monocytic or myelomonocytic leukemia, including both acute and chronic forms, in the late 1960s.8 Lysozyme-induced nephropathy (LyN) is a rare and underrecognized cause of AKI in a subset of these patients, particularly those with CMML.3,7,9 Kidney International Reports (2016) -, -–-
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157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
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211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264
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D Santoriello et al.: Lysozyme-Induced Nephropathy
Figure 1. A low-power view demonstrates widespread proximal tubular degenerative changes and interstitial edema. (a) A glomerulus appears unremarkable (hematoxylin and eosin, 200). (b) At higher magnification, many proximal tubular cells are distended by numerous small intracytoplasmic PASþ granules (periodic acid–Schiff, 400). (c) Immunohistochemial staining for lysozyme shows intense granular reactivity in the distribution of proximal tubular cell cytoplasm (immunoperoxidase, 100). (d) No significant staining is seen in a paired negative control obtained from an allograft postreperfusion biopsy (immunoperoxidase, 100). (e) On ultrastructural evaluation, proximal tubular cells contain abundant membrane-bound vacuoles (8000). (f) On closer inspection, the vacuoles in proximal tubules contain clumped, degenerating organellar debris, consistent with autophagolysosomes (25,000). (g) In rare cells, the autophagolysomes appear to form larger, membrane-bound aggregates (6,000), corresponding to the (h) scattered large round eosinophilic inclusions seen in a minority of cells (hematoxylin and eosin, 600).
Lysozyme is a small cationic protein (molecular weight, 15 kDa) that is freely filtered by the glomerulus.8 Lysozyme is reabsorbed in the proximal tubule, where it is taken up by endocytosis and catabolized in
phagolysosomes.10 Despite the proximal tubules’ high absorptive capacity for lysozyme,11,12 marked overproduction of lysozyme in patients with monocytic leukemias may exceed the transport maximum, leading
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265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318
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319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
D Santoriello et al.: Lysozyme-Induced Nephropathy
to nephrotic-range nonalbumin proteinuria, as appears to have occurred in this case.13,14 By protein electrophoresis, lysozyme migrates in the gamma (g) region. Therefore, the presence of nonalbumin proteinuria with an increased g-globulin fraction but without detectable monoclonal bands by immunofixation electrophoresis may be a useful indicator of lysozymuria in the appropriate clinical context.15 Unfortunately, these studies were not performed. In the setting of monocytic and myelomonocytic leukemias, including chronic and acute forms, a steady proportion of the filtered load of lysozyme accumulates in proximal tubular cells, allowing the kidney to act as a reservoir for circulating lysozyme.12 There appears to be a threshold above which the concentration of lysozyme, a lytic enzyme with bactericidal properties,7 becomes toxic to proximal tubular cells, leading to the development of acute tubular injury and AKI. Indeed, lysozymuria can impair proximal tubular cell function,16 leading to renal insufficiency, hypokalemia secondary to renal potassium wasting, and low-level tubular albuminuria, but not Fanconi syndrome.9,17 However, elevated serum and urine lysozyme levels commonly occur in patients with monocytic and myelomonocytic leukemias and are usually not associated with the development of AKI.16,18 Therefore, kidney biopsy is needed to establish a definitive diagnosis of LyN. Descriptions of the pathologic findings in LyN are limited. An early report described tubular degenerative changes accompanied by hyaline droplets in proximal tubules, but also described hyaline droplets in patients with monocytic or myelomonocytic leukemia and intact renal function.16 A more recent report described coarse protein granules in proximal tubules by light microscopy, corresponding with the ultrastructural finding of large and prominent but relatively isomorphic lysosomes.7 In the case reported herein, the direct toxicity of lysozyme was associated with the distinctive ultrastructural finding of abundant autophagolysosomes containing degenerating organellar debris and strong immunostaining of proximal tubules for lysozyme. Similar ultrastructural findings have been described in rats transplanted with chloroleukemia, a myelomonoblastic tumor that also secretes large amounts of lysozyme, leading to lysozymuria.19 Other causes of AKI should be excluded in patients with CMML. Laboratory evaluation of our patient also revealed hyperuricemia, mild hyperphosphatemia, and mild hypocalcemia, raising the possibility of concurrent tumor lysis syndrome. However, no intratubular deposition of uric acid crystals or calcium phosphate salts was seen on kidney biopsy. Nonetheless, elevated serum uric acid levels may still have contributed to 4
Table 2. Lysozyme-induced nephropathy (LyN): teaching points
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Lysozyme is a small cationic protein produced by monocytes that is freely filtered by
the glomerulus and reabsorbed by proximal tubules. Overproduction of lysozyme in patients with chronic monocytic or myelomonocytic
leukemia may lead to nephrotic-range proteinuria (lysozymuria). Lysozyme-induced nephropathy is a rare cause of acute kidney injury in patients with
chronic monocytic or myelomonocytic leukemia. Lysozyme-induced nephropathy is characterized by acute tubular injury with abundant
cytoplasmic granular inclusions that stain strongly for lysozyme and have an ultrastructural appearance consistent with that of autophagolysosomes.
