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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Nephrogenic fibrosing dermopathy (NFD) is a newly recognized cutaneous fibrotic disorder occurring in individuals with end-stage renal disease (ESRD). The aim of the present study was to describe the clinical and histopathologic features of 9 new cases and to characterize the inflammatory cells and expression of transforming growth factor β1 (TGFβ1) in affected skin.

Methods

Clinical and laboratory assessments, including serology and pulmonary function studies, were performed in 9 patients undergoing long-term dialysis (8 hemodialysis; 1 peritoneal dialysis) for ESRD of diverse etiologies. Skin, fascia, striated muscles, lungs, and heart were examined by histopathology. Inflammatory cells were characterized by immunophenotyping using specific monoclonal antibodies. TGFβ1 expression was determined by in situ hybridization.

Results

All patients displayed cutaneous features resembling both systemic sclerosis and diffuse fasciitis, with severe loss of motion and flexion contractures in multiple joints. Six patients displayed woody induration of the muscles of the legs, thighs, and forearms. Five of the 6 patients with lung involvement had a reduced diffusion capacity for carbon monoxide on pulmonary function testing. Marked elevations of the erythrocyte sedimentation rate and/or C-reactive protein level were found in 6 patients. Antinuclear antibodies were present at low titers in 4 patients. Histopathologic studies indicated that in addition to the dermis, the fibrotic process affected the subcutaneous tissue, fascia, striated muscles, lungs, and myocardium. Large numbers of CD68+/factor XIIIa+ dendritic cells and increased expression of TGFβ1 were found in affected skin and muscle.

Conclusion

Our findings indicate that the fibrotic process of NFD affects not only the dermis, but also the subcutaneous tissues, fascia, and other organs, including striated muscles, heart, and lungs. We therefore believe this is a systemic fibrosing process, and we suggest that dialysis-associated systemic fibrosis would be a better term for the condition.

Nephrogenic fibrosing dermopathy (NFD) is a newly recognized cutaneous fibrotic disorder occurring in individuals with end-stage renal disease (ESRD) (1, 2). With a few exceptions, the nearly 100 patients described to date were undergoing long-term hemodialysis or peritoneal dialysis and many had received kidney transplants. Although the etiologic factor(s) responsible for NFD is not known, renal insufficiency and dialysis appear to be intimately involved (for review, see ref. 3).

Initially, this fibrotic process was considered a form of scleromyxedema (1); however, subsequent studies indicated that the disease was unique (2). The histopathologic changes in affected skin included marked proliferation of spindle-shaped fibroblasts, the presence of numerous dendritic cells, and the accumulation of mucinous material and thick collagen bundles. Except for the severe cutaneous induration and thickening, no systemic involvement was described in the initial reports (1, 2). However, more recently, systemic features, including cardiomyopathy and pulmonary fibrosis (4), and fibrotic infiltration of the diaphragm, the psoas muscle, the kidney tubules, and the rete testes (5), have been documented.

We report here the clinical features of 9 new cases, the findings of histopathologic studies of skin, fascia, striated muscles, lungs, and heart, as well as the characterization of inflammatory cells and transforming growth factor β1 (TGFβ1) expression in affected skin, fascia, and striated muscles. Our studies indicate that the fibrotic process is not confined to the dermis, since severe fibrosis of the subcutaneous tissue and the fascia was universally present. Furthermore, some patients had fibrosis of the striated muscles, myocardium, lung parenchyma, and adventitia of lung arterioles. A marked accumulation of CD68+/factor XIIIa+ dendritic cells and an increased expression of TGFβ1 were observed in affected skin and muscle.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients.

Seven patients with a severe fibrotic disease occurring in the setting of ESRD treated with chronic hemodialysis or peritoneal dialysis were evaluated at the Scleroderma Center of Thomas Jefferson University between March 2002 and January 2003. In addition, a patient evaluated at the Veterans Affairs Medical Center (VAMC) in St. Louis, and a patient evaluated at the Robert Wood Johnson Medical School in Camden were also studied. All patients were receiving standard therapy for ESRD. None of the patients have been previously described.

