Med Mol Morphol DOI 10.1007/s00795-015-0114-3
CASE REPORT
The reduced expression of proximal tubular transporters in acquired Fanconi syndrome with j light chain deposition Akihiro Tojo1 • Kensuke Asaba1 • Satoshi Kinugasa1 • Yoichiro Ikeda1 Yukako Shintani2 • Masashi Fukayama2 • Masaomi Nangaku1
•
Received: 12 May 2015 / Accepted: 23 June 2015 Ó The Japanese Society for Clinical Molecular Morphology 2015
Abstract In a case of acquired Fanconi syndrome associated with smoldering myeloma, we confirmed the deposition of protease-resistant j light chain proteins in a proximal tubular injury and found the decreased expression of apical tubular transporters including sodium glucose cotransporter, sodium phosphate co-transporter, uric acid transporter 1, and a decrease of Na?/K?-ATPase in the basolateral membrane. The protease-resistant kappa light chain has a pathological role in the expression of tubular transporters in the proximal tubule and causes Fanconi syndrome associated with smoldering myeloma. Keywords Fanconi syndrome j light chain Myeloma Proximal tubule SGLT2 NaPi-IIa URAT1
significance or smoldering myeloma is rare [2, 3]. Light chains are normally filtered through the glomerulus, absorbed by megalin or cubilin in the proximal tubule and removed by catabolism in proximal tubular epithelial cells. Some kappa light chain proteins produced in patients with myeloma are protease-resistant and accumulate and form crystalline structures within the proximal tubule [2, 4]. We revealed that the urine of a patient with Fanconi syndrome associated with smoldering myeloma contained a monoclonal kappa light chain that was resistant to protease treatment. We also noted the suppression of sodium phosphate co-transporter, sodium glucose co-transporter, uric acid transporter and the Na?/K?-ATPase pump.
Clinical case Introduction Acquired Fanconi syndrome is a disorder of the proximal tubular reabsorption of phosphate, glucose, uric acid, amino acid, low molecular weight proteins, and bicarbonate. It often occurs in association with amyloidosis, multiple myeloma, paroxysmal nocturnal hemoglobinuria, heavy metal poisoning (including lead and cadmium), and drug use (including antiretroviral drugs and ifosfamide) [1]. However, acquired Fanconi syndrome showing osteomalacia through monoclonal gammopathy of undetermined & Akihiro Tojo [email protected] 1
Division of Nephrology and Endocrinology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2
Department of Pathology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
A 64-year-old woman with complaints of back pain and muscle weakness was referred to our hospital for evaluation of hypophosphatemia and low bone mineral density with multiple incomplete fractures, which were observed during a 10-year follow-up for chronic kidney disease. A physical examination revealed no obvious abnormalities. Laboratory studies showed hypokalemia (2.9 mEq/L) with normal sodium level (142 mEq/L), hypouricemia (1.9 mg/dL) with an increased fractional excretion of uric acid (FEUA 45 %), hypophosphatemia (2.0 mg/dL) with reduced tubular reabsorption (TmP/GFR 0.8 mg/dL), glycosuria (3?) with a normal plasma glucose level (95 mg/dL), aminoaciduria and hyperchloremic metabolic acidosis with a normal anion gap (pH7.337, HCO315.7 mmol/L, CL- 117 mEq/L). Bone radiographs showed no osteolytic lesions. Dual-energy X-ray absorptiometry examination showed T scores of -2.8 in the femoral region. The patient was diagnosed with
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osteomalacia caused by Fanconi syndrome, but she had no particular family history or past history of bone or mineral disorders, and no history of exposure to toxic agents or heavy metals. A monoclonal kappa light chain was detected in her serum and urine, and bone marrow showed a 6.1 % infiltration by atypical plasma cells, which led to a diagnosis of smoldering multiple myeloma. Treatment with phosphate and potassium supplements and calcitriol immediately ameliorated her back pain and weakness. A renal biopsy was performed because the patient’s renal dysfunction continued with an elevation of serum creatinine level to 2.8 mg/dL.