AKI in our patient, as hyperuricemia may reduce renal plasma flow and may inhibit proximal tubular cell proliferation.20 Although unlikely in the case reported herein, alternative etiologies of lysozymuria should be considered. Heavy lysozymuria may occur rarely as a form of overflow proteinuria in the setting of other disease states associated with macrophage activation and overproduction of lysozyme (including sarcoidosis) or at very low levels in the setting of tubular proteinuria related to proximal tubular cell dysfunction.7,9 The treatment of CMML is initially supportive; however, the development of leukemia-associated organ damage may be an indication for more aggressive treatment, including cytoreductive therapy or use of hypomethylating agents.21 Variable improvement in kidney function has been described in patients receiving CMML-specific chemotherapy who have direct leukemic infiltration or overproduction of lysozyme by the malignant cells.22 Although our patient had a good hematologic response to chemotherapy, he remained dialysis dependent. CONCLUSION In conclusion, acute and chronic monocytic and myelomonocytic neoplasms, most notably CMML, are associated with overproduction of lysozyme, resulting in elevated serum and urine levels. Lysozyme is freely filtered by the glomerulus, and can be associated with nephrotic-range lysozymuria. Lysozyme accumulates in proximal tubular cells, and there is a threshold at which this accumulation is associated with renal potassium wasting, toxic proximal tubular injury, and AKI. LyN is a rare and likely underrecognized etiology of AKI that has a distinctive pathologic appearance characterized by ATN, with increased cytoplasmic granules in proximal tubular cells at the light microscopic level, corresponding to abundant phagolysosomes containing partially digested organellar debris at the ultrastructural level, with strong proximal tubular cytoplasmic staining for lysozyme. Recognition of this rare etiology of AKI may guide chemotherapeutic intervention in the management of acute and chronic monocytic and myelomonocytic leukemias. Kidney International Reports (2016) -, -–-
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Q5
373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426
NEPHROLOGY ROUNDS
D Santoriello et al.: Lysozyme-Induced Nephropathy
427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
11. Cojocel C, Baumann K. Renal handling of endogenous lysozyme in the rat. Ren Physiol. 1983;6:258–265.
DISCLOSURE All the authors declared no competing interests.
