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Rene C Bakker,

Leiden University Medical Center. Leiden, The Netherlands



The subject of chronic rejection of allografted kidneys has received increasing attention in recent years and newer morphometric tools have been developed to demonstrate this condition. The electron microscopic evaluation of the basement membrane of the peritubular capillaries is most likely a valuable new tool. The significance of a positive staining for C4d is not fully clear. These newer tests may suggest the presence of chronic rejection but do not exclude a co-existing toxic influence of a calcineurin inhibitor.

The impact of chronic cyclosporine nephrotoxicity

The specific understanding of the importance of chronic CsA nephrotoxicity (CsAT) in renal transplantation has long been hampered by the lack of specific and sensitive markers of this condition and the absence of studies with long term follow-up. CsAT may affect the allografted kidney rather slowly and it may take many years before the real impact is evident in clinical trials. Data derived from studies on patients with various autoimmune disease or solid organ transplants other than a kidney treated with CsA have indicated a relative high incidence of chronic CsAT 1-11. In heart transplant patient’s end-stage renal failure has been observed in 6-10%, the frequency of which apparently increases with a longer period of follow-up 12. Heart transplant patients receive relatively high doses of CsA because of fear of rejection. However, renal functional or structural changes have also been observed frequently in patients on lower doses of CsA treated for autoimmune disease. Two studies examined patients with psoriasis and included pre and post treatment protocol biopsies 8;11. Biopsies taken at 1 year showed de novo interstitial fibrosis in more than 40% of patients in the study of Svarstad 8. Zachariae reported a histological follow-up of 25 patients 11. Seventeen patients had normal baseline histology and at two years all had histological changes compatible with chronic CsAT. At four years all studied biopsies (n=11) displayed moderate to severe fibrosis.

More direct evidence of the importance of chronic CsAT after allograft transplantation has come from recently analyzed data from two azathioprine conversion studies with 15 years of follow-up 13, and two studies that evaluated the conversion to MMF in patients with established aspecific chronic allograft nephropathy (CAN) 14;15.

The fifteen year data from our open-label prospective randomized study that compared cyclosporine continuation with conversion to azathioprine three months after transplantation showed a higher risk of CAN in the group that continued cyclosporine (relative risk 4.3, 95% CI: 1.4 to 12.9) 16;17. We studied predominantly Caucasian recipients, generally well matched for HLA antigens. A better death censored graft survival was observed in the azathioprine group after two years post-transplantation

The 15 years death censored graft survival was 81.9 vs. 69.2 % (p=0.012). These results are in line with data from a not yet published multicenter Australian conversion study with an identical follow-up. In this study a highly significant difference in mean graft survival was found favoring CsA usage shorter than 6 months (13.3 vs. 11.8 years, p <0.01)13.

Sparing of cyclosporine in patients with established CAN has been studied with MMF as the added agent. 14. The slope in GFR decline pre- and post-intervention was compared. Fifty to sixty percent of the patients treated with either reduced dose of CsA (n=67) or tacrolimus (n=33) showed an improvement in the rate of decline and more than 90% of the patients that were withdrawn from CNI therapy (n=18). A small risk of AR was noted. A second ongoing multicenter study examined the withdrawal of CsA in patients with gradually declining allograft function. After 6 months stabilization or improvement was seen more often (58% vs. 32%) in the patients that converted to MMF (n=73) as compared to the ones that continued on CsA (N=70)15.

The pathologic diagnosis of chronic CsA nephrotoxicity

The lesions that are most frequently found in native kidneys with chronic CsAT are non-specific and include: tubular atrophy, interstitial fibrosis, slight mononuclear cell infiltration, Bowman’s capsule basement membrane thickening, glomerular collapse, global sclerosis and non-specific arteriolar hyalinosis as seen in hypertension or diabetes. Glomerular thrombosis or necrosis and signs of arteriolar thrombotic microangiopathy are very rarely observed with the use of lower doses of CsA 18, 19. Also tubular alterations including honeycomb vacuolization of the proximal tubular epithelium, giant mitochondria and micro-calcifications or minor changes of endothelial or smooth muscle cells occur infrequently 19. The most specific marker of CsAT is a nodular hyaline insudation in the periphery of small arterioles either patchy or circumferential designated as peripheral nodular hyaline degeneration (PNHD) 19-21. Arterioles up to two layers of smooth muscle cells are involved. Electron microscopy have suggested that the deposits of PNHD occur at sites of myocyte necrosis 19;22;23. Immunofluorescence staining is often positive for IgM and C3 19, representing aspecific binding of plasma proteins. PNHD must be differentiated from the arteriolar hyaline changes as seen in diabetes or long-standing hypertension, where the hyaline insudation occurs on the inside of the SMC layer 19. The differentiation may be difficult on light microscopy, especially when the lesions are more pronounced. PNHD may regress or even completely vanish upon reduction of dose or withdrawal of CsA 24-27.

The hallmark of irreversible chronic CsAT is the occurrence of tubulointerstitial and glomerular changes including segmental and global glomerulosclerosis, interstitial fibrosis and tubular atrophy 11;19;22;28-30. The tubulointerstitial changes may be found before renal function is impaired 4, and in a rat model even before PNHD occurs 31. The severity of the tubulointerstitial changes correlates well with the degree of glomerular sclerosis 22. Non affected glomeruli are hypertrophied 32-34, and presumably preserve function by hyperfiltration.

In a series of 192 patients treated with CsA for various autoimmune diseases 26% of the biopsies showed interstitial fibrosis, however PNHD was only noted in 4% 35. Aspecific arteriolar hyalinosis was observed more frequently. In another study with protocol biopsies of uveitis patients a high incidence of arteriolar hyaline changes was found that increased steadily with time 11. However, in this study it was not clear if the specific PNHD lesion was scored. In 1994, an international advisory board of nephropathologists with extensive experience in the evaluation of kidney biopsies of patients on CsA, found that the reproducibility and diagnostic reliability of the evaluation of arteriolar lesions including PNHD was poor 35. In contrast, the interobserver variation on tubulointerstitial changes was low.

The diagnosis of chronic CsAT in allografted kidneys is therefore difficult. The nature of changes in the vascular and glomerular compartments may sometimes be suggestive of an etiologic factor in graft deterioration but do exclude concommitant chronic CsAT. Allograft glomerulopathy with reduplication of the GBM indicates an allodependent insult at the level the glomeruli and is found in a minority of late biopsies 36-41. Concentric intimal thickening of arteries and arterioles may result from chronic rejection but is less specific 40;42;43. A high degree of multilayering of the peritubular capillary basement membrane of is thought to indicate chronic rejection. When PNHD is found CsAT should be considered. Striped fibrosis in renal allografts is non specific 44. It is clear that the pathologic diagnosis of chronic CsAT in allografted kidneys still needs further improvement by finding a more specific and sensitive marker.

Newer tools in studying the cause of chronic allograft nephropathy

Because the tubulointerstitial compartment is most frequently involved in chronic CsAT we decided to study the interstitial matrix and searched for specific markers at the molecular level. We studied biopsy samples from two well defined groups of patients suffering from either chronic allograft rejection or chronic CsAT. Various matrix molecules were evaluated by computerized immunohistochemistry and also mRNA expression was studied by RT-PCR .

In the immunohistochemistry study we examined the proteins collagen I, III, and IV, collagen IV 3 and laminin 2 in biopsies of allografted kidneys with chronic CsAT (n=17), chronic rejection (CR) (n=12), or chronic allograft nephropathy (CAN) (n=19). Alpha-smooth muscle actin (SMA) expression was also examined. Tissue from normal native kidneys was used as control (n=11) 45.

Biopsy samples were studied by routine light microscopy and after immunostaining. The mean interstitial fibrosis score was significantly higher of the CR and CAN group than of the chronic CsAT group. The cortical tubulointerstitial areas of the CR or CAN groups, but not of the chronic CsAT group, contained more collagen I than normal controls. A difference was already noted in biopsies with mild fibrosis. Collagens III, IV and IV3 were increased in all groups. Collagen III accumulation was greater in the CR or CAN group than in the chronic CsAT group. ROC curve analysis demonstrated that collagen I staining had the best discriminatory value in differentiating CR from chronic CsAT with a sensitivity of 63% and specificity 94% at a cut-off area value of 19%. Laminin 2 staining did not differentiate CR from CsAT. Increased SMA staining did not differ between the three groups. We concluded that during chronic CsAT tubulointerstitial collagens III and IV accumulate preferentially and that an early increase in deposition of collagen I along with collagens III and IV is more specific for CR.

In the mRNA study we examined mRNA levels of transforming growth factor (TGF-) and the extracellular matrix (ECM) molecules collagen I1, III1, IV3, decorin, fibronectin, and laminin 2 by real-time PCR (Klaas Koop et al, submitted for publication) . In both patient groups, the mean mRNA levels of TGF- and of the ECM molecules, except those of fibronectin, were significantly elevated compared to those in controls. The increase of laminin 2 and TGF- mRNA levels was significantly higher in the CsA toxicity group (23.0 and 10.4 fold) than in the chronic rejection group (6.1 and 3.4 fold). ROC-curve analysis showed that with a 15.6 fold increase in laminin 2 mRNA expression as cut off point, the presence of CsA toxicity could be predicted with an 87 percent sensitivity and an 88 percent specificity.

What do these two studies tell us ? Do we have new tools to evaluate the presence of CR or chronic CsAT? We think that the results of the first study showed that indeed differences could be found in the composition of the tubulointerstitial matrix related the cause of allograft deterioration. The study also refuted data from an earlier study that claimed that positive staining for collagen IV 3 and laminin 2 at the proximal tubular basement membrane indicates CR. Our study suggested that early accumulation of collagen I is a marker of chronic rejection, however no specific marker for chronic CsAT was found as an increase in staining for collagen III and IV also occurred in CR. The study was small and done on selected population without longitudinal follow-up. Therefore we think that although the results are promising and stimulate further research, they do not have a meaning for daily clinical practice yet.

In the mRNA study however we did find in the laminin 2 mRNA level a marker for CsAT. The study was also small and done on selected population and lacked longitudinal follow-up. The increase of this marker may not be a surprise when one realizes that the laminin 2 protein is normally found in vessel walls within the kidney and that CsA has been shown to be toxic for arterioles. Further research has to show how valuable this possible new marker of chronic CsAT is.

We conclude that the pathologic recognition of chronic CsA nephrotoxicity in allografted kidneys is still imperfect and needs further improvement. Differences in the composition of the tubulointerstitial matrix can be found in kidney allografts that suffer from either CR or chronic CsAT. An increase in laminin 2 mRNA is found in allografts suffering from chronic CsAT. Further research has to show how valuable immunohistochemical staining for matrix components and the measurement of mRNA laminin 2 is.

Figure 1. Graft survival censored for death with a functioning graft of the patients that stayed on the drug they were initially assigned to. The vertical dashed lines indicate the start of the study three months post-transplantation (left), and the start of the period of survival benefit in the azathioprine arm of the study two years post-transplantation (right).


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