Materials and methods
was washed with a buffer solution containing 20 mM Trishydrochloride, 150 mM sodium hydrochloride, and 1 % NP-40 (pH7.5). The precipitate in the SDS sample buffer was boiled for 3 min. 2 lL was then applied on a 15/20 % gel. The sample was then electrophoretically transferred to a PVP membrane. After blocking with 5 % dry milk, the membrane was incubated with polyclonal rabbit anti-human kappa light chain antibody at 1:500 dilution and with anti-rabbit immunoglobulin (Dako, Glostrup, Denmark) at 1:2000 dilution. The bands were detected with the diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan) with hydrogen peroxide and nickel chloride. Each 250 ng of human kappa light chain protein (Abcam, Tokyo, Japan) in 100 lL of PBS was also processed the same way and compared with the patient’s urine.
Immunohistochemistry
Results Paraffin-embedded tissue was processed for immunohistochemistry, as previously described [5]. Sections (2 lm in thickness) were dewaxed and incubated with citrate buffer (pH6) at 100 °C for 20 min to retrieve the antigens. The sections were then incubated with 3 % H2O2 and blocking serum followed by polyclonal antibodies against sodium glucose co-transporter 2 (SGLT2, Abcam, Tokyo, Japan), uric acid transporter SLC22A12 (URAT1, MBL, Nagoya, Japan), sodium phosphate co-transporter IIa (NaP-IIa, Abcam, Tokyo, Japan), Na?/K? ATPase a1 (Upstate biotechnology, NY, USA), or human kappa light chains (Dako, Glostrup, Denmark) at 1:200 dilution. The sections were subsequently incubated a horseradish peroxidase– conjugated secondary antibody against anti-rabbit IgG (Dako, Glostrup, Denmark) at 1:50 dilution, and horseradish peroxidase labeling was detected using the DAB reaction. The sections were then counterstained with periodic acid-Schiff staining before being examined under a light microscope. Non-malignant areas from the resected kidney of a patient with renal cell carcinoma served as a control. Proteolysis of the urinary kappa light chain and Western blotting According to the previously described methods of protease treatment [6], the patient’s urine samples (100 lL) were incubated with three different proteases including cathepsin B (R&D Systems, Minneapolis, MN, USA), trypsin (Wako, Osaka, Japan), and pepsin (Tokyo Chemical Industry, Tokyo, Japan) overnight in a water bath at 37 °C. The samples were then immunoprecipitated using antihuman kappa light chain antibodies (Dako, Glostrup, Denmark) at 1:200 dilution and protein A. The precipitate
123
Renal biopsy findings A renal biopsy revealed that 6 (24 %) of 25 glomeruli showed global sclerosis with 3 (12 %) showing mesangial cell proliferation, and a 10 % area of interstitial fibrosis. No immune deposits were identified in the glomeruli by immunofluorescence microscopy or electron microscopy. The proximal tubule and the tubular casts were positive for kappa light chain (Fig. 1) and weakly positive for lambda light chain. An electron microscopic observation of the proximal tubule revealed intracytoplasmic needle-shaped electron-dense crystalline structures (Fig. 1) and some mitochondria became smaller round shape with irregular cristae, suggesting reduced ATP production. Protease resistance of urinary kappa light chain In contrast with the kappa light chain protein, the kappa light chain in the patient’s urine was resistant to three proteases including cathepsin B, trypsin, and pepsin, and showed 23 kD of the original kappa light chain and its digested protein in a 12 kD band even after overnight treatment with the proteases (Fig. 2). The expression of transporters in the proximal tubule The expressions of SGLT2, NaPi-IIa, and URAT1 in the apical membrane of the proximal tubule was reduced in comparison to the normal control kidney (Fig. 3). The expression of Na?/K?-ATPase expression was also reduced in the basolateral membrane of the patient’s proximal tubule (Fig. 3).