REFERENCES 1. Salahudeen AK, Doshi SM, Pawar T, et al. Incidence rate, clinical correlates, and outcomes of AKI in patients admitted to a comprehensive cancer center. Clin J Am Soc Nephrol. 2013;8:347–354. 2. Christiansen CF, Johansen MB, Langeberg WJ, et al. Incidence of acute kidney injury in cancer patients: a Danish population-based cohort study. Euro J Intern Med. 2011;22: 399–406. 3. Ganguli A, Sawinski D, Berns JS. Kidney diseases associated with haematological cancers. Nat Rev Nephrol. 2015;1:478–490. 4. Canet E, Zafrani L, Lambert J, et al. Acute kidney injury in patients with newly diagnosed high-grade hematological malignancies: impact on remission and survival. PLoS One. 2013;8:e55870. 5. Luciano RL, Brewster UC. Kidney involvement in leukemia and lymphoma. Adv Chronic Kidney Dis. 2014;21:27–35. 6. Perazella MA, Eisen RN, Frederick WG, Brown E. Renal failure and severe hypokalemia associated with acute myelomonocytic leukemia. Am J Kidney Dis. 1993;22:462–467. 7. Patel TV, Rennke HG, Sloan JM, et al. A forgotten cause of kidney injury in chronic myelomonocytic leukemia. Am J Kidney Dis. 2009;54:159–164. 8. Osserman EF, Lawlor DP. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med. 1966;124:921–952. 9. Muggia FM, Heinemann HO, Farhangi M, Osserman EF. Lysozymuria and renal tubular dysfunction in monocytic and myelomonocytic leukemia. Am J Med. 1969;47:351–366. 10. Ottosen PD, Bode F, Madsen KM, Maunsbach AB. Renal handling of lysozyme in the rat. Kidney Int. 1979;15:246–254.
12. Osserman EF, Canfield RE, Beychok S, Columbia University. Institute of Cancer Research. Lysozyme [proceedings]. New York: Academic Press; 1974. 13. Aguado MJ, Garcia de Bustos J, Ojeda E, et al. Proteinuria caused by lysozymuria mimics nephrotic syndrome. Nephron. 2000;86:183. 14. Mok CC, Tam SC, Kwong YL. Pseudonephrotic syndrome caused by lysozymuria. Ann Intern Med. 1994;121:818. 15. Levinson SS, Elin RJ, Yam L. Light chain proteinuria and lysozymuria in a patient with acute monocytic leukemia. Clin Chem. 2002;48:1131–1132. 16. Pruzanski W, Platts ME. Serum and urinary proteins, lysozyme (muramidase), and renal dysfunction in mono- and myelomonocytic leukemia. J Clin Invest. 1970;49:1694–1708. 17. Pickering TG, Catovsky D. Hypokalaemia and raised lysozyme levels in acute myeloid leukaemia. Q J Med. 1973;42: 677–682. 18. Wiernik PH, Serpick AA. Clinical significance of serum and urinary muramidase activity in leukemia and other hematologic malignancies. Am J Med. 1969;46:330–343. 19. Klockars M, Azar HA, Hermida R, et al. The relationship of lysozyme to the nephropathy in chloroleukemic rats and the effects of lysozyme loading on normal rat kidneys. Cancer Res. 1974;34:47–60. 20. Wilson FP, Berns JS. Tumor lysis syndrome: new challenges and recent advances. Adv Chronic Kidney Dis. 2014;21:18–26. 21. Onida F, Barosi G, Leone G, et al. Management recommendations for chronic myelomonocytic leukemia: consensus statements from the SIE, SIES, GITMO groups. Haematologica. 2013;98:1344–1352. 22. Aribi A, Borthakur G, Ravandi F, et al. Activity of decitabine, a hypomethylating agent, in chronic myelomonocytic leukeQ6 mia. Cancer. 2007;109:713–717.
