Annals of Clinical & Laboratory Science 38:163-167 (2008)
© 2008 Association of Clinical Scientists
Nephrotic Range Proteinuria: Rare Manifestation of Scleroderma Renal Crisis
Manish Nepal1,
Roshan Mainali2,
Charles M. Schworer3,
William Difilippo2,
Ping L. Zhang4 and
Michael F. Schultz2
1 Division of Internal Medicine, 2 Department of Nephrology, 3 Weis Center for Research, and 4 Division of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania
Address correspondence to Manish Nepal, M.D., Department of Internal Medicine, Geisinger Medical Center, 100 N. Academy Ave., Danville, PA 17822, USA; tel 570 271 6164; e-mail mnepal1{at}geisinger.edu.
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Abstract
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The nephrotic range of proteinuria is uncommon in scleroderma renal crisis. This 46-yr-old woman with a medical history of scleroderma presented with very high blood pressure, a sudden elevation of serum creatinine, and proteinuria in the nephrotic range. Renal biopsy revealed onion-skin type of arterial changes with necrosis, confirming the presence of scleroderma nephropathy. Electron microscopy showed diffuse fusion of foot processes. Immunohistochemical staining (IHC) revealed increased expression in glomeruli of phosphorylated mammalian target of rapamycin (p-mTOR). These findings suggest that fusion of foot processes and activation of mammalian target of rapamycin-dependent pathways in podocytes are most likely responsible for the severe proteinuria in this patient with scleroderma nephropathy.
Keywords: scleroderma renal crisis, proteinuria, phosphorylated mammalian target of rapamycin
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Introduction
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Scleroderma is an autoimmune connective tissue disorder characterized by fibrosis, degenerative changes, and vascular lesions in skin, joints, and multiple internal organs, which may occur in diffuse or limited forms [1]. One of the most dangerous manifestations of scleroderma is scleroderma renal crisis, characterized by sudden, accelerated, or malignant hypertension followed by rapidly progressive renal failure [2]. Proteinuria may be present but rarely in the nephrotic range.
We report a patient with scleroderma renal crisis presenting with acute renal failure, severe hypertension, and nephrotic range proteinuria. We performed electron microscopy of her renal biopsy specimen and we stained the renal biopsy for the phosphorylated (and activated) form of mammalian target of rapamycin (p-mTOR), a key element of growth factor pathways, in order to identify potential glomerular changes responsible for the nephrotic range of proteinuria.
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Case Report
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A 46-yr-old Caucasian woman had a week of intermittent headaches, lethargy, and worsening shortness of breath. She had experienced Raynaud’s phenomenon for several years but had negative scleroderma tests (anti-centromere antibody, complement assays, and RN-smooth antibody, scl70). She had never had heart burn, dyspeptic symptoms, calcinosis, hardening of the skin (face, hand, extremities, or chest), or telangiectasia. She presented with severe shortness of breath, chest discomfort, and anuria. On initial examination, her systolic blood pressure was 190 mmHg, respiratory rate 34/min, and pulse 100 beats/min. Her chest X-ray showed marked pulmonary edema. She had elevated serum levels of cardiac enzymes and brain natriuretic peptide. Coagulation studies were within normal limits. Serum urea nitrogen was 69 mg/dl and serum creatinine was 6.1 mg/dl. A 2-dimensional echocardiographic study showed reduced left ventricular systolic function and a wall motion abnormality with basal sparing, suggesting a cardiomyopathy of non-ischemic etiology.
Hypertension was controlled with iv enalapril and urgent hemodialysis, and it was subsequently managed with oral enalapril, cardizem, and clonidine. With these treatments, the patient felt better, the elevated levels of cardiac enzymes were normalized, the pulmonary edema improved, and echocardiography showed normal ventricular function. However, she remained anuric and renal function failed to improve. Her urinary protein: creatinine ratio was 3.64 and 3.06 (normal ratio = <0.2) in 2 collections, 5 days apart with the patient on a renal diet, indicating a nephrotic range of proteinuria. On repeat testing, the serum antinuclear antigen test was positive but the anti-centromere antibody, smooth muscle antibody, complement 3, complement 4, and cryoglobulin assays were negative. She underwent a renal biopsy to determine her kidney pathology.