Med Mol Morphol Fig. 1 Electron microscopy and the immunostaining of renal biopsy sample for kappa light chain. a An electron micrograph illustrating the cytoplasmic needle-shaped inclusions in the proximal tubule (yellow arrow). Bar indicates 1 lm. b A light micrograph of Epon section showing needle-shape crystals (yellow arrow). Bar indicates 10 lm. c An immunostaining for kappa light chain detected kappa light chain in the proximal tubules. The scale bars indicate 15 lm
Fig. 2 The protease resistance of the urinary kappa (j) light chain. The patient’s urinary j light chain and commercially available purified kappa light chain protein were digested with cathepsin B (Cat), trypsin (Tri), pepsin (Pep), or with no protease (No), and a Western blot analysis was performed to detect j light chain. The 23 kD j light chain form the patient’s urine was resistant to cathepsin B, trypsin and pepsin and 12 kD products remained, whereas the purified j light chain protein (purchased) was completely degraded by trypsin and pepsin without 12kD products
Discussion In this case report, we have clearly demonstrated the reduced expression of the apical transporters including SGLT2, NaPi-IIa, and URAT1 and of the basolateral Na?/
K?-ATPase pump in the proximal tubule in a patient with acquired Fanconi syndrome. The suppressed expression of the apical transporters and the basolateral pump generating Na? gradient to facilitate solute transport can explain our patient’s glycosuria, hypophosphatemia, hypouricemia, and amino aciduria. Most of proximal tubular transport is Na?-coupled and depending on Na?-gradient produced by basolateral Na?/K?-ATPase pump. Thus, as a mechanism of tubular dysfunction, the reduced expression of Na?/K?ATPase [7] or its activity with decreased ATP level [8], and the reduced expression of Na? phosphate transporter [9] have been reported in the primary cultured proximal tubular cells of Fanconi syndrome models. However, to our knowledge, no reports have described the presence of all of these transporters in an immunohistochemical analysis of a renal biopsy sample from a patient with acquired Fanconi syndrome. Acquired hypophosphatemic osteomalacia has been rarely reported as a complication of multiple myeloma, MGUS, lymphoplasmacytic lymphoma, and chronic lymphocytic leukemia. In these cases, hypophosphatemic osteomalacia appeared in relation to the urinary secretion of a monoclonal immunoglobulin light chain that formed crystals inside the cells of the proximal tubule. The precise pathophysiology of the light chain in Fanconi syndrome is still unknown. We demonstrated that kappa light chain is deposited in the proximal tubule, which is consistent with the previous case reports that have shown the predominance of kappa light chain deposition [10]. Furthermore, as confirmed in our case, the urinary light chains of patients with Fanconi syndrome have been shown to be resistant to degradation by lysosomal proteases including cathepsin, pepsin and trypsin. The light chains from patients with Fanconi syndrome associated with myeloma have a unique biochemical characteristic in part of the variable domain
123
Med Mol Morphol Fig. 3 Immunostaining for sodium glucose co-transporter 2 (SGLT2), sodium phosphate cotransporter IIa (NaPi-IIa), uric acid transporter 1 (URAT1) and sodium potassium ATPase (Na?/K?-ATPase). P indicates proximal tubules that can be identified by the brush border membrane. The scale bars indicate 50 lm
[10], which accumulates in the lysosomes as intracytoplasmic needle- or rhomboid-shaped crystals [11, 12] and which presumably impairs the membrane recycling of tubular transporters. The accumulation of kappa light chain is nephrotoxic and has been shown to reduce both the activity and gene expression of Na?/K?-ATPase pump in the primary cultures of rat proximal tubule cells [7]. Cystinosis, a lysosomal storage disease, is the most common inherited cause of Fanconi syndrome. The main mechanisms of the impaired tubular reabsorption investigated in the cultured human renal cells incubated with
123
cysteine dimethyl ester are the decreased intracellular ATP production via a lower intracellular phosphate concentration [8, 13] with the reduced expression of Na?-phosphate co-transporter [14]. Similar to cysteine, the lysosomal accumulation of kappa light chain also causes the reduction of ATP production and the inhibition of Na?-dependent transporters. Despite sodium reabsorption in the proximal tubule is damaged, serum sodium level is maintained at a normal level, because the sodium reabsorption in the loop of Henle and distal nephron remain intact and compensated the proximal tubular damage in Fanconi syndrome.
Med Mol Morphol
Among patients with multiple myeloma, this complication seems to occur in only a small subset of patients with kappa light chain-excreting myeloma and is usually benign and slowly progressive. In most cases, hypophosphatemic osteomalacia responds to supportive treatment with phosphate supplements; however, the improvement of tubular function after myeloma treatment has only rarely been reported. Ma et al. [15] studied 32 cases of patients with MGUS or smoldering myeloma, and found that end-stage renal disease developed in 5 patients (15.6 %) during an average follow-up period of 65 months. In our case, prednisolone treatment, which was administered to reduce light chain production, did not improve renal function and tubular reabsorption.