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Lysozyme-Induced Nephropathy Dominick Santoriello1, Laurie May Andal2, Robert Cox3, Vivette D. D’Agati1 and Glen S. Markowitz1
Q11
1 Department of Pathology & Cell Biology, Columbia University Medical Center, New York, New York, USA; 2Department of Medicine, Division of Nephrology, Christiana Care Health System, Newark, Delaware, USA; and 3Department of Medicine, Division of Nephrology, VA Medical Center, Wilmington, Delaware, USA
Correspondence: Dominick Santoriello, Department of Pathology, Columbia University, College of Physicians and Surgeons, Renal Pathology Laboratory, Room VC14-224, 630 W 168th Street, New York, NY 10032, USA. E-mail: [email protected]
Kidney Int Rep (2016) -, -–-; http://dx.doi.org/10.1016/j.ekir.2016.09.002 ª 2016 International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
INTRODUCTION eukemia is among the most common malignancies associated with hospital admissions for acute kidney injury (AKI).1,2 AKI develops in up to one-third of patients affected by hematologic malignancy, particularly in older patients with prior chronic kidney disease (CKD),3 and has been shown to have an adverse effect on long-term prognosis in this patient population.4 Intravascular volume depletion, tumor lysis syndrome, and drug-induced acute tubular injury are among the most common causes of leukemiaassociated AKI.4,5 Radiologic studies may be useful to exclude obstructive uropathy secondary to retroperitoneal lymphadenopathy or tumorous masses. Direct infiltration of the renal parenchyma by leukemic cells, although a common finding at autopsy,5 is only rarely associated with the development of symptomatic AKI.4 Rare cases of AKI secondary to intravascular leukostasis have also been described, particularly in patients with white blood cell counts greater than 100,000/mm3.6 Lysozyme-induced nephropathy (LyN) is a rare and underrecognized complication of chronic myelomonocytic leukemia (CMML) and other forms of monocytic leukemia in which lysozyme, a small cationic protein, is released into the circulation, filtered by the glomerulus and reabsorbed by the proximal tubule, causing toxic tubular injury.7
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CASE PRESENTATION Clinical History and Initial Laboratory Data A 69-year-old Caucasian man with a history of CKD (baseline serum creatinine, 2.0 mg/dl; estimated glomerular filtration rate, 35 ml/min/1.73 m2) and wellcontrolled HIV infection (CD4 count, 500 cells/mm3; viral load, <20 copies/ml) presented to the emergency department after 10 days of watery diarrhea. Two months prior, he had been diagnosed with CMML, but
had not commenced treatment. Maintenance antiretroviral therapy included darunavir, emtricitibine, and ritonavir. Physical examination findings were notable for a blood pressure of 119/63 mm Hg, splenomegaly, and the absence of edema. Initial laboratory evaluation (Table 1) revealed markedly elevated serum creatinine (10.9 mg/dl; estimated glomerular filtration rate, 5 ml/min/1.73 m2) associated with oliguria. Complete blood count revealed leukocytosis, anemia, and thrombocytopenia. Urinalysis revealed 3þ protein by dipstick. Urine protein:creatinine ratio was 6.7 g/g on a spot measurement. Urinary microscopy, performed on a specimen collected after Foley catheter insertion, showed 21 to 30 red blood cells and 31 to 40 white blood cells per high-power field, but no cellular casts or crystals. Serologic workup results were negative (Table 1). The patient showed no improvement in renal function with volume resuscitation. In light of the additional laboratory findings of hyperuricemia with hyperphosphatemia, hypocalcemia, and elevated lactate dehydrogenase, the patient was started on dialysis and allopurinol for suspected tumor lysis syndrome. A kidney biopsy was performed on the 10th hospital day. Kidney Biopsy Results The sampling for light microscopy included 16 glomeruli, 2 of which were globally sclerotic. The remaining 14 glomeruli appeared largely unremarkable, and no lesions of focal segmental glomerulosclerosis were identified. The predominant abnormalities involved proximal tubular epithelial cells, which exhibited widespread degenerative changes including luminal ectasia, cytoplasmic simplification and vacuolization, irregular luminal profiles, loss of brush border, enlarged nuclei with prominent nucleoli, and focal apoptotic figures (Figure 1a). The proximal tubular injury was accompanied by mild interstitial edema and
