CHRONIC TRANSPLANT NEPHROPATHY: Insights into the new decade.

 Miguel Hueso, Lluis Espinosa & Jordi Bover

Nephrology Department

Hospital Prínceps d’Espanya, CSUB

L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain

Correspondence: Jordi Bover 24177jbs@wanadoo.es

 

Introduction and Definitions Etiology TGF-beta
TGF in protocol and diagnostic biopsies Vascular lesions Final considerations and DNA chips

 

While Nephrology is entering into the new millennium, chronic transplant nephropathy (CTN) leads the concerns for the upcoming future in renal transplantation since it remains as the most common cause of graft failure after the first year of follow-up (57.4%) (1). During the last decades, a noticeable and progressive decrement in the incidence of acute rejection has been accomplished; however, it has only had a mild impact on graft-half life, which marginally improved from 8 years in the 80’s to 10 years in the 90’s (1). This concern is even greater if we bear in mind the persistent shortage of donors.

Chronic transplant nephropathy, as defined according to the Banff classification (2), is characterized by the presence of INTERSTITIAL FIBROSIS and TUBULAR ATROPHY or loss, as well as vascular and glomerular changes. However, the grading of severity is only based on tubule and interstitial lesions since these features are the most accurately sampled. Quantification is then based on the percentage of cortical parenchyma involved. As expected, tubulointerstitial changes have been shown to correlate with ulterior graft survival similarly as described before in native kidneys even in glomerular diseases (2-5). CTN is often confused with the more vague and general term “chronic rejection”. Clinically, chronic rejection is characterized by a progressive decline in renal function, proteinuria and/or hypertension once other causes (i.e. obstructive uropathy, de novo o recurrent glomerulonephritis,...) have been ruled out. Histologically, chronic rejection characteristically exhibits VASCULAR CHANGES usually accompanied by interstitial fibrosis. Among these vascular features, fibrous intimal thickening, the presence of disruptions of the elastica, inflammatory cells in the fibrotic intima, and also a proliferation of myofibroblasts in the expanded intima as well as the formation of a second neointima are considered hallmarks of chronic rejection (2). In any case, both CTN and chronic rejection share histological features with other entities (i.e. chronic cyclosporine toxicity, atherosclerosis, donor’s preexistent vascular or interstitial lesions) which sometimes jeopardize an accurate histologic differential diagnosis. Finally, and from the ethimological point of view, the term chronic rejection implies allogenic mechanisms of injury. Thus, since it is often impossible to define the precise mechanisms leading to chronic allograft damage, we think that only CTN or CTN + vasculopathy following Banff’s terminology should be used to describe histological findings. Moreover, it is necessary to stress that assessment of basal donor’s graft histological picture should be somehow mandatory in the future.

 No treatment has been shown to be capable of interfering with the natural history of CTN but, during the last years, several drugs and strategies have been promising. It is now well known that  CTN may already be present in protocol biopsies before it is clinically apparent, and there are data which suggest that protocol biopsies may be a useful tool to design strategies with the aim of interfering with the progressive course of the disease (6) (Serón-CIN2000). Thus, early histological changes may be used as surrogate markers in new trials (6) (Serón-CIN2000). Chronic transplant nephropathy may  already be present in as much as 5-50% of protocol biopsies (7), and as mentioned before, it has been shown that CTN as well as CTN + vasculopathy (chronic rejection) may predict graft outcome (6,7) (Serón-CIN2000).  

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The etiology of CTN remains elusive; nevertheless, both immunological and non-immunological risk factors have been clearly implicated (8). Among IMMUNOLOGICAL factors, acute rejection (AR) episodes (9) and severity (10) are currently considered the main risk factors for the development of CTN. In favor of the immunological origin, second transplants carry a higher risk for CTN (11). Immunohistochemistry of kidney grafts with CTN shows enhanced expression of cell adhesion molecules, invasion with macrophages and T cells, whereas pretransplant immunization with donor antigens accelerates the process. Manoeuvers aimed at the induction of tolerance -such as simultaneous blocking both CD28/B7 and CD40/CD154 (CD40L)- prolong allograft survival with an absence of CTN lesions (12,13), and experimental disruption of the B-cell lineage, CD4+ cells or macrophages inhibit the development of vasculopathy (8). Low-grade immune reaction against classical HLA antigens or against cryptic antigens exposed during previous tissue damage may result in excessive fibrosis (8,14).