Conclusion The expression of the proximal tubular transporters, including SGLT2, NaPi-IIa, URAT1 and Na?/K?-ATPase was reduced in a patient with Fanconi syndrome associated with smoldering multiple myeloma with hypophosphate osteomalacia. The deposition of protease-resistant kappa light chain in the proximal tubule may damage the tubular expression of these transporters. Acknowledgments This work was partly supported by a grant-inaid for scientific research from the Japan Science Promotion Foundation to A.T. (C-23591214), and a scholarship donation from the Chugai Pharmaceutical Co. Ltd. to AT. Conflict of interest The authors declare that no conflicts of interests were associated with the present study.
References 1. Haque SK, Ariceta G, Batlle D (2012) Proximal renal tubular acidosis: a not so rare disorder of multiple etiologies. Nephrol Dial Transplant 27:4273–4287 2. Lee DB, Drinkard JP, Rosen VJ, Gonick HC (1972) The adult Fanconi syndrome: observations on etiology, morphology, renal function and mineral metabolism in three patients. Medicine (Baltimore) 51:107–138
3. Hashimoto T, Arakawa K, Ohta Y, Suehiro T, Uesugi N, Nakayama M, Tsuchihashi T (2007) Acquired fanconi syndrome with osteomalacia secondary to monoclonal gammopathy of undetermined significance. Intern Med 46:241–245 4. Lacy MQ, Gertz MA (1999) Acquired Fanconi’s syndrome associated with monoclonal gammopathies. Hematol Oncol Clin North Am 13:1273–1280 5. Tojo A, Onozato ML, Fujita T (2006) Role of macula densa neuronal nitric oxide synthase in renal diseases. Med Mol Morphol 39:2–7 6. Leboulleux M, Lelongt B, Mougenot B, Touchard G, Makdassi R, Rocca A, Noel LH, Ronco PM, Aucouturier P (1995) Protease resistance and binding of Ig light chains in myeloma-associated tubulopathies. Kidney Int 48:72–79 7. Guan S, el-Dahr S, Batuman SDipp (1999) Inhibition of Na-KATPase activity and gene expression by a myeloma light chain in proximal tubule cells. J Investig Med 47:496–501 8. Coor C, Salmon RF, Quigley R, Marver D, Baum M (1991) Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. J Clin Invest 87:955–961 9. Taub ML, Springate JE, Cutuli F (2011) Reduced phosphate transport in the renal proximal tubule cells in cystinosis is due to decreased expression of transporters rather than an energy defect. Biochem Biophys Res Commun 407:355–359 10. El Hamel C, Thierry A, Trouillas P, Bridoux F, Carrion C, Quellard N, Goujon JM, Aldigier JC, Gombert JM, Cogne MT, Touchard G (2010) Crystal-storing histiocytosis with renal Fanconi syndrome: pathological and molecular characteristics compared with classical myeloma-associated Fanconi syndrome. Nephrol Dial Transplant 25:2982–2990 11. Stokes MB, Aronoff B, Siegel DD, Agati VD (2006) Dysproteinemia-related nephropathy associated with crystal-storing histiocytosis. Kidney Int 70:597–602 12. Herrera GA (2009) Renal lesions associated with plasma cell dyscrasias: practical approach to diagnosis, new concepts, and challenges. Arch Pathol Lab Med 133:249–267 13. Baum M (1998) The Fanconi syndrome of cystinosis: insights into the pathophysiology. Pediatr Nephrol 12:492–497 14. Cetinkaya I, Schlatter E, Hirsch JR, Herter P, Harms E, Kleta R (2002) Inhibition of Na(?)-dependent transporters in cystineloaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis. J Am Soc Nephrol 13:2085–2093 15. Ma CX, Lacy MQ, Rompala JF, Dispenzieri A, Rajkumar SV, Greipp PR, Fonseca R, Kyle RA, Gertz MA (2004) Acquired Fanconi syndrome is an indolent disorder in the absence of overt multiple myeloma. Blood 104:40–42
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CASE REPORT
The reduced expression of proximal tubular transporters in acquired Fanconi syndrome with j light chain deposition Akihiro Tojo1 • Kensuke Asaba1 • Satoshi Kinugasa1 • Yoichiro Ikeda1 Yukako Shintani2 • Masashi Fukayama2 • Masaomi Nangaku1
•
Received: 12 May 2015 / Accepted: 23 June 2015 Ó The Japanese Society for Clinical Molecular Morphology 2015
Abstract In a case of acquired Fanconi syndrome associated with smoldering myeloma, we confirmed the deposition of protease-resistant j light chain proteins in a proximal tubular injury and found the decreased expression of apical tubular transporters including sodium glucose cotransporter, sodium phosphate co-transporter, uric acid transporter 1, and a decrease of Na?/K?-ATPase in the basolateral membrane. The protease-resistant kappa light chain has a pathological role in the expression of tubular transporters in the proximal tubule and causes Fanconi syndrome associated with smoldering myeloma. Keywords Fanconi syndrome j light chain Myeloma Proximal tubule SGLT2 NaPi-IIa URAT1
significance or smoldering myeloma is rare [2, 3]. Light chains are normally filtered through the glomerulus, absorbed by megalin or cubilin in the proximal tubule and removed by catabolism in proximal tubular epithelial cells. Some kappa light chain proteins produced in patients with myeloma are protease-resistant and accumulate and form crystalline structures within the proximal tubule [2, 4]. We revealed that the urine of a patient with Fanconi syndrome associated with smoldering myeloma contained a monoclonal kappa light chain that was resistant to protease treatment. We also noted the suppression of sodium phosphate co-transporter, sodium glucose co-transporter, uric acid transporter and the Na?/K?-ATPase pump.