Assessment of the renal biopsy by light microscopy showed lobulations in all glomeruli with thickened capillary loops and mild mesangial expansion. Arteries had an onion-skin appearance with edematous endothelial expansion, narrowed arterial lumen, fibrinoid necrosis, and thrombi. A thioflavin–T stained section was negative, ruling out amyloidosis. Fig. 1A
shows lobulated glomeruli, fibrinoid necrosis in arterioles, and arteries with narrowed lumen. An arteriole displaying prominent edematous changes with a nearly occluded lumen is shown in Fig. 1B. Immunofluorescent microscopy showed positive fibrinogen staining in arterioles, confirming arteriolar necrosis (Fig. 1C). Other stains for immunoreactants including IgG, IgA, IgM, C3, C1q, and kappa or lambda light chains were negative.
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Fig. 1. Pathologic changes in the renal biopsy. A. Lobulated glomerulus with necrosis in arteries (arrow) (H&E, x400). B. Edematous subendothelial areas (arrow) with narrowed lumen (H&E, x400). C. Positive fibrinogen staining in arteriole (immunofluorescent staining for fibrinogen, x400). D. Wrinkled glomerular capillary loops with diffuse fusion of foot processes with subendothelial lucent expansion (short arrow) and subendothelial fibrin deposition (long arrow) (electron microscopy, x2500). E. A glomerulus showing control levels of cytoplasmic and nuclear p-mTOR in podocytes of glomeruli and some tubular epithelium (p-mTOR staining, x400). F. The current biopsy demonstrating prominent p-mTOR staining in tubular epithelial cells and various types of cells in the glomerulus (p-mTOR staining, x400).
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Electron microscopy of the renal biopsy showed wrinkled glomerular capillary loops with subendothelial lucent spaces and focal subendothelial fibrin deposition, indicating ischemic glomerular changes (Fig. 1D
). There was diffuse fusion of foot processes along glomerular capillary loops (Fig. 1D). The overall pathologic findings of obliterative and necrotizing arteriopathy and glomerular ischemia were consistent with scleroderma nephropathy.
One of the biopsy sections was evaluated for the presence of p-mTOR by immunohistochemistry using an antibody to the Ser2448-phosphorylated mTOR (Cell Signaling, Danvers, MA). In order to minimize the staining background using antibodies against p-mTOR, a semi-automatic method for phosphorylated protein antibodies was developed. Paraffin-embedded tissues were sectioned, placed on glass slides, and dried at 60°C for 1–2 hr. They were deparaffinized and underwent antigen retrieval. Slides were placed in a solution containing 0.1M citric acid and 0.1M sodium citrate and were heated in a microwave oven until the solution began to boil. Once boiling was reached, the slides were heated for 10 min, followed by a 20 min cooling period outside the microwave. The slides were then placed in 0.05 M Tris-HCl, 0.05% Tween-20 (TBST buffer) for 5 min (Tris-HCl from Dako Cytomation; Tween-20 from EM Science). The tissue was treated with 3% H2O2 for 5 min and then rinsed with TBST buffer. A few drops of diluted normal blocking serum (Vectastain kit, Vector Laboratories) were placed on the tissue and incubated at room temperature for 1 hr. The serum was then carefully removed by blotting and the slides were incubated with primary antibody overnight at 4°C. The following day the tissues were rinsed well with TBST buffer for a minimum of 5 min. The rest of the staining procedure took place on a DAKO Autostainer. The apparatus was programmed to treat each slide with diluted biotinylated secondary antibody solution (Vectastain kit, Vector Laboratories) for 30 min. The slides were rinsed with TBST buffer and incubated with Vectastain Elite antigen-binding complex reagent (Vectastain Kit, Vector Laboratories) for 30 min. The slides were rinsed with TBST buffer and incubated with 3,3'-diamino-benzidine chromogen solution (DAKO EnVision+ System Kit) for 10 min. The slides were removed from the autostainer and rinsed with distilled water. They were then counterstained with Gill II hematoxylin (Thermo Shandon) for 20 sec, treated with xylene for 10–15 sec, and cover slipped. When compared to 10 control sections from transplant protocol renal biopsies that exhibited nuclear p-mTOR in some podocytes (Fig. 1E), the glomeruli from this patient’s biopsy (Fig. 1F) showed intense p-mTOR staining in a majority of glomerular cells.