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D Santoriello et al.: Lysozyme-Induced Nephropathy
Table 1. Initial laboratory findings Parameter SCr (mg/dl) eGFR (ml/min/1.73 m2)
Value (reference range) 10.9 (0.6–1.2) 5 (>90)
Serum urea nitrogen (mg/dl)
72 (8–20)
Serum potassium (mmol/l)
4.4 (3.5–5.5)
Serum uric acid (mg/dl)
25 (3.8–8.0)
Serum phosphorus (mg/dl)
9.7 (2.4–4.1)
Serum calcium (mg/dl)
7.1 (8.5–10.2)
Serum albumin (g/dl)
2.5 (3.5–5.1)
LDH (IU/l) Hemoglobin (g/dl) WBC count ( 103/ml)
1100 (140–280) 9 (12.0–16.0) 67.4 (4.5–13.5)
Differential blood count (%) Neutrophils
48 (40–70)
Monocytes
39 (0–10)
Lymphocytes
9 (20–50)
Eosinophils
0 (0–6)
Urine dipstick protein Urine RBC (/hpf) Urine WBC (/hpf) Spot urine PCR (g/g)
31–40 (0–2)a 6.7 (<0.3)
Urine culture
No growth
C3 (mg/dl)
124 (88–165)
C4 (mg/dl)
37 (14–44)
ANA Q7
3þ 21–30 (none)a
Neg (neg)
MPO-ANCA
<1:20 (<1:20)
PR3-ANCA
<1:20 (<1:20)
Hepatitis C antibody
Neg (neg)
Anti-GBM antibody
Neg (neg)
Hepatitis B core antigen
Neg (neg)
Serum cryoglobulins
Neg (neg)
SPEP
No M-spike
ANA, antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; anti-GBM, antiglomerular basement membrane; eGFR, estimated glomerular filtration rate; hpf, high-power field; LDH, lactate dehydrogenase; Neg, negative; PCR, protein:creatinine ratio; RBC, red blood cell; SCr, serum creatinine; SPEP, serum protein electrophoresis WBC, white blood cell. Conversion factors for units: SCr in mg/dl to mmol/l, 88.4; SUN in mg/dl to mmol/l, 0.357. a Results obtained from Foley cathetercollected urine.
mild interstitial inflammation composed of lymphocytes and monocytes. Many proximal tubular cells had hypereosinophilic granular cytoplasm owing to the presence of abundant intracytoplasmic, PAS-positive granulules (Figure 1b). Scattered larger rounded, eosinophilic inclusions that were moderately periodic acid–Schiff positive and nonargyrophilic with the Jones methenamine silver stain were also seen (Figure 1h). Immunohistochemical staining for lysozyme (Lysozyme EP134; RabMAb, Rocklin, CA) revealed strong positivity in the distribution of the proximal tubular cell cytoplasm (Figure 1c, d). No atypical casts or intracytoplasmic crystalline-type inclusions were seen. Mild tubular atrophy and interstitial fibrosis involved 15% of the cortex sampled. Immunofluorescence staining for IgG, IgM, IgA, C3, C1, fibrinogen, albumin, and kappa (k) and lambda (l) light chains revealed no significant positivity. Immunofluorescence staining for k and l light chains 2
repeated on pronase-digested paraffin tissue sections again was negative, providing evidence against light chain proximal tubuloapthy. On ultrastructural evaluation, glomeruli exhibited no significant abnormalities. Specifically, there was only 10% foot process effacement, and no electron-dense deposits were seen. Proximal tubules displayed diffuse degenerative changes including loss of apical brush border, cytoplasmic simplification, dilatation of the endoplasmic reticulum, and intraluminal cellular debris. The most distinctive finding was abundant membranebound cytoplasmic vacuoles containing clumped, electron-dense, degenerating cellular organellar debris, consistent with autophagolysosomes (Figure 1e, f). The autophagolysosomes were diffusely distributed in proximal tubular epithelia and in some instances formed larger, membrane-bound aggregates that approached the size of the nucleus (Figure 1g). No intracellular crystals or dysmorphic mitochondria were identified. Diagnosis A diagnosis of lysozyme-induced nephropathy (LyN) was made. Clinical Follow-up Additional testing revealed markedly elevated serum lysozyme levels (101 mg/ml; nl range 2.7–9.4) and lysozymuria (>11 mg/mL; nl <3), supporting a diagnosis of lysozyme-induced nephropathy. The patient was started on chemotherapy with azacitidine, resulting in normalization of his white blood cell count. Serum lysozyme levels remained elevated 4 months after initiation of chemotherapy, but ultimately decreased to within reference range. The