Clinical case Introduction Acquired Fanconi syndrome is a disorder of the proximal tubular reabsorption of phosphate, glucose, uric acid, amino acid, low molecular weight proteins, and bicarbonate. It often occurs in association with amyloidosis, multiple myeloma, paroxysmal nocturnal hemoglobinuria, heavy metal poisoning (including lead and cadmium), and drug use (including antiretroviral drugs and ifosfamide) [1]. However, acquired Fanconi syndrome showing osteomalacia through monoclonal gammopathy of undetermined & Akihiro Tojo [email protected] 1
Division of Nephrology and Endocrinology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2
Department of Pathology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
A 64-year-old woman with complaints of back pain and muscle weakness was referred to our hospital for evaluation of hypophosphatemia and low bone mineral density with multiple incomplete fractures, which were observed during a 10-year follow-up for chronic kidney disease. A physical examination revealed no obvious abnormalities. Laboratory studies showed hypokalemia (2.9 mEq/L) with normal sodium level (142 mEq/L), hypouricemia (1.9 mg/dL) with an increased fractional excretion of uric acid (FEUA 45 %), hypophosphatemia (2.0 mg/dL) with reduced tubular reabsorption (TmP/GFR 0.8 mg/dL), glycosuria (3?) with a normal plasma glucose level (95 mg/dL), aminoaciduria and hyperchloremic metabolic acidosis with a normal anion gap (pH7.337, HCO315.7 mmol/L, CL- 117 mEq/L). Bone radiographs showed no osteolytic lesions. Dual-energy X-ray absorptiometry examination showed T scores of -2.8 in the femoral region. The patient was diagnosed with
123
Med Mol Morphol
osteomalacia caused by Fanconi syndrome, but she had no particular family history or past history of bone or mineral disorders, and no history of exposure to toxic agents or heavy metals. A monoclonal kappa light chain was detected in her serum and urine, and bone marrow showed a 6.1 % infiltration by atypical plasma cells, which led to a diagnosis of smoldering multiple myeloma. Treatment with phosphate and potassium supplements and calcitriol immediately ameliorated her back pain and weakness. A renal biopsy was performed because the patient’s renal dysfunction continued with an elevation of serum creatinine level to 2.8 mg/dL.
Materials and methods
was washed with a buffer solution containing 20 mM Trishydrochloride, 150 mM sodium hydrochloride, and 1 % NP-40 (pH7.5). The precipitate in the SDS sample buffer was boiled for 3 min. 2 lL was then applied on a 15/20 % gel. The sample was then electrophoretically transferred to a PVP membrane. After blocking with 5 % dry milk, the membrane was incubated with polyclonal rabbit anti-human kappa light chain antibody at 1:500 dilution and with anti-rabbit immunoglobulin (Dako, Glostrup, Denmark) at 1:2000 dilution. The bands were detected with the diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan) with hydrogen peroxide and nickel chloride. Each 250 ng of human kappa light chain protein (Abcam, Tokyo, Japan) in 100 lL of PBS was also processed the same way and compared with the patient’s urine.