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Discussion
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Renal involvement in scleroderma can be divided into scleroderma renal crisis (SRC) and non-renal crisis. In 1952, Moore and Sheehan [3] first described SRC characterized by renal insufficiency (serum creatinine 
2.0 mg/dl or doubling of serum creatinine above the value at baseline, in the absence of any other defined cause), malignant hypertension (systolic blood pressure 160 mm Hg and diastolic blood pressure of 110 mm Hg on at least 2 occasions, a minimum of 12 hr apart), and persistent urinary abnormalities or evidence of microangiopathic hemolytic anemia. Some renal involvement is estimated to occur in 60 to 80% of patients with scleroderma [5,6]. SRC occurred in 10% of a University of Pittsburgh cohort of 1118 scleroderma patients followed between 1972 and 1990. The diffuse form occurred 10 times more frequently [6]. Langevitz et al [7] reported SRC in 3% of 243 scleroderma patients, suggesting that the incidence of SRC is declining. This trend may be due to early diagnosis and treatment with angiotensin converting enzyme inhibitors.
Dramatic improvement in survival has been shown with therapy using angiotensin converting enzyme inhibitors for scleroderma renal crisis [9]. However, irreversible kidney damage and death still occur if the diagnosis of renal crisis is delayed or if angiotensin converting enzyme inhibitors are not used aggressively. Patients with early diffuse scleroderma are at greatest risk for scleroderma renal crisis and should be encouraged to monitor their own blood pressure [6]. Some patients in renal crisis present without Raynaud’s phenomenon or skin thickening, but usually have swollen hands or legs, fatigue, arthralgia, carpal tunnel syndrome, palpable tendon friction rubs, or anti-topoisomerase antibody [10,11]. Anti-RNA polymerase III is highly specific for scleroderma, and renal crisis occurs in 25% to 33% of patients who have it [11]. SRC usually develops early in the course of the disease with >70% of cases occurring within 4 yr of diagnosis, mostly in patients with the diffuse form of scleroderma [12].
Patients with scleroderma and SRC have the combination of proteinuria, azotemia, and hypertensive urgency. Patients with isolated proteinuria have poorer prognosis than patients without proteinuria [13,14]. In a study of 210 patients, 47% with proteinuria died within 20 yr, compared to 10% of those without proteinuria [13]. Nephrotic range proteinuria is rare and was reported once, but not in a case of SRC [15]. Our patient with serology-negative scleroderma disease developed SRC. The changes in arteries and subendothelial expansion clearly indicated ischemic insults to the kidney, and the diffuse fusion of foot processes may represent the changes responsible for nephrotic proteinuria. However, a definite link between ischemia and fusion of the foot processes is uncertain.
Proteinuria usually results from either fusion of foot processes of visceral epithelium (podocytes) in glomeruli (such as minimal change disease or focal segmental glomerulosclerosis) or deposition of immune complexes in glomeruli (such as membranous glomerulopathy or lupus nephritis). Sirolimus is an inhibitor of mTOR and is also a new immune suppression agent that has the advantage of low nephrotoxicity when compared to calcineurin inhibitors. However, in recent years, several studies have documented increased proteinuria in renal transplant patients after their conversion from calcineurin inhibitors to sirolimus (rapamycin) [16–23]. The conversion to sirolimus can even induce focal segmental glomerulosclerosis [23]. Withdrawing sirolimus halts progression of proteinuria or causes reduction in proteinuria [16,19]. Since proteinuria is known to be toxic to renal tubules, impairing renal function over time [24,25], the belief that sirolimus lacks nephrotoxicity effects has been challenged [26,27]. In our hands, the presence of p-mTOR and its downstream signal p-p70S6K has been demonstrated in podocytes of normal glomeruli (unpublished data). Therefore the m-TOR growth pathway in glomeruli may play an important role in regulating glomerular filtration, including the protein components. In the current case, prominent expression of phosphorylated mTOR in glomeruli may represent a key element promoting diffuse fusion of podocyte foot processes, resulting in severe proteinuria. These changes may represent an adaptation process in response to hypoxic insult.
This case illustrates the rare presentation of scleroderma renal crisis with nephrotic range proteinuria. Scleroderma renal crisis is usually associated with proteinuria, but nephrotic range proteinuria is rare. Regulating the level of mTOR phosphorylation in glomeruli may be a key link between the renal ischemic insults and the formation of foot processes responsible for the nephrotic proteinuria.
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Abstract
Introduction