 Interestingly, there is now a growing body of evidence that subclinical episodes of AR (just detected on the basis of protocol biopsies) may be harmful for the allograft and that these otherwise undetectable AR episodes may deserve anti-rejection treatment (15) (Rush-CIN2000). Nevertheless, it is not yet known whether treatment of subclinical AR will lead to a decrease in the incidence of CTN.

Conversely, the decrement of the incidence of AR with the advent of new immunosuppressive treatments has been accompanied by only a marginal improvement of graft survival. This apparent contradiction may be explained by the fact that donor age is progressively increasing due to the shortage of donors and, important enough,  that calcineurin inhibitors may induce chronic nephrotoxicity which resembles CTN. It is well known that heart and liver allograft recipients may develop progressive renal scarring. The reduction of CsA doses together with the introduction of MMF resulted in an early improvement in renal function –at least partially mediated by hemodynamic factors- and significantly ameliorated the degree of loss of renal function (16,17). It has also been suggested that the most recent immunosuppressive treatments such as mycophenolate mofetil (MMF) or sirolimus may prevent CTN; however, although there are some promising recent experimental data (18,19) it has not been proven and a longer follow-up is mandatory to determine if promises turn into reality in human transplantation.

Several NON-IMMUNOLOGICAL mechanisms have been described as risk factors for CTN: Brain-death injury, ischemia-reperfusion injury, delayed graft function (influenced by donor age, cold ischemia time, nephrotoxic drugs,...), differences in recipient/donor body surface area, increased filtration of toxic macromolecules (11,20-22).  

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 Whatever the main initial insult is, repetitive injuries over a short-period of time, parenchymal stress, a low-degree of chronic inflammation, may all trigger endothelial and parenchymal injury, expression of adhesion molecules favoring cell recruiting or myofibroblast proliferation, transformation and migration (perhaps epithelial-mesenchymal cell transdifferentiation) and an increased release of a myriad of chemokynes, cytokines, growth factors and enzymes. Biological pathways are somehow redundant and all these different insults may lead by distinct means to excessive scar formation. It has been hypothesized that an excessive production of fibrogenic cytokines such as transforming growth factor beta (TGF-b) may be the common end of this cascade of events (23). Genotipically, high TGF-b producers seem at increased risk of losing their grafts late after transplantation (24).

Transforming growth factor-b1 (TGF-b1) is a multifunctional cytokine which plays a pivotal role in extracellular matrix deposition and degradation. Physiological levels of TGF-b1 are essential for normal development, tissue repair and maintenance of organ functions. Nonetheless, a sustained overexpression of TGF-b1 has been implicated in the pathogenesis of fibrotic diseases both in experimental and human studies (23,25,26). On the other hand, several reports have focused on the protective actions of TGF-b1 against injury by exerting immunosuppressive and  anti-inflammatory actions (27).

Increased expression of TGF-b protein and mRNA levels (by means of immunochemistry and Northern-blots or PCR) have been related to experimental kidney and allograft fibrosis (28-30). In human biopsies from patients undergoing diagnostic evaluation, it has been described increased TGF-b protein levels in both acute and chronic rejection (28), and recently they have been significantly associated with an increased rate of decline in renal function (31). Sharma et al (32) also found an association between the qualitative expression of TGF-b1 mRNA and the presence of either fibrosis or chronic allograft rejection in biopsies obtained for diagnostic purposes as well.

 Despite these results suggest that TGF-b1 could play an important pathogenic role in the development of CTN, most of the human studies have been reported in diagnostic biopsies; thus, late in the course of the disease when therapeutical maneuvers are likely to be unsuccessful. Interistingly, Campistol et al have shown that losartan may decrease plasma TGF-b1 levels in patients with CTN; however, we do not know yet whether the progression of the disease may be altered at this stage (33) (Campistol-CIN2000).