Immunohistochemistry
Results Paraffin-embedded tissue was processed for immunohistochemistry, as previously described [5]. Sections (2 lm in thickness) were dewaxed and incubated with citrate buffer (pH6) at 100 °C for 20 min to retrieve the antigens. The sections were then incubated with 3 % H2O2 and blocking serum followed by polyclonal antibodies against sodium glucose co-transporter 2 (SGLT2, Abcam, Tokyo, Japan), uric acid transporter SLC22A12 (URAT1, MBL, Nagoya, Japan), sodium phosphate co-transporter IIa (NaP-IIa, Abcam, Tokyo, Japan), Na?/K? ATPase a1 (Upstate biotechnology, NY, USA), or human kappa light chains (Dako, Glostrup, Denmark) at 1:200 dilution. The sections were subsequently incubated a horseradish peroxidase– conjugated secondary antibody against anti-rabbit IgG (Dako, Glostrup, Denmark) at 1:50 dilution, and horseradish peroxidase labeling was detected using the DAB reaction. The sections were then counterstained with periodic acid-Schiff staining before being examined under a light microscope. Non-malignant areas from the resected kidney of a patient with renal cell carcinoma served as a control. Proteolysis of the urinary kappa light chain and Western blotting According to the previously described methods of protease treatment [6], the patient’s urine samples (100 lL) were incubated with three different proteases including cathepsin B (R&D Systems, Minneapolis, MN, USA), trypsin (Wako, Osaka, Japan), and pepsin (Tokyo Chemical Industry, Tokyo, Japan) overnight in a water bath at 37 °C. The samples were then immunoprecipitated using antihuman kappa light chain antibodies (Dako, Glostrup, Denmark) at 1:200 dilution and protein A. The precipitate
123
Renal biopsy findings A renal biopsy revealed that 6 (24 %) of 25 glomeruli showed global sclerosis with 3 (12 %) showing mesangial cell proliferation, and a 10 % area of interstitial fibrosis. No immune deposits were identified in the glomeruli by immunofluorescence microscopy or electron microscopy. The proximal tubule and the tubular casts were positive for kappa light chain (Fig. 1) and weakly positive for lambda light chain. An electron microscopic observation of the proximal tubule revealed intracytoplasmic needle-shaped electron-dense crystalline structures (Fig. 1) and some mitochondria became smaller round shape with irregular cristae, suggesting reduced ATP production. Protease resistance of urinary kappa light chain In contrast with the kappa light chain protein, the kappa light chain in the patient’s urine was resistant to three proteases including cathepsin B, trypsin, and pepsin, and showed 23 kD of the original kappa light chain and its digested protein in a 12 kD band even after overnight treatment with the proteases (Fig. 2). The expression of transporters in the proximal tubule The expressions of SGLT2, NaPi-IIa, and URAT1 in the apical membrane of the proximal tubule was reduced in comparison to the normal control kidney (Fig. 3). The expression of Na?/K?-ATPase expression was also reduced in the basolateral membrane of the patient’s proximal tubule (Fig. 3).
Med Mol Morphol Fig. 1 Electron microscopy and the immunostaining of renal biopsy sample for kappa light chain. a An electron micrograph illustrating the cytoplasmic needle-shaped inclusions in the proximal tubule (yellow arrow). Bar indicates 1 lm. b A light micrograph of Epon section showing needle-shape crystals (yellow arrow). Bar indicates 10 lm. c An immunostaining for kappa light chain detected kappa light chain in the proximal tubules. The scale bars indicate 15 lm
Fig. 2 The protease resistance of the urinary kappa (j) light chain. The patient’s urinary j light chain and commercially available purified kappa light chain protein were digested with cathepsin B (Cat), trypsin (Tri), pepsin (Pep), or with no protease (No), and a Western blot analysis was performed to detect j light chain. The 23 kD j light chain form the patient’s urine was resistant to cathepsin B, trypsin and pepsin and 12 kD products remained, whereas the purified j light chain protein (purchased) was completely degraded by trypsin and pepsin without 12kD products
Discussion In this case report, we have clearly demonstrated the reduced expression of the apical transporters including SGLT2, NaPi-IIa, and URAT1 and of the basolateral Na?/
