The advantages conferred by renal transplant, such as the improved quality of life and survival, are compromised by the reduced half-life of the transplanted kidney to a decade because of chronic allograft injury, which is the leading cause of transplant loss. There has been a significant evolution in the concept of the nomenclature, grading of histologic changes, diagnostic markers, and the theories of the pathogenesis of chronic allograft injury in the past decade. This review sought to consolidate the published literature that contributes toward under­standing the changing concepts and pathogenesis of the chronic allograft injury, which has implications to managing and preventing chronic allograft injury in experimental and clinical settings.

Key words : Chronic rejection, Pathogenesis, Renal transplant, Transplant fibrosis, Risk factors

Introduction

A renal transplant (RT) is the best form of treatment for most patients with stage 5 chronic kidney disease, because this improves the quality of life, patient survival, and is cost-effective.¹ Despite advancements in immunosuppressive agents and treatment of acute rejection, chronic allograft injury (CAI) continues to remain the leading cause of late RT loss. Interplay of both immunologic and non-immunologic factors in the pathogenesis of CAI has posed significant problems in preventing and treating CAI; hence, attention is focused on understanding the pathogenesis of RT fibrogenesis and interventional strategies to prevent and treat CAI.²

Chronic allograft injury is characterized by a relatively slow but variable rate of decline in renal function after the first 3 months of an RT, often in combination with proteinuria and hypertension. It is important to assess other causes of transplant dysfunction in the differential diagnosis of CAI, such as rejection (acute, subclinical, and chronic), calcineurin inhibitor (CNI) nephrotoxicity, glomerulonephritis (recurrent and de novo), nephrosclerosis (secondary to old donor age, recipient hypertension, hyper­lipidemia, and smoking) and other possibilities including ureteric obstruction, BK virus nephropathy, and transplant renal artery stenosis.³

There has been significant evolution in the concepts of the nomenclature, grading of histologic changes, risk factors, diagnostic markers, and theories of the pathogenesis of CAI in the past decade, which have implications to its prevention and management. This review seeks to consolidate the published literature that contributes toward understanding of the changing concepts of CAI, which has significant implications to research and clinical practice.

Literature Search Strategy

The literature search was carried out in PubMed and relevant Web sites using the words “renal transplant,” “chronic allograft nephropathy,” “transplant fibrosis,” “chronic allograft injury,” “chronic rejection,” “chronic allo­graft damage,” and “prevention.” Relevant ref­erences were compiled in EndNote software (Ver­sion X 