patient remained dialysis dependent from the time of kidney biopsy, and expired 18 months later in the setting of relapsing leukemia with blast crisis. DISCUSSION CMML is a rare, aggressive, malignant, hematopoietic neoplasm that most commonly affects patients over the age of 65 years and is characterized by peripheral blood monocytosis with myelodysplastic features. Lysozyme, also known as muramidase, is a lytic enzyme with bactericidal properties that is synthesized by monocytes and can be produced in large amounts by neoplastic cells of monocyte lineage.7 Increased serum and urine lysozyme levels (lysozymuria) were first described in patients with monocytic or myelomonocytic leukemia, including both acute and chronic forms, in the late 1960s.8 Lysozyme-induced nephropathy (LyN) is a rare and underrecognized cause of AKI in a subset of these patients, particularly those with CMML.3,7,9 Kidney International Reports (2016) -, -–-
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D Santoriello et al.: Lysozyme-Induced Nephropathy
Figure 1. A low-power view demonstrates widespread proximal tubular degenerative changes and interstitial edema. (a) A glomerulus appears unremarkable (hematoxylin and eosin, 200). (b) At higher magnification, many proximal tubular cells are distended by numerous small intracytoplasmic PASþ granules (periodic acid–Schiff, 400). (c) Immunohistochemial staining for lysozyme shows intense granular reactivity in the distribution of proximal tubular cell cytoplasm (immunoperoxidase, 100). (d) No significant staining is seen in a paired negative control obtained from an allograft postreperfusion biopsy (immunoperoxidase, 100). (e) On ultrastructural evaluation, proximal tubular cells contain abundant membrane-bound vacuoles (8000). (f) On closer inspection, the vacuoles in proximal tubules contain clumped, degenerating organellar debris, consistent with autophagolysosomes (25,000). (g) In rare cells, the autophagolysomes appear to form larger, membrane-bound aggregates (6,000), corresponding to the (h) scattered large round eosinophilic inclusions seen in a minority of cells (hematoxylin and eosin, 600).
Lysozyme is a small cationic protein (molecular weight, 15 kDa) that is freely filtered by the glomerulus.8 Lysozyme is reabsorbed in the proximal tubule, where it is taken up by endocytosis and catabolized in
phagolysosomes.10 Despite the proximal tubules’ high absorptive capacity for lysozyme,11,12 marked overproduction of lysozyme in patients with monocytic leukemias may exceed the transport maximum, leading
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D Santoriello et al.: Lysozyme-Induced Nephropathy
to nephrotic-range nonalbumin proteinuria, as appears to have occurred in this case.13,14 By protein electrophoresis, lysozyme migrates in the gamma (g) region. Therefore, the presence of nonalbumin proteinuria with an increased g-globulin fraction but without detectable monoclonal bands by immunofixation electrophoresis may be a useful indicator of lysozymuria in the appropriate clinical context.15 Unfortunately, these studies were not performed. In the setting of monocytic and myelomonocytic leukemias, including chronic and acute forms, a steady proportion of the filtered load of lysozyme accumulates in proximal tubular cells, allowing the kidney to act as a reservoir for circulating lysozyme.12 There appears to be a threshold above which the concentration of lysozyme, a lytic enzyme with bactericidal properties,7 becomes toxic to proximal tubular cells, leading to the development of acute tubular injury and AKI. Indeed, lysozymuria can impair proximal tubular cell function,16 leading to renal insufficiency, hypokalemia secondary to renal potassium wasting, and low-level tubular albuminuria, but not Fanconi syndrome.9,17 However, elevated serum and urine lysozyme levels commonly occur in patients with monocytic and myelomonocytic leukemias and are usually not associated with the development of AKI.16,18 Therefore, kidney biopsy is needed to establish a definitive diagnosis of LyN. Descriptions of the pathologic findings in LyN are limited. An early report described tubular degenerative changes accompanied by hyaline droplets in proximal tubules, but also described hyaline droplets in patients with monocytic or myelomonocytic leukemia and intact renal function.16 A more recent report described coarse protein granules in proximal tubules by light microscopy, corresponding with the ultrastructural finding of large and prominent but relatively isomorphic lysosomes.7 In the case reported herein, the direct toxicity of lysozyme was associated with the distinctive ultrastructural finding of abundant autophagolysosomes containing degenerating organellar debris and strong immunostaining of proximal tubules for lysozyme. Similar ultrastructural findings have been described in rats transplanted with chloroleukemia, a myelomonoblastic tumor that also secretes large amounts of lysozyme, leading to lysozymuria.19 Other causes of AKI should be excluded in patients with CMML. Laboratory evaluation of our patient also revealed hyperuricemia, mild hyperphosphatemia, and mild hypocalcemia, raising the possibility of concurrent tumor lysis syndrome. However, no intratubular deposition of uric acid crystals or calcium phosphate salts was seen on kidney biopsy. Nonetheless, elevated serum uric acid levels may still have contributed to 4
Table 2. Lysozyme-induced nephropathy (LyN): teaching points
Q8
Lysozyme is a small cationic protein produced by monocytes that is freely filtered by
the glomerulus and reabsorbed by proximal tubules. Overproduction of lysozyme in patients with chronic monocytic or myelomonocytic
leukemia may lead to nephrotic-range proteinuria (lysozymuria). Lysozyme-induced nephropathy is a rare cause of acute kidney injury in patients with
chronic monocytic or myelomonocytic leukemia. Lysozyme-induced nephropathy is characterized by acute tubular injury with abundant
cytoplasmic granular inclusions that stain strongly for lysozyme and have an ultrastructural appearance consistent with that of autophagolysosomes.
AKI in our patient, as hyperuricemia may reduce renal plasma flow and may inhibit proximal tubular cell proliferation.20 Although unlikely in the case reported herein, alternative etiologies of lysozymuria should be considered. Heavy lysozymuria may occur rarely as a form of overflow proteinuria in the setting of other disease states associated with macrophage activation and overproduction of lysozyme (including sarcoidosis) or at very low levels in the setting of tubular proteinuria related to proximal tubular cell dysfunction.7,9 The treatment of CMML is initially supportive; however, the development of leukemia-associated organ damage may be an indication for more aggressive treatment, including cytoreductive therapy or use of hypomethylating agents.21 Variable improvement in kidney function has been described in patients receiving CMML-specific chemotherapy who have direct leukemic infiltration or overproduction of lysozyme by the malignant cells.22 Although our patient had a good hematologic response to chemotherapy, he remained dialysis dependent. CONCLUSION In conclusion, acute and chronic monocytic and myelomonocytic neoplasms, most notably CMML, are associated with overproduction of lysozyme, resulting in elevated serum and urine levels. Lysozyme is freely filtered by the glomerulus, and can be associated with nephrotic-range lysozymuria. Lysozyme accumulates in proximal tubular cells, and there is a threshold at which this accumulation is associated with renal potassium wasting, toxic proximal tubular injury, and AKI. LyN is a rare and likely underrecognized etiology of AKI that has a distinctive pathologic appearance characterized by ATN, with increased cytoplasmic granules in proximal tubular cells at the light microscopic level, corresponding to abundant phagolysosomes containing partially digested organellar debris at the ultrastructural level, with strong proximal tubular cytoplasmic staining for lysozyme. Recognition of this rare etiology of AKI may guide chemotherapeutic intervention in the management of acute and chronic monocytic and myelomonocytic leukemias. Kidney International Reports (2016) -, -–-
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11. Cojocel C, Baumann K. Renal handling of endogenous lysozyme in the rat. Ren Physiol. 1983;6:258–265.
DISCLOSURE All the authors declared no competing interests.
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