DIABETIC NEPHROPATHY is a major

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1 KIDNEY BIOPSY TEACHING CASES Nodular Glomerulopathy in a 50-Year-Old Diabetic Man Erika R. Bracamonte, MD, Peter Hullman, MD, and Kelly D. Smith, MD, PhD INDEX WORDS: Diabetes; membranoproliferative glomerulonephritis; cryoglobulin; nodular mesangiosclerosis; hyaline; immune deposits. DIABETIC NEPHROPATHY is a major cause of end-stage renal disease (ESRD) in the United States. The number of individuals initiating treatment for ESRD has grown exponentially during the past 2 decades, with the majority of this increase attributable to patients with diabetes and hypertension. 1 Nephrotic syndrome commonly occurs in patients with longstanding diabetic nephropathy and usually is a sign of incipient progression to end-stage renal failure. Typical morphological changes of advanced diabetic nephropathy associated with nephrotic syndrome are those of nodular glomerulosclerosis and diffuse thickening of the glomerular basement membranes. Nodular glomerulosclerosis is a pattern of injury that can be seen in patients with diabetic glomerulopathy, but also is present in those with a variety of other disease processes. We report an interesting cause of nodular glomerulosclerosis in a patient with diabetes who presented with nephrotic syndrome. CASE REPORT Clinical History A 50-year-old man presented with cough and swelling in his feet and legs and was discovered to have proteinuria (20 g/24 h) with hematuria. His past medical history was notable for type 2 diabetes with peripheral neuropathy and retinopathy, obesity, and hypertension. Diabetes was diagnosed in this patient approximately 8 years ago when he presented with peripheral neuropathy. Although he had been treated initially with metformin hydrochloride (Glucophage; Bristol- Meyers Squibb, New York, NY), he stopped this medication 4 years ago and attempted to control his diabetes with dietary modifications. He denied signs or symptoms of collagen vascular disease, intravenous drug use, heroin or cocaine abuse, or known risk factors for hepatitis or acquired immunodeficiency syndrome. His father and 2 siblings had type 2 diabetes, but there was no family history of renal disease. His only current medication was lisinopril (40 mg/d). Physical examination showed an obese middle-aged man with blood pressure of 178/82 mm Hg, bilateral retinopathy, and 2 to 3 edema of the lower extremities. Pertinent laboratory data included the following values: urine protein, g/24 h; serum creatinine, 1.8 mg/dl (159 mol/l); creatinine clearance, 48.6 ml/min (0.81 ml/s); albumin, 2.8 g/dl (28 g/l); urinalysis with ph 6, specific gravity of 1.020, 3 protein, 3 blood, trace glucose, and 15 to 20 red blood cells; white blood cell count, / L ( /L); hemoglobin, 11.8 g/dl (118 g/l); and hematocrit, 34.5%. Additional laboratory tests showed normal values for electrolytes and liver function test results. Serological studies were negative for hepatitis B and C and human immunodeficiency virus. Antinuclear antibody serological test results were negative, and rapid plasma reagin was nonreactive. C3 and C4 levels were normal. Rheumatoid factor was positive. Serum protein electrophoresis was negative. It was thought that the patient likely had diabetic nephropathy, but the degree of proteinuria and hematuria was concerning for superimposed glomerulopathy. Diuresis with furosemide (Lasix, Sanofi-Aventis, Bridgewater, NJ; 40 mg orally every morning) was initiated, and a diagnostic renal biopsy was performed. Renal Biopsy Examination of sections from all submitted material showed approximately 22 to 23 glomeruli, most of which demonstrated diffuse and focally nodular mesangial sclerosis, with some glomeruli showing segmental sclerosis and approximately 10% to 15% showing global sclerosis. Several glomeruli demonstrated prominent mesangiolysis. In some glomeruli, there were massive accumulations of hyaline-like material that occluded capillary loops and lined subendothelial portions of peripheral capillary basement membranes, with some eosinophilic deposits partially surrounded by circulating leukocytes (Fig 1). The hyaline-like deposits were heterogeneous, with some globular deposits appearing distinct from hyaline and showing stronger staining with eosin, periodic acid Schiff, and fuchsin (Fig 2). Tubular parenchyma showed a moderate degree of patchy interstitial fibrosis, tubular atrophy, and tubular basement From the Department of Pathology, University of Washington Medical Center, Seattle, WA; and Alaska Kidney and DM Associates, Anchorage, AK. Received June 12, 2006; accepted in revised form August 8, Originally published online as doi: /j.ajkd on November 6, Support: None. Potential conflicts of interest: None. Address reprint requests to Kelly D. Smith, MD, PhD, Department of Pathology, University of Washington Medical Center, Box , 1959 NE Pacific, Seattle, WA kelsmith@u.washington.edu 2006 by the National Kidney Foundation, Inc /06/ $32.00/0 doi: /j.ajkd American Journal of Kidney Diseases, Vol 48, No 6 (December), 2006: pp

2 BRACAMONTE ET AL 999 only vague substructure in some areas and no definitive microtubule-like structures (Fig 4B). Glomerular basement membranes were additionally thickened by the homogeneous expansion of the lamina densa. Podocyte foot processes were diffusely effaced. The mesangium was expanded by increased matrix and discrete immune-type electron-dense deposits, similar to those seen in capillary walls. Interpretation and Final Diagnosis Pathological changes were most consistent with cryoglobulinemic glomerulonephritis, likely caused by the presence of type III cryoglobulins. The lack of staining for IgG in immune deposits was considered to be caused by masking of IgG by polyclonal IgM rheumatoid factor. In addition, the biopsy specimen showed several morphological changes consistent with coexistent diabetic nephropathy, including thickening of glomerular basement membranes, mesangial sclerosis, tubular basement membrane thickening, and microvascular disease. Clinical Follow-Up A cryocrit was ordered and determined to be positive. A clinical workup for occult malignancy was negative. Fig 1. Prominent nodular mesangial expansion and accumulation of hyaline-like material in capillary loops (Jones silver stain; original magnification [A] 400 and [B] 600). membrane thickening. Scattered interstitial inflammatory cell infiltrates were present throughout the cortex, with numerous intermixed plasma cells. Sections of small arteries showed a moderate degree of intimal sclerosis, and arterioles showed prominent subendothelial hyaline accumulation. Examination of frozen sections from all material submitted for immunofluorescence microscopy showed approximately 24 glomeruli, of which 8 were completely sclerosed. Glomeruli demonstrated strong staining of the mesangium and capillary walls, often in a globular distribution, for immunoglobulin M (IgM; 4 ) and and light chains (4 and 3, respectively; Fig 3). There was modest mesangial staining for C3 (negative to 1 ) and IgA (1 ). No significant staining was present for IgG, C1q, fibrinogen, or albumin. Ultrastructural examination of 2 glomeruli showed prominent thickening of capillary loops due to the accumulation of massive subendothelial immune-type electron-dense deposits (Fig 4A). Some deposits were surrounded by matrix material and lamellar basement membrane material. Several deposits were directly adjacent to the endothelial cell cytoplasm, whereas others appeared to completely occlude the capillary lumina. Deposits appeared mostly granular, with DISCUSSION This biopsy specimen shows pathological changes classified under the broad category of nodular glomerulopathy. Many different glomerular diseases can cause nodular lesions within glomeruli. These entities can be difficult to distinguish at low magnification (Table 1) and often require special stains and ancillary studies, such as immunofluorescence and electron microscopy, to accurately define the pathological diagnosis. The main histological feature that distinguishes membranoproliferative glomerulonephritis (MPGN) from diabetic nephropathy is the presence of immune deposits, which usually can be appreciated by using light microscopy. Tinctorial properties of immune deposits are distinct from hyaline and discerned most readily on Masson trichrome stain, for which immune deposits are fuchsinophilic (red) and hyaline usually stains blue. In addition, immune deposits in MPGN accumulate in the subendothelial space of the glomerular capillary wall and often are associated with remodeling of the glomerular basement membrane. This frequently results in double-contoured capillary walls, best seen on silver stains (commonly referred to as splitting or tram-tracking ). Similar glomerular basement membrane changes also can occur in chronic thrombotic microangiopathy, easily differentiated from MPGN by using immunofluores-

3 1000 NODULAR GLOMERULOPATHY IN DIABETES Fig 2. Tinctorial properties of the hyaline-like material in glomeruli. (A) Accumulation of eosinophilic subendothelial material frequently occludes capillary lumina (Jones silver stain; original magnification 400). Globules of intracapillary material are (B) eosinophilic on hematoxylin and eosin stain, (C) strongly periodic acid Schiff positive, and (D) fuschinophilic on Masson trichrome stain (original magnification: [B-D] 400). Fig 3. Immunofluorescence staining of glomeruli. Strong, frequently globular staining of mesangium and capillary walls for IgM (4 ; original magnification 200), light chain (4 ; original magnification 200), and light chain (3 ; original magnification 200). No significant staining is appreciated for IgG (original magnification 200).

4 BRACAMONTE ET AL 1001 Fig 4. Electron microscopy of immune deposits in glomeruli. (A) Accumulation of massive subendothelial immune-type electron-dense deposits. Deposits also were identified in the mesangium (electron micrograph; original magnification 3,200). (B) High-power magnification of immune deposits shows no definite microtubular-like substructure (electron micrograph; original magnification 40,000). cence and electron microscopy. Glomerular leukocytic infiltrates are more common in MPGN, but these can be variable. Diabetic nephropathy also can have features of increased glomerular cellularity, mesangiolysis, and prominent hyaline accumulation, which can mimic MPGN. In patients with diabetes, these findings may be associated with poor glycemic control and hypertension. Immunofluorescence microscopy is essential in diagnosing MPGN and typically shows IgG, IgM, C3, and C1q deposition in capillary walls and mesangium. Staining for immunoglobulin light chains is useful to distinguish polyclonal from monoclonal processes. When interpreting immunofluorescence staining, one must be aware of nonspecific accumulations of IgM, C3, and C1q in areas of sclerosis and hyaline accumulation, also commonly present in patients with diabetic nephropathy, and not confuse this with immune-complex deposition. Electron microscopy shows characteristic discrete subendothelial and mesangial electrondense deposits in MPGN, and intramembranous and subepithelial deposits also may be present. Diabetic nephropathy lacks discrete immune deposits, but can have prominent accumulations of electron-dense hyaline material, usually illdefined, but occasionally challenging to accurately distinguish from immune deposits. Additional ultrastructural features of diabetic nephropathy include diffuse homogeneous expansion of the glomerular capillary basement membrane lamina densa and expansion of the mesangium by matrix. There is no known causal link between diabetes and immune-complex mediated glomerulonephritides, although several recent studies found that a surprisingly high percentage (10% to 20%) Table 1. Differential Diagnosis for Nodular Glomerulopathy Disease Diabetic nephropathy Monoclonal immunoglobulin deposition disease MPGN Thrombotic microangiopathy Fibrillary and immunotactoid glomerulopathies Amyloidosis Idiopathic nodular glomerulosclerosis Fibronectin glomerulopathy Collagenofibrotic glomerulopathy Cause Type 1 or 2 diabetes mellitus Lymphoproliferative disorder Infection, autoimmune, idiopathic Drugs, toxins, infection, autoimmune, idiopathic Lymphoproliferative disorders, idiopathic Lymphoproliferative disorder, inflammation, others Idiopathic Hereditary Idiopathic

5 1002 NODULAR GLOMERULOPATHY IN DIABETES Table 2. Classification of MPGN Type I Type II (dense deposit disease) Type III Pathology Subendothelial and mesangial electron-dense deposits Intramembranous and mesangial electron-dense deposits, positive for C3 Subendothelial and subepithelial and mesangial electron-dense deposits Cause Immune complexes, autoimmunity Loss of C3 convertase regulatory activity, C3 nephritic factor (autoantibody), factor H deficiency Immune complexes, autoimmunity of biopsy specimens from patients with diabetic nephropathy also show postinfectious glomerulonephritis. 2 Limited literature exists upon which to base prognostic predictions for patients with diabetes and superimposed immune-complex glomerulonephritis. One study concluded that patients with superimposed MPGN have decreased kidney survival. 3 In addition, older patients ( 50 years) with diabetes and acute postinfectious glomerulonephritis appear to progress more rapidly to ESRD. 2 No significant difference in decrease in renal function has been reported for patients with diabetic nephropathy and superimposed IgA nephropathy, membranous glomerulonephritis, or minimal change disease, 3,4 although short follow-up periods and small numbers of patients are limiting factors in these studies. We recently reviewed the pathogenesis of MPGN. 5 MPGN is classified into 3 morphological variants (Table 2). Type I and III diseases are considered by many to be highly related. Cryoglobulinemic glomerulonephritis usually presents as type I MPGN. Cryoglobulins are divided into 3 types based on their molecular composition (Table 3). This case is an example of a specific class of MPGN: namely, cryoglobulinemic glomerulonephritis, with polyclonal rheumatoid factor (type III cryoglobulins) and no association with hepatitis B or C virus infection. In this disease, rheumatoid factor is thought to have a central role in the disease process by binding serum IgG and creating immune complexes that are fixed by complement and bound to cell-surface receptors. This results in the accumulation of immune complexes within the microvasculature in both intraluminal and subendothelial compartments. Immune complexes are recognized by cell-surface receptors, 5 triggering inflammatory responses. A recent model for rheumatoid factor production emerged from mouse models of innate immunity. Immune complexes of IgG and chromatin are capable of stimulating the proliferation of rheumatoid factor producing B cells. This suggests that certain antigens produced in the course of infections or autoimmune diseases may con- Table 3. Cryoglobulins Composition Causes Type I Monoclonal immunoglobulin (usually IgM or IgG) 1. Waldenström macroglobulinemia 2. Leukemia/lymphoma/myeloma Idiopathic nonmalignant monoclonal cryoglobulinemia Type II Type III Mixed cryoglobulins: monoclonal rheumatoid factor (usually IgM) and polyclonal IgG Mixed cryoglobulins: polyclonal rheumatoid factor (usually IgM) and polyclonal IgG *Type II and III cryoglobulins share the same causes. 1. Infections* Hepatitis B/C Other viral, parasitic and bacterial 2. Autoimmune diseases* Systemic lupus erythematosus Rheumatoid arthritis Polyarteritis nodosa Sjögren syndrome Scleroderma 3. Mixed essential cryoglobulinemias*

6 BRACAMONTE ET AL 1003 tain Toll-like receptor or other agonist activities that promote autoreactive B-cell proliferation. 6-8 Future studies of the pathogenesis of rheumatoid factor and MPGN should elucidate additional therapeutic targets and strategies to treat these disorders. Currently, treatment for patients with MPGN consists of therapies aimed at eradicating the infectious agent, when possible, such as use of interferon alfa in those with hepatitis C virus associated disease. Other strategies are aimed at treating the inflammation with steroids or such cytotoxic drugs as cyclophosphamide and, recently, anti tumor necrosis factor therapies 9 or eliminating cryoglobulins with plasmapheresis, rituximab (a monoclonal anti-cd20 antibody), or a combination of these therapies A recent prospective study in Italy 12 showed that rituximab treatment improved symptoms and clinical parameters (quantitative cryocrit and C4 levels) in 15 patients who had essential mixed cryoglobulinemia unresponsive to steroids, immunosuppressive therapy, or plasma exchange. Although the combination of MPGN or cryoglobulinemic glomerulonephritis and diabetes is unusual, clinical experience indicates that these patients progress more rapidly to ESRD. New therapeutics and a better understanding of the pathogenesis of MPGN may offer alternatives to treat this disease. REFERENCES 1. Jones CA, Krolewski AS, Rogus J, Xue JL, Collins A, Warram JH: Epidemic of end-stage renal disease in people with diabetes in the United States population: Do we know the cause? Kidney Int 67: , Haas M: Postinfectious glomerulonephritis complicating diabetic nephropathy: A frequent association, but how clinically important? Hum Pathol 34: , Chihara J, Takebayashi S, Taguchi T, Yokoyama K, Harada T, Naito S: Glomerulonephritis in diabetic patients and its effect on the prognosis. Nephron 43:45-49, Mak SK, Wong PN, Lo KY, Tong GM, Wong AK: Prospective study on renal outcome of IgA nephropathy superimposed on diabetic glomerulosclerosis in type 2 diabetic patients. Nephrol Dial Transplant 16: , Smith KD, Alpers CE: Pathogenic mechanisms in membranoproliferative glomerulonephritis. Curr Opin Nephrol Hypertens 14: , Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Shlomchik MJ, Marshak-Rothstein A: Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416: , Marshak-Rothstein A, Busconi L, Lau CM, et al: Comparison of CpG s-odns, chromatin immune complexes, and dsdna fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells. J Endotoxin Res 10: , Viglianti GA, Lau CM, Hanley TM, Miko BA, Shlomchik MJ, Marshak-Rothstein A: Activation of autoreactive B cells by CpG dsdna. Immunity 19: , Koukoulaki M, Abeygunasekara SC, Smith KG, Jayne DR: Remission of refractory hepatitis C-negative cryoglobulinaemic vasculitis after rituximab and infliximab. Nephrol Dial Transplant 20: , Garini G, Allegri L, Vaglio A, Buzio C: Hepatitis C virus-related cryoglobulinemia and glomerulonephritis: Pathogenesis and therapeutic strategies. Ann Ital Med Int 20:71-80, Dammacco F, Sansonno D, Piccoli C, Tucci FA, Racanelli V: The cryoglobulins: An overview. Eur J Clin Invest 31: , Zaja F, De Vita S, Mazzaro C, et al: Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: , 2003