K?-ATPase pump in the proximal tubule in a patient with acquired Fanconi syndrome. The suppressed expression of the apical transporters and the basolateral pump generating Na? gradient to facilitate solute transport can explain our patient’s glycosuria, hypophosphatemia, hypouricemia, and amino aciduria. Most of proximal tubular transport is Na?-coupled and depending on Na?-gradient produced by basolateral Na?/K?-ATPase pump. Thus, as a mechanism of tubular dysfunction, the reduced expression of Na?/K?ATPase [7] or its activity with decreased ATP level [8], and the reduced expression of Na? phosphate transporter [9] have been reported in the primary cultured proximal tubular cells of Fanconi syndrome models. However, to our knowledge, no reports have described the presence of all of these transporters in an immunohistochemical analysis of a renal biopsy sample from a patient with acquired Fanconi syndrome. Acquired hypophosphatemic osteomalacia has been rarely reported as a complication of multiple myeloma, MGUS, lymphoplasmacytic lymphoma, and chronic lymphocytic leukemia. In these cases, hypophosphatemic osteomalacia appeared in relation to the urinary secretion of a monoclonal immunoglobulin light chain that formed crystals inside the cells of the proximal tubule. The precise pathophysiology of the light chain in Fanconi syndrome is still unknown. We demonstrated that kappa light chain is deposited in the proximal tubule, which is consistent with the previous case reports that have shown the predominance of kappa light chain deposition [10]. Furthermore, as confirmed in our case, the urinary light chains of patients with Fanconi syndrome have been shown to be resistant to degradation by lysosomal proteases including cathepsin, pepsin and trypsin. The light chains from patients with Fanconi syndrome associated with myeloma have a unique biochemical characteristic in part of the variable domain
123
Med Mol Morphol Fig. 3 Immunostaining for sodium glucose co-transporter 2 (SGLT2), sodium phosphate cotransporter IIa (NaPi-IIa), uric acid transporter 1 (URAT1) and sodium potassium ATPase (Na?/K?-ATPase). P indicates proximal tubules that can be identified by the brush border membrane. The scale bars indicate 50 lm
[10], which accumulates in the lysosomes as intracytoplasmic needle- or rhomboid-shaped crystals [11, 12] and which presumably impairs the membrane recycling of tubular transporters. The accumulation of kappa light chain is nephrotoxic and has been shown to reduce both the activity and gene expression of Na?/K?-ATPase pump in the primary cultures of rat proximal tubule cells [7]. Cystinosis, a lysosomal storage disease, is the most common inherited cause of Fanconi syndrome. The main mechanisms of the impaired tubular reabsorption investigated in the cultured human renal cells incubated with
123
cysteine dimethyl ester are the decreased intracellular ATP production via a lower intracellular phosphate concentration [8, 13] with the reduced expression of Na?-phosphate co-transporter [14]. Similar to cysteine, the lysosomal accumulation of kappa light chain also causes the reduction of ATP production and the inhibition of Na?-dependent transporters. Despite sodium reabsorption in the proximal tubule is damaged, serum sodium level is maintained at a normal level, because the sodium reabsorption in the loop of Henle and distal nephron remain intact and compensated the proximal tubular damage in Fanconi syndrome.
Med Mol Morphol
Among patients with multiple myeloma, this complication seems to occur in only a small subset of patients with kappa light chain-excreting myeloma and is usually benign and slowly progressive. In most cases, hypophosphatemic osteomalacia responds to supportive treatment with phosphate supplements; however, the improvement of tubular function after myeloma treatment has only rarely been reported. Ma et al. [15] studied 32 cases of patients with MGUS or smoldering myeloma, and found that end-stage renal disease developed in 5 patients (15.6 %) during an average follow-up period of 65 months. In our case, prednisolone treatment, which was administered to reduce light chain production, did not improve renal function and tubular reabsorption.
Conclusion The expression of the proximal tubular transporters, including SGLT2, NaPi-IIa, URAT1 and Na?/K?-ATPase was reduced in a patient with Fanconi syndrome associated with smoldering multiple myeloma with hypophosphate osteomalacia. The deposition of protease-resistant kappa light chain in the proximal tubule may damage the tubular expression of these transporters. Acknowledgments This work was partly supported by a grant-inaid for scientific research from the Japan Science Promotion Foundation to A.T. (C-23591214), and a scholarship donation from the Chugai Pharmaceutical Co. Ltd. to AT. Conflict of interest The authors declare that no conflicts of interests were associated with the present study.
References 1. Haque SK, Ariceta G, Batlle D (2012) Proximal renal tubular acidosis: a not so rare disorder of multiple etiologies. Nephrol Dial Transplant 27:4273–4287 2. Lee DB, Drinkard JP, Rosen VJ, Gonick HC (1972) The adult Fanconi syndrome: observations on etiology, morphology, renal function and mineral metabolism in three patients. Medicine (Baltimore) 51:107–138
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