Full-thickness skin biopsies including fascia (7 patients), a skin punch biopsy (1 patient), and muscle biopsies (4 patients) were obtained for diagnostic purposes. Skin biopsies and specimens obtained at autopsy were available from the patient whose case was followed at the VAMC in St. Louis. Discarded tissues were examined following staining with hematoxylin and eosin and with Masson's trichrome. Some samples were stained with periodic acid–Schiff, Giemsa, Alcian blue, colloidal gold, and Verhoeff–van Gieson stains.

Immunophenotyping.

Immunophenotyping of tissue sections was performed with monoclonal antibodies specific for CD3, CD4, CD8, CD20, CD25, CD45RA, CD45RO, CD56, CD68, HLA–DR/DQ/DP, and factor XIIIa. The sections were deparaffinized, and the primary antibody was diluted and applied to the sections. The unbound antibody was washed off with 3 changes of phosphate buffered saline (PBS) for 2 minutes each. The secondary antibody was Cy-2–conjugated rabbit anti-mouse Ig, which was diluted 1:50 in PBS and applied for 40 minutes. The unbound antibody was washed off, and sections were mounted with 4′,6-diamidino-2-phenylindole and examined with a Nikon epifluorescence microscope with a triple-band filter. In individual tissue sections, the number of cells per mm2 of tissue was determined with the ImageJ 1-26 t software (NIH Image, National Institutes of Health, Bethesda, MD; online at: http://rsb.info.nih.gov/ij/).

TGFβ1 in situ hybridizations.

Paraffin-embedded sections were examined for TGFβ1 expression by use of digoxigenin-labeled TGFβ1 complementary DNA (cDNA; R&D, Minneapolis, MN). Following processing, the sections were incubated with alkaline phosphatase–conjugated antidigoxigenin antibody (diluted 1:100 in PBS) for 90 minutes at room temperature. The bound probe was detected with a BCIP/nitroblue tetrazolium alkaline phosphatase substrate kit IV (30 minutes at 37°C; Vector, Burlingame, CA). Sections were counterstained with methyl green. A semiquantitative assessment of the intensity of TGFβ1 cDNA hybridizations was used, where 0 = no hybridization, + = minimal hybridization, ++ = moderate hybridization, and +++ = very strong hybridization. All control (normal) skin specimens examined were rated 0.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Clinical features of the study patients.

The demographic characteristics, relevant clinical features, and results of laboratory tests and ancillary studies are shown in Table 1. All patients developed swelling of the arms and legs as well as pruritus and a burning sensation in the skin, which was followed by severe and progressive skin induration with clinical features that resembled the cutaneous involvement of both systemic sclerosis and diffuse fasciitis. There was sclerodactyly and thickening, induration, and loss of skin appendages of the dorsum of the hands and the lower legs, but the face was spared. The affected skin on the arms, legs, thighs, and abdomen displayed a brawny induration, thickening, “peau d'orange” appearance, deep grooves (“groove sign”), and furrowing. The induration initially affected the feet, lower legs, and hands, and in most patients, it progressed proximally to involve the upper legs, thighs, and forearms. The trunk and/or the abdomen was involved in 4 patients. All patients had severe loss of motion and flexion contractures of multiple joints; 3 patients required the use of a wheelchair. A remarkable thickening and shortening of the digital flexor tendons and of the palmar fascia was observed in 2 patients. Six patients displayed a severe woody induration of the muscles of the legs, thighs, and forearms. None of the patients had typical Raynaud's phenomenon.

Table 1. Demographic data, relevant renal disease history, clinical features, survival, findings of selected laboratory tests, quantification of inflammatory cells, and TGFβ1 in situ hybridization results
FeaturePatient 1Patient 2Patient 3Patient 4Patient 5Patient 6Patient 7Patient 8Patient 9
  • *

    Each renal transplant failed and required removal.

  • Gastrointestinal (GI) involvement in each patient consisted of difficulty swallowing solid foods.

  • TLC = total lung capacity; DLCO = diffusion capacity for carbon monoxide; ND = not done.

  • §

    Normal levels of creatine phosphokinase (CPK) are 25–215 IU/liter. Normal levels of aldolase are 2.0–7.0 units/liter.

  • Antinuclear antibody (ANA) patterns were homogeneous (homo.) or nucleolar (nucleo.).

  • #

    ACA = anticentromere antibody.

  • **

    Normal erythrocyte sedimentation rates (ESRs) are <30 mm/hour. Normal C-reactive protein levels are <0.8 μg/ml.

  • ††

    The number of positive cells per mm2 of tissue was determined with the Image J 1–26 t software.

  • ‡‡

    Two separate skin biopsies were analyzed.

  • §§

    Transforming growth factor β1 cDNA hybridizations were scored semiquantitatively (0 = no hybridization, + = minimal, ++ = moderate, and +++ = very strong hybridization). All control (normal) skin specimens examined were scored as 0.

Age/sex55/F52/M74/M47/M55/M52/F53/F42/F33/M
Renal diseasePolycystic kidneysChronic glomerulo-sclerosisDiabetic nephrosclerosisDiabetic nephrosclerosisPolycystic kidneysDiabetic nephrosclerosisChronic glomerulo-nephritisChronic glomerulo-nephritisChronic nephritis
Dialysis type (duration to onset of dermopathy)Hemodialysis (2–3 mos.)Hemodialysis (16 mos.)Hemodialysis (14 mos.)Hemodialysis (2 mos.)Hemodialysis (3 years)Hemodialysis (6 mos.)Hemodialysis (22 years)Peritoneal dialysis (10 years)Hemodialysis (17 years)
Renal transplant*YesNoNoNoNoYes (×2)Yes (×3)Yes (×2)No
Skin sclerosis/puckeringYes/yesYes/yesYes/yesYes/yesYes/yesYes/yesYes/yesYes/yesYes/yes
Raynaud's phenomenonNoNoNoNoNoNoNoNoNo
GI involvementNoNoNoNoYesYesYesNoNo
Lung involvement (% predicted TLC/DLCO)No (ND)Yes (65/75)No (ND)Yes (64/66)Yes (86/59.5)Yes (71/49)Yes (85/64)Yes (81/60)No (ND)
Muscle indurationYesNoYesYesYesYesYesNoNo
Joint flexion contracturesYesYesYesYesYesYesYesYesYes
SurvivalDeceasedAliveDeceasedAliveDeceasedAliveAliveAliveAlive
CPK/aldolase§ND5/4.7ND14/3.42/ND22/9.034/9<20/6.8ND
ANA titer (pattern)1:160 (homo.)Neg.1:320 (homo.)Neg.Neg.1:80 (homo.)1:160 (homo.); 1:160 (nucleo.)Neg.Neg.
ACA/Scl-70#Neg./neg.Neg./neg.Neg./neg.Neg./neg.Neg./neg.Neg./neg.Neg./neg.Neg./neg.Neg./neg.
ESR, mm/hour/CRP, IU/liter**58/11.322/1.3110/ND73/ND68/ND20/<567/10.4109/3.540/4.6
CD68,/mm2††38.4ND40.783.230.5/23.9‡‡NDNDNDND
Factor XIIIa,/mm2††37.3ND43.676.732.9/22.7‡‡NDNDNDND
TGFβ1§§+++ND++++++++/+++‡‡NDNDNDND

Very high erythrocyte sedimentation rates and/or C-reactive protein levels were observed in 6 patients. There was no eosinophilia, dysproteinemia, or elevation of the creatine phosphokinase or aldolase levels in any of the patients tested. Antinuclear antibodies (by immunofluorescence) were present at low titers (<1:360) with a homogeneous pattern in sera from 4 patients. Serum from 1 of these 4 patients also displayed a nucleolar pattern. Tests for anticentromere, anti–Scl-70, and anti–RNA polymerase antibodies gave negative results in all patients tested. Five of the 6 patients with lung involvement had a reduction in the diffusion capacity for carbon monoxide and in the total lung capacity on pulmonary function studies, but findings for all other parameters were normal. Radiographs and high-resolution computed tomography scans of the chest yielded normal results in all patients examined.

All patients had a progressive course of the fibrotic process. Three patients experienced a severe deterioration of their clinical condition, which led to their demise within 2 years of onset. Two of them died of cardiopulmonary failure, and 1 died of an infection following therapy with a tumor necrosis factor α inhibitor.

Histopathologic features.

The histopathologic features of affected tissues are shown in Figure 1. Skin biopsy sections demonstrated marked fibrosis of the papillary and deep dermis, with thick fibrous tissue tracts extending across the adipose tissue into the fascia, which was severely thickened. The adipose tissue was encroached within the fibrous tracts, yielding a microlobular architecture. Mucin accumulation in the clefts between the collagen bundles was notable (results not shown). Numerous spindle-shaped and elongated fibroblasts were present throughout the dermis and fascia.

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Figure 1. Histopathologic features of affected tissues. A and B, Full-thickness skin biopsy sections, showing marked thickening of the entire dermis and subcutaneous tissues, with severe accumulation of collagen and thick tracts of fibrous tissue extending into the adipose layer, resulting in microlobular architecture and thickening of the fascia. C, Biopsy section of striated muscle, showing remarkable accumulation of fibrotic tissue in the interfascicular septae and surrounding muscle fibers. D, Biopsy section of myocardium, showing thickening of the pericardium and accumulation of large amounts of fibrous tissue surrounding and between myocardial fibers. E, Biopsy section of lung parenchyma, showing interstitial fibrosis with severe distortion of the alveolar architecture and a moderate inflammatory cell infiltrate. F, Biopsy section of small pulmonary arterioles, showing marked thickening and fibrosis of the adventitial layer. Sections were stained with hematoxylin and eosin (A) or with trichrome (B–F). (Original magnification × 60 in A and B; × 100 in C–F.)

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Histopathologic examination of the muscles showed infiltration of the perimysium and endomysium with fibrotic tissue and muscle fiber atrophy. The myocardium was also affected by a similar fibrotic process. The lungs showed mild interstitial fibrosis, with scanty inflammatory cell infiltration. A pronounced increase in the thickness of the adventitial layer of small and medium-size arterioles, with marked accumulation of fibrous tissue, was observed in the lung and heart.

Immunophenotype of inflammatory cells.

Five samples of skin (2 samples were obtained from patient 5) were analyzed by immunohistochemistry for inflammatory T cells (CD4, CD8, CD45RO, CD45RA), activated T cells (CD25, class II), and other inflammatory cells (natural killer cells [CD56], macrophages [CD68], B cells [CD20], and dendritic cells [factor XIIIa]). The inflammatory infiltrates contained a large number of CD68+/factor XIIIa+ dendritic cells, which did not express class II major histocompatibility complex molecules (Figure 2 and Table 1). Abundant CD68+/factor XIIIa+ cells were also observed infiltrating the affected muscles along the tracts of fibrotic tissue in the interfascicular and interfibrillar spaces (Figure 2).

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Figure 2. Immunophenotyping of inflammatory cells and in situ hybridization of transforming growth factor β1 (TGFβ1) mRNA. A, Fluorescence staining for factor XIIIa in skin. Note the marked accumulation of factor XIIIa+ yellow/green fluorescent cells (dendritic cells) throughout the dermis, adipose tissue, and fascia. B, Fluorescence staining for factor XIIIa in muscle. Numerous factor XIIIa+ green/yellow fluorescent cells are present in striated muscle. The same cells that stained in A and B also stained positively for CD68. C and D, In situ hybridizations for TGFβ1 in the dermis (top) and the subcutaneous tissue and fascia (bottom) of skin biopsy sections from a normal individual (C) and from an affected area of a patient (D). Note the marked increase in TGFβ1 transcripts in the affected skin from the patient compared with normal skin. (Original magnification × 100.)

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In situ hybridization for TGFβ1 messenger RNA (mRNA).

In situ hybridizations for TGFβ1 showed a marked increase in TGFβ1 mRNA levels diffusely distributed throughout the skin and fascia in all samples examined (Figure 2 and Table 1). In patient 4, who had pronounced muscle involvement, increased TGFβ1 mRNA expression was found between the muscle fibers in the endomysium and perimysium (results not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A newly recognized disorder characterized by severe cutaneous fibrosis associated with ESRD and chronic dialysis has been recently described (1–3). The disorder has been named NFD. Although the initial reports and numerous subsequent descriptions (for review, see ref. 3) considered it to be a purely cutaneous disorder, our studies indicate that the fibrotic process affects the subcutaneous tissue, fascia, and other organs, including striated muscles, heart, and lungs, and therefore, it should be considered a systemic fibrosing process. It should be noted, however, that a shortcoming of the present study is that we did not perform a comparison of the extent or severity of the systemic fibrotic process in this cohort with a comparable group of patients with ESRD who did not have skin fibrosis. While the interstitial lung fibrosis was only moderate in the tissue samples we studied, a notable finding was adventitial and perivascular fibrosis in small and medium-sized arterioles of the lungs. Although less well recognized than the medial and intimal fibroproliferative lesions typically found in the pulmonary vasculature of patients with systemic sclerosis and pulmonary hypertension, adventitial fibrosis has also been described in these patients (6, 7). Adventitial and perivascular fibrosis in small myocardial vessels were also observed in the present study.

The systemic nature of this unique fibrotic process was further demonstrated by the substantial reduction in diffusion capacity for carbon monoxide in 5 of the 6 patients who had lung involvement, the presence of antinuclear autoantibodies in 4 patients, and the remarkable elevation of the erythrocyte sedimentation rate and/or serum C reactive protein levels in 6 patients. The occurrence of antinuclear antibodies is of substantial interest because it suggests that the fibrotic process per se may result in an autoimmune reaction, perhaps by the unmasking of hidden epitopes, the production of neoepitopes caused by protein modifications, such as protease cleavage or complex formation with a putative etiologic agent, or as described previously, as a result of apoptotic cell death (8). Alternatively, autoantibodies may have developed by a process of molecular mimicry, owing to structural similarities between the putative etiologic agent and endogenous proteins.

Although the etiology and pathogenesis of this severe fibrotic process remain elusive, our observations indicate that CD68+/factor XIIIa+ dendritic cells and TGFβ1 are intimately involved, since there was a remarkable increase in CD68+/factor XIII+ cells and in the expression of TGFβ1. It is possible that the prominent presence of dendritic cells may represent a response of the host in an attempt to eliminate a noxious putative etiologic agent. The high levels of TGFβ1 expression suggest that activation of the TGFβ1 pathway may be ultimately responsible for the remarkable tissue fibrosis. Since TGFβ is expressed in dendritic cells (9) and is involved in the regulation of the complex process of dendritic cell maturation (10, 11), it is possible that the causative agent(s) resulted in increased expression of this growth factor as part of the response of the dendritic cells to the noxious agent. The TGFβ produced by these cells is, in turn, responsible for both the fibrotic process and the expansion and enhancement or initiation of antigen-presenting functions of additional dendritic cells, establishing a vicious circle that results in their accumulation in affected tissues. If this possibility proves to be correct, therapeutic approaches aimed at removing TGFβ or counteracting its profibrotic effects may be an effective treatment for this currently incurable disease.

The term NFD has become accepted for this novel disorder; however, we believe that this term is a misnomer for two reasons. First, the term NFD implies that the kidneys cause the disease. If chronic renal disease is the cause of this disorder, it is difficult to explain why it is so rare and why it was not observed prior to 1997. Second, the disorder is not just a cutaneous disease, as implied by the term NFD. It is a systemic disease as well, with the fibrotic process affecting the muscles, myocardium, lungs, kidneys, and testes, and very likely other organs as well. Therefore, we suggest that the term dialysis-associated systemic fibrosis be used until a clearer understanding of the etiology and pathogenesis of the disease becomes apparent.

Whatever nomenclature is applied for this novel disorder, the study of its etiology and pathogenesis will undoubtedly provide valuable clues to the pathogenesis of systemic sclerosis, diffuse fasciitis, and other fibrotic diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors gratefully acknowledge the following physicians who referred the patients for study: Janet F. Burkholder, MD, Pierre Minerva, MD, James Burke, MD, Richard Mitchell, MD, and William J. Gaughan, MD. The expert assistance of Dr. Sonsoles Piera with the illustrations and of Kate Salmon with preparation of the manuscript is gratefully acknowledged.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES