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Keywords:

  • CD46/MCP;
  • hemolytic uremic syndrome;
  • microchimerism;
  • renal transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References

Mutations in the gene of the membrane cofactor protein (MCP/CD46), a complement regulatory protein, were recently described as a cause of hemolytic uremic syndrome (HUS). MCP is a transmembrane glycoprotein expressed in kidneys; therefore, the transplantation of a normal kidney should not be complicated by HUS recurrence. However, we report the case of a 32-year-old woman with an MCP mutation who developed a recurrence of HUS after renal transplantation. We found that she had vascular microchimerism of endothelial cells. We suggest that recurrence may be favored by vascular microchimerism, in which the mutated protein is produced in the in the kidney graft by endothelial cells originating from recipient.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References

Mutation in membrane cofactor protein (MCP/CD46), a protein regulating complement activation, is now a well-recognized cause of familial and sporadic atypical hemolytic uremic syndrome (HUS), unrelated to HUS associated with shigatoxin-secreting bacteria (1–3). Other atypical nonshigatoxin-associated HUS cases have also been described. These cases present defects in the regulation of the complement activation pathway, for example in Factor H, a soluble protein that inhibits the complement activation cascade. Factor H deficiency is complicated by a high recurrence rate of HUS—almost 50%—after kidney transplantation (4). MCP, in contrast to Factor H, is a transmembrane glycoprotein expressed mostly in kidneys acting locally as a cofactor with Factor I to activate complement (C3b and C4b fractions) catabolism; MCP also regulates the complement activation pathway but is produced and exerts its effects locally. Therefore, complement dysregulation related to MCP mutation should be corrected by the transplantation of a normal kidney. Only three siblings with an identified MCP mutation and who have been treated by kidney transplant have been reported: clinical evolution was favorable without a recurrence of HUS in the allograft (5). Here, we report the case of a patient with a novel MCP mutation. The patient suffered a recurrence after renal transplantation, indicating that some MCP mutations may be at risk factors for HUS recurrence. Analyses of graft biopsies revealed endothelial cells of recipient origin, indicating vascular microchimerism. This also suggests that the production of mutated MCP protein in the kidney graft by endothelial cells originating from recipient may explain the recurrence of HUS.

Case Report

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References

A 32-year-old woman had with HUS 10 years ago, which lead to severe acute renal failure, requiring dialysis. She had had two pregnancies, each complicated by pre-eclampsia, and gave birth to two healthy children. The patient had no infection related to shigatoxin-producing bacteria or other infectious agents at the onset of HUS, and was not taking any drugs. Plasma infusions were unsuccessful and she was treated by periodic hemodialysis. She had three brothers and three sisters. One of her sisters was also on dialysis therapy from the age of 29 years as a result of HUS; she died suddenly 4 years later of unknown causes. None of the patient's other siblings or children had HUS.

Biological investigations were performed to identify predisposing genetic or immunological factors for HUS. Complement C3 and C4 levels were within the normal ranges. Tests for anti-nuclear, anti-double strand DNA and anti-phospholipid antibodies were negative. The Von Willebrand factor-cleaving protease (ADAMTS-13) activity was within the normal range of a normal population (80% of the activity of control patients). Factor H and Factor I genes were sequenced, but no mutations were detected. We then analyzed her MCP/CD46 gene. We identified a heterozygous T to G mutation at position +2 of exon 2 (IVS2 + 2T > G) (Figure 1, panel B), which involved the donor splice site in intron 2. RT-PCR, using primers from exon 1 (forward primer) and exon 4 (reverse primer), of the MCP transcript resulted in a smaller fragment of 235 bp (Figure 1, panel C). Direct sequencing of this fragment after gel purification revealed that the first 45 bp from exon 2 was spliced onto exon 3 (Figure 1, panel D). Thus, this nucleotide change results in the selection of an alternative splice site (TTG gtaaac). This splice site is located in exon 2, and its use results in the deletion of 144 bp and 48 amino acids, which are in phase with the wild type sequence of the protein.

image

Figure 1. Genetic analysis and expression of CD46. Panel A: MCP distribution—Flow cytometry histograms of MCP in PBMCs from the proband (dark line, arrow) and from a representative healthy control (light line) stained with anti-CD46 Phycoerythrin (PE)-conjugated mAb (Serotec, U.K.). Dashed line represents the staining of proband granulocytes with an isotype control (Serotec). The mean fluorescence intensity of the proband PBMC was 319 with anti-CD46. Panel B: Genetic analysis—DNA sequencing electropherograms demonstrating the mutation identified in the MCP-deficient proband. The DNA sequence of a healthy control individual is shown. Normal control (Ctrl) and patient (P) DNA sequences and corresponding amino acids are shown. A splice-site mutation at the +2 position downstream from exon 2 of the 100% conserved sequence is indicated. Materials and Methods: Total RNA was extracted from patient and healthy control leukocytes and isolated using the QIAamp RNA Blood Mini kit (Qiagen). A mixture of oligo (dT) primers and random hexamers was used to synthesize the first strand of cDNA from leukocyte-derived total RNA, according to the manufacturer's (Promega) protocols. Oligonucleotide primers for amplification and sequencing the part of the hMCP cDNA encompassing exons 1 through 4 were as follows: MCP-1f (5′-TGTTGCTGCTGTACTCCTTCT-3′) and MCP-4r (5′- aggatcagtagcaatttggag-3′). Panel C: Amplification of the fragment encompassing exon 2—The cDNA was prepared as described for panel B. With the 2 primers described previously, cDNAs were amplified and analyzed by electrophoresis on agarose gel (2%). cDNA was visualized using ethidium bromide. Amplification of cDNA containing the wild-type allele was expected to result in a 379-bp fragment. A second band (235 bp) was also obtained in samples from the reported case. Panel D: DNA sequencing of the isolated fragment from agarose gel. The corresponding amino-acid sequence is indicated.

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FACS analysis of the patient's peripheral blood mononuclear cells (PBMCs) showed that surface expression of CD46 was lower in patients than normal subjects (Figure 1A). Only one of the patient's sisters, who had never developed HUS, was available for testing; no mutation was detected in her MCP gene.

The patient received a kidney transplant from a woman who suffered brain death following cardiac arrest after a pulmonary embolism. The immunosuppressive regimen consisted of an induction therapy with anti-thymoglobulin and solumedrol. The maintenance therapy consisted of steroids, mycophenolate mofetil and sirolimus. The patient recovered renal function immediately after transplantation. Her serum creatinine concentration was 99 μmol/L on day 7, and there was no significant proteinuria. Sirolimus through levels were maintained between 12 and 16 ng/mL. A graft biopsy was performed 2 months later, because of sudden nephrotic-range proteinuria without graft dysfunction or signs of hemolysis. Lesions corresponding to thrombotic microangiopathy were observed. Optical microscopy revealed intraluminal fibrin thrombi in two arterioles and capillaries of one glomerulus (Figure 2). Other glomeruli were ischemic. The samples tested negative for the C3 fraction and all immunoglobulins by immunofluorescent staining. Donor-specific antibodies were absent as assessed by ELISA on sera from the pre-engraftment period or at the date of HUS and the samples were negative for C4d staining, ruling out the possibility of antibody-mediated rejection. HUS recurrence in the engraft kidney was diagnosed and the patient was treated by plasma exchange, followed by intravenous globulins. The immunosuppressive treatment was not modified. Proteinuria progressively regressed, and two further kidney biopsies were performed (after 1 and after 3 months): no signs of thrombotic microangiopathy were detected.

image

Figure 2. HUS recurrence 2 months post transplantation. Light microscopy of the kidney biopsy performed 2 months after kidney transplantation showing lesions typical of thrombotic microangiopathy: intraluminal fibrin thrombi in arterioles and glomerular capillaries. Masson's trichrome coloration (×40 magnification).

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The patient experienced a second relapse of HUS 2 years later. She had refractory hypertension, mild acute renal failure with an increase in serum creatinine concentrations from 100 to 180 μmol/L, but no significant proteinuria. Kidney biopsies revealed glomerular capillary thrombosis, mesangiolysis and intimal swelling in the arteries. No donor-specific antibodies were detected by ELISA. She received a second course of plasma exchange and the sirolimus dose was halved. Sirolimus trough levels decreased from 12 to 6 ng/mL. Renal function returned to normal baseline levels and hypertension was controlled by anti-hypertensive therapy involving angiotensin converting enzyme inhibitors.

MCP is produced in the kidney and exerts its effects locally; therefore, HUS recurrence may have been due to endothelial microchimerism of the transplanted kidney. However, the demonstration of microchimerism by detecting chromosome Y (in case of male recipient transplanted with a female transplant) or AB blood group antigens would not have been informative, as both the donor and recipient were women with blood group A. Therefore we took advantage of the presence of an HLA class I mismatch to identify microchimerism. The A10 HLA antigen was expressed by the recipient, but not by the donor. Staining with a human anti-HLA A10 antibody was sporadically positive in the biopsy (Figure 3A). The same frozen sections were stained with a mouse IgG1 anti-human PECAM/CD31 monoclonal antibody, specific for human endothelial cells, to confirm that A10-positive cells were endothelial cells and not leucocytes adhering to the endothelium (Figure 3B). Overlaying the two immunofluorescent staining images confirmed that cells expressing recipient-type HLA antigens were endothelial cells (Figure 3C).

image

Figure 3. Endothelial microchimerism. Six μ-thick cryostat sections of the kidney biopsy performed at the first HUS recurrence were incubated with two primary monoclonal antibodies: human anti-HLA A10 (Human serum anti-HLA-A10, provided from the Etablissement Français du Sang, Hôpital Henri Mondor, Créteil, France) and Phycoerythrine conjugated mouse IgG1 anti-human-CD31 (Diaclone, France). For the anti-A10 staining, the secondary antibody was a Fluorescein Isothiocyanate-conjugated mouse anti-human IgG (Sigma Aldrich, France). (A, B, C) Upper panels represent a control graft biopsy. (D, E, F) Lower panels represent our patient. Recipient and donor were both HLA A10 negative. Panels A and C show the staining with anti-HLA A10 (green fluorescence) and panels B and E the staining with anti-CD31 (red fluorescence). Panels C and F are the overlays of the two corresponding images (For all panels, ×25 magnification).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References

Various teams have demonstrated that there is a genetic predisposition to atypical HUS involving complement-regulating components: Factor H, CD46 (or MCP for membrane cofactor Protein) and Factor I (1,2,4,6–12). These three proteins are involved in the regulation of the alternative pathway of the complement activation pathway. Their inactivation or decreases in their concentration favor complement activation and a predisposition to the development of HUS. Membrane cofactor protein (MCP, CD46) is a transmembrane complement regulator that is widely produced, particularly in endothelial cells of the kidney (13). It acts as a local cofactor to Factor I, which cleaves C3b and C4b deposited on target membranes, and thereby inhibits complement activation. Mutated MCP has a decreased capacity to bind C3b and, therefore, a decreased ability to regulate complement activation. Upon exposure to a trigger of the complement cascade, the reduced cofactor activity of mutated MCP may result in insufficient local protection of renal endothelial cells and may consequently lead to HUS (6,12).

Several mutations of MCP have been reported to be associated with HUS (3). The most common mutation of CD46 reported involves the second intron and is associated with a decrease in its production at the cell surface. This mutation was not present in our patient. However, the patient had a heterozygotic mutation in the second exon, which is thought to involve the splice site: the thymidine (T) in position +2 is replaced by guanosine (G). We confirmed the presence of a transcript using RT-PCR and primers for exon 1 and 4. The transcript was indeed shorter than the wild-type (minus 144 bp) due to alternative splicing in exon 2. This transcript is associated with a weaker expression of CD46 at the cell surface, as demonstrated by the FACS analysis of PBMCs. Thus, this alternative splicing appears to impair the production or the trafficking of CD46 at the cell surface. This might explain why under normal conditions the patient did not suffer HUS, but under certain particular conditions there were triggers that may have overwhelmed the weakened regulatory properties of the affected CD46 leading to the development of HUS.

MCP is produced in the kidney and acts locally. Therefore, transplanting a normal kidney (with normal MCP production) would be expected to prevent a recurrence of HUS (1,2). By contrast, mutation factor I, which is produced in the liver, is not cured by renal transplantation and is associated with HUS recurrence. We surprisingly observed HUS recurrence in our patient. The very low incidence of MCP mutations in the general population, the absence of HUS history in the donor, and the absence of HUS episodes involving the controlateral kidney of the recipient largely exclude the possibility that the donor also carried a MCP mutation, which have been responsible for the recurrence of HUS. HUS is also observed in cases of vascular rejection, mostly antibody-mediated. No histological lesions compatible with humoral rejection (conventional analysis and C4d immunostaining), or donor-specific antibodies were detected using ELISA in our patient, indicating that HUS was not associated with an acute humoral rejection. HUS has also been reported as a consequence of the use of immunosuppressive drugs. As the most commonly described molecules are calcineurin inhibitors (cyclosporine A or tacrolimus) (14). Then, we decided to use an alternate treatment: sirolimus. Sirolimus belongs to the macrolide antibiotic family and binds to the same intracellular FKB12 protein than tacrolimus. Despite these similarities, sirolimus does not display the same nephrotoxicity profile as calcineurin inhibitors. Some authors have reported the resolution of HUS in transplant patients due to calcineurin inhibitors after switching to sirolimus (14). However, more recently few cases of HUS associated with sirolimus treatment have been reported. However, no study of predisposing factors of HUS involving the regulatory pathway of complement activation has been performed (15–17). We cannot exclude the possibility that sirolimus was directly or indirectly associated with HUS. However, sirolimus trough levels were strictly within the normal range and HUS disappeared after the first course of plasma exchange, even though the dose and trough levels of sirolimus were unchanged. Additionally, no HUS relapse was observed over a period of 2 years with similar sirolimus trough levels.

Another possible explanation for the recurrence of HUS is the replacement of some graft endothelial cells by recipient endothelial cells; these cells would have the patient's genotypic background and produce the mutated MCP protein. This endothelial microchimerism has been suggested by Medawar as a mechanism for graft adaptation. Such endothelial microchimerism has previously been recognized and described in human organ transplantation (18). Microchimerism, as observed by Lagaaij and colleagues, appears to be a mechanism for repair, occurring after the injury of endothelial cells. Chimerism occurred significantly more frequently in female than in male recipients, suggesting a role for hormonal factors (19). Consequently, we looked for endothelial microchimerism in the kidney graft as an explanation for the production of the mutated MCP and for HUS recurrence in our case. As donor and recipient were both women sharing the same blood group, we took advantage of the HLA class 1 mismatch between donor and recipient, and tested whether endothelial cells from the graft expressed the restricted recipient antigen (A10). Some vessels in the graft—essentially peritubular capillaries—expressed the HLA A10 antigen; this confirms the occurrence of microchimerism in our patient and provides an explanation for the recurrence of HUS. Therefore, microchimerism may constitute a susceptibility factor for HUS recurrence. Indeed, the HUS associated with a MCP mutation was detected in our patient after childhood and evolved with relapses. The triggers generally remain unknown; however, patients with genetic predisposition to developing HUS may require a trigger to develop a relapse. In our patient, sirolimus (despite trough levels in the normal range) may have been a potential trigger for HUS due to renal microchimerism of endothelial cells from the recipient (with mutated MCP) invading the graft. We, therefore, decided to decrease the dose after the second relapse.

In conclusion, our observation indicates that HUS due to MCP mutation may relapse following renal transplantation and we suggest that it could be favored by endothelial microchimerism of the kidney by recipient cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References

We thank Dr. Ketty Lee, Etablissement Français du Sang, Hôpital Henri Mondor, Créteil, France, for her technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case Report
  5. Discussion
  6. Acknowledgments
  7. References
  • 1
    Noris M, Brioschi S, Caprioli J et al. Familial haemolytic uraemic syndrome and an MCP mutation. Lancet 2003; 362: 15421547.
  • 2
    Richards A, Kemp EJ, Liszewski MK et al. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci U S A 2003; 100: 1296612971.
  • 3
    Fremeaux-Bacchi V, Moulton EA, Kavanagh D et al. Genetic and functional analyses of membrane cofactor protein (CD46) mutations in atypical hemolytic uremic syndrome. J Am Soc Nephrol 2006; 17: 20172025.
  • 4
    Neumann HP, Salzmann M, Bohnert-Iwan B et al. Haemolytic uraemic syndrome and mutations of the factor H gene: A registry-based study of German speaking countries. J Med Genet 2003; 40: 676681.
  • 5
    Pirson Y, Lefebvre C, Arnout C, Van Ypersele de Strihou C. Hemolytic uremic syndrome in three adult siblings: A familial study and evolution. Clin Nephrol 1987; 28: 250255.
  • 6
    Goodship TH, Liszewski MK, Kemp EJ, Richards A, Atkinson JP. Mutations in CD46, a complement regulatory protein, predispose to atypical HUS. Trends Mol Med 2004; 10: 226231.
  • 7
    Nilsson SC, Karpman D, Vaziri-Sani F et al. A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation. Mol Immunol 2007; 44: 18351844.
  • 8
    Richards A, Kathryn Liszewski M, Kavanagh D et al. Implications of the initial mutations in membrane cofactor protein (MCP; CD46) leading to atypical hemolytic uremic syndrome. Mol Immunol 2007; 44: 111122.
  • 9
    Caprioli J, Noris M, Brioschi S et al. Genetics of HUS: The impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 2006; 108: 12671279.
  • 10
    Fremeaux-Bacchi V, Kemp EJ, Goodship JA et al. The development of atypical HUS is influenced by susceptibility factors in factor H and membrane cofactor protein-evidence from two independent cohorts. J Med Genet 2005; 42: 852856.
  • 11
    Dragon-Durey MA, Fremeaux-Bacchi V. Atypical haemolytic uraemic syndrome and mutations in complement regulator genes. Springer Semin Immunopathol 2005; 27: 359374.
  • 12
    Noris M, Remuzzi G. Hemolytic uremic syndrome. J Am Soc Nephrol 2005; 16: 10351050.
  • 13
    Ichida S, Yuzawa Y, Okada H, Yoshioka K, Matsuo S. Localization of the complement regulatory proteins in the normal human kidney. Kidney Int 1994; 46: 8996.
  • 14
    Schwimmer J, Nadasdy TA, Spitalnik PF, Kaplan KL, Zand MS. De novo thrombotic microangiopathy in renal transplant recipients: A comparison of hemolytic uremic syndrome with localized renal thrombotic microangiopathy. Am J Kidney Dis 2003; 41: 471419.
  • 15
    Barone GW, Gurley BJ, Abul-Ezz SR, Gokden N. Sirolimus-induced thrombotic microangiopathy in a renal transplant recipient. Am J Kidney Dis 2003; 42: 202206.
  • 16
    Saikali JA, Truong LD, Suki WN. Sirolimus may promote thrombotic microangiopathy. Am J Transplant 2003; 3: 229230.
  • 17
    Florman S, Benchimol C, Lieberman K, Burrows L, Bromberg JS. Fulminant recurrence of atypical hemolytic uremic syndrome during a calcineurin inhibitor-free immunosuppression regimen. Pediatr Transplant 2002; 6: 352325.
  • 18
    Lagaaij EL, Cramer-Knijnenburg GF, Van Kemenade FJ, Van Es LA, Bruijn JA, Van Krieken JH. Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet 2001; 357: 3337.
  • 19
    Van Poelgeest EP, Baelde HJ, Lagaaij EL et al. Endothelial cell chimerism occurs more often and earlier in female than in male recipients of kidney transplants. Kidney Int 2005; 68: 847853.