On the other hand, some contradictory results on renal TGF-b expression have also been reported. Despite we have to take into account that technical and methodological problems may lay behind immunohistochemistry (34),  Lantz et al (35) did not find differences in TGF-b protein expression among rejected grafts and they only detected a major difference between trasplanted and non-trasplanted kidneys. Horvath (36) et al described by Northern blot analysis that the expression of mRNA encoding for different TGF-b isoforms, including TGF-b1, was decreased in 10 chronically rejected renal cortex samples compared to normal controls in surgically removed kidneys. Recently,  Mannon et al have observed that the severity of CTN is independent of the level of TGF-b expression within the allograft in an experimental mouse model, despite they had observed that TGF-b was markedly enhanced in control allografts compared with isografts (30). In this latter study, the severity of chronic rejection was shown to be independent of TGF-b1 expression within the allograft.  

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Since it  is not known whether early protocol biopsies with CTN have increased TGF-b1 mRNA levels and no data are currently available on the direct relationship between fibrosis and intragraft TGF-b1 mRNA expression in clinically stable patients during the early posttransplantation period, we have been performing protocol biopsies 149 ± 7 days (range 95-210) and 429 ± 9 days (range 342-596)  after transplantation and we have measured intragraft renal TGF-b1 mRNA expression using real-time quantitative PCR (Taq-Man® ABI PRISM 7700, Perkin Elmer).

We observed that all biopsies expressed TGF-b1 and we did not find significant differences among the different Banff’s histological diagnosis (Table 1).  

  5-month protocol biopsies         TGF-b1  / GAPDH   
Normal                                     (n=18)         1.15 ± 0.32  
Borderline changes                     (n=6)                0.98 ± 0.26  
CTN                                          (n=1)                0.83  

CTN + Borderline changes         (n=2)             

 2.14 ± 1.80

 NS

  14-month protocol biopsies                           TGF-b1  / GAPDH   
Normal                                     (n=19)        1.29 ± 0.29  
Borderline changes                     (n=5)                 1.25 ± 0.27  
CTN                                         (n=16)    0.78 ± 0.11

CTN + Borderline changes         (n=11)             

 1.99 ± 0.93  

     NS

Table 1. Intragraft transforming growth factor-b1 (TGF-b1) mRNA expression in 5-month and 14-month protocol biopsies according to Banff histological criteria. Results are expressed as mean ± SEM. The TGF-b1 / GAPDH ratio represents the relative abundance of TGF- b1 expression once normalized with the expression of a normal non-grafted kidney. N = number of samples, GAPDH = house-keeping gene, CTN = Chronic transplant nephropathy. NS = non significant differences (Kruskal-Wallis test).

 

Since TGF-b1 mRNA levels were not a correlate of the time at what biopsy was performed (Figure 1) ...

....we grouped them in order to increase the power of statistical analysis but differences were not yet apparent (Table 2).  

 

 

 
Banff  in protocol biopsies                          TGF-b1  / GAPDH   
Normal                                     (n=37)        1.22 ± 0.21  
Borderline changes                   (n=11)                 1.10 ± 0.18  
CTN                                         (n=17)   0.78 ± 0.10

CTN + Borderline changes         (n=13)             

 2.01 ± 0.80  

  NS

We did not find either significant differences when we grouped the protocol biopsies according to the presence, absence or degree of tubular atrophy, interstitial infiltrate (p= 0.15); presence or absence of hyaline arteriolar lesions  or chronic vascular changes. From the clinical point of view, we did not find any correlation between TGF-b1 mRNA expression and mean arterial pressure, creatinine or proteinuria at the time of biopsy, nor any clinical parameter during a follow up of 4.1 ± 0.2 years (range 1.9-6.1 years). No association between TGF-b1 mRNA expression and mycophenolate mofetil or CsA use was detected in this subset of patients.


Interestingly enough, when we compared intragraft expression of TGF-b1 mRNA according to the indication of biopsy (either protocol or diagnostic) in patients with CTN, it was significantly higher in diagnostic biopsies (0.78 ± 0.10 vs 2.04 ± 0.51,  respectively; p=0.04) performed for worsening renal function and/or proteinuria. The comparison between characteristics of patients with CTN according to the indication of biopsy is shown in Table 3.  

 

CTN (protocol) n=17  

CTN (diagnostic) n=9    

p
Donor age (years)    32 ± 4                          31 ± 6     ns
Donor sex (M/F) 5/2       8/1    ns
Recipient age (years) 48 ± 3  39 ± 4               0.07  
Recipient sex (M/F)  9/8       7/2   ns  
BSA(m2)              1.72 ± 0.05    1.70 ± 0.04     ns  

Number transplant (1/2)

13/4     8/1                      ns     
PRA  >20%               2/15        1/8               ns  

HCV serology (neg/pos) 

 13/4       7/2                ns

Etiology of ESRD

    Glomerular

    Interstitial

    APKD                            Nephrosclerosis           

    Unknown                   

 

  

3

2

4

2

6

  

4

2

1

1

1

  

 

 

ns

 
 HLA DR mismatches     0.65 ± 0.12       0.7 ± 0.2       ns

HLA DR identities    

1.00 ± 0.12        1.0 ± 0.2       ns

HLA A mismatches   

0.88 ± 0.19    0.8 ± 0.2       ns
HLA B mismatches    1.06 ± 0.16    1.2 ± 0.1        ns

CIT (hours)

23  ± 2    23 ± 3              ns
DGF (yes/no)          6/11       0/9                    0.02
AR (yes/no)           5/12         5/4                   ns

AR (0/1/2 episodes) 

12/4/1     4/5/0                ns

Time of biopsy (days) 

 416 ± 23   1878 ± 364 <0.001  
sCreatinine (mmol/l) 127 ± 7    236 ± 26      0.001  
Proteinuria (mg/24h) 0.25 ± 0.04   1.48 ± 0.37     0.02  

Blood Pressure:

SBP     

DBP 

MBP                   

  

144 ± 6       

 81 ± 2        

102 ± 3                              

   

161 ± 7           

93 ± 5    

116 ± 5                                 

  

0.06  

0.05

0.02

ACEI (yes/no) 2/15    5/4                   0.03  

MMF (yes/no)         

 3/14        0/9    ns
CsA (yes/no) 16/1 9/9 ns

BSA = body surface area, PRA= Panel reactive antibodies, HCV= Hepatitis C virus, ESRD= end-stage renal disease, APKD= Adult polycystic kidney disease, CIT = cold ischemia time,  DGF = Delayed graft function, AR = Acute rejection, HTA = Hypertension, SBP-MBP-MBP= systolic, diastolic and mean blood pressure, ACEI = angiotensin converting enzime inhibitors, CsA= Cyclosporine, MMF = mycophenolate mofetil,

 

Our results demonstrate that CTN may already be present early in the allograft without a detectable increase in TGF-b1 mRNA levels measured by real-time quantitative PCR. Thus,  a sustained overexpression of TGF-b1 mRNA might not be a prerequisite for the development of CTN and factors different than TGF-b1 may play a more important etiological role.

An increased expression of TGF-b1 either by immunochemistry, qualitative PCR or plasma levels has been described in diagnostic biopsies displaying CTN (28,32,33). However, our results would suggest that this detected overexpression of TGF-b1 mRNA in diagnostic biopsies may not be directly related to the initial development of CTN but it could be rather a consequence reflecting an ongoing reparative process. As mentioned before, it is well known that a common feature of CTN is that it develops in grafts which have undergone previous damage (22). Moreover, fibrosis is a complex process involving the interaction between multiple humoral factors and cell types (14,22). Accordingly, it has been recently described that differences in TGF-b1 expression did not prevent loss of graft function, affect the course nor alter the pathological characteristics of chronic rejection in an experimental model (30). Furthermore, the severity of chronic rejection was shown to be independent of TGF-b1 expression within the allograft (28).

Nevertheless, although mRNA and protein expression generally correlate, we can not completely rule out a role of TGF-b1 as a mediator in the development of interstitial fibrosis and CTN earlier after renal transplantation. Moreover,  the activity of TGF-b1 is under a complex regulation not only at the level of gene transcription but also at the level of mRNA stability, protein secretion, conversion from a latent to an active form, the presence of soluble or cellular TGF-b1 -binding molecules and through modulation of its receptors (26,37,38).  Moreover, it has been suggested that local activation of TGF-b1 may be a more important determinant of subsequent graft fibrosis than the actual level of production of the latent protein (34).

Thus, we can conclude that TGF-b mRNA is not a proper molecular surrogate marker of early CTN and that an increased expression of TGF-b may be secondary. Nevertheless, this decade is going to witness a great effort in the blockade of this potential effector at different levels since a lack of down-regulation of TGF-b1  after normal tissue repair may accelerate the natural history of the disease. Nonetheless, we consider that expectations should be moderated. Thus, the use of antibodies against TGF-b does not completely prevent renal fibrosis and some experimental models show that knock-out mice showed multi-organ inflammation including the kidney (27,39). TGF-b is a physiological means of repair and we should still limit persistent known and yet unknown immunological and non-immunological injuries which actually trigger and maintain this cascade of  events.  

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Despite the main characteristic of CTN is fibrosis and attention is lately focused on mediators of extracellular matrix accumulation, it is now known that VASCULAR LESIONS carry the worst prognosis for the graft (6). It is known that endothelial and smooth muscle cells react to injury producing cytokines which cause vessel wall proliferation and vascular sclerosis. Thus, glomerular and tubulointerstitial lesions may be secondary as well. On the other hand, it is undoubtful that lipid metabolism plays an important role in the development of these lesions (40-42) and our group has demonstrated that hypercholesterolemia may be an independent risk factor for the development of graft CTN + vasculopathy (6) (Serón-CIN2000). It is likely that these vascular lesions share many links with the quite unknown pathophysiology of atherosclerosis. In fact, in the context of heart transplantation, chronic rejection is described as accelerated atherosclerosis. We think that our relevant prognostic data points to a tight link between the pathophysiology of atherosclerosis and CTN.

Finally, other molecules and cytokines have also been involved in CTN and vasculopathy (endothelin, PDGF, bFGF, IFN-g, AII, filtered macromolecules, VEGF, HGF...).  

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We have seen that the most common approach to the pathogenesis of CTN focuses on targeting of a single molecule (or many of them with a moving target). However, we should also bear in mind that there are other physiopathological theories, not directly linked with the cytokine excess theory (interference in the tissue repair following injury –heat-shock proteins-, loss of supporting extracellular matrix architecture, premature senescence theory –Hayflick limit-, apoptosis, ...), which  could result in excessive fibrosis in grafts which have undergone previous damage (43,44). The problem of CTN and vasculopathy is of such a complexity that we consider that an unbiased gene study by using the most modern tools is mandatory and the derived results may be much more promising in the near future.

Thus, the complete sequence genome of several organisms has been already analyzed and the Human Genome Project is scheduled to finish before 2003.
These data may be used as a new powerful tool to pursue candidate genes for different diseases, depict differential patterns of gene expression in various conditions or the identification of targets for therapeutical development. Recent progress of DNA technology allows us to handle thousands of genes simultaneously in microarrays, and it is likely that clusters of genes more than unique transcripts may better help us to understand mechanisms of human diseases.
These DNA-chip expression probe arrays have set out-of-date several less sensible and cumbersome methods such as comparative and subtractive hybridization, sequencing of cDNA libraries, Serial Analysis of gene expression (SAGE) and so on. This is possible because these arrays contain oligonucleotide probes for thousands of genes and hybridisation signal intensities represent relative amounts of mRNA. Thus, Takenaka et al have shown that this approach may provide basis of normal renal gene expression and could be applied to prepare kidney specific or nephron segment specific microarrays in the future (45). We are currently following a similar approach in renal transplantation, comparing different histological and clinical situations. Despite any procedure carries with it some drawbacks (individual vs pooled differential expression, the presence of many expressed sequence tags (EST) as well as other technical and methodological considerations), we think that the unbiased nature and speed of this technique may soon prompt us to a new insight in both renal physiology and pathology. “Functional genomics” have finally come true

Acknowledgements: This work is supported by grants from FISS 96/0785, FISS 99/0842 and Novartis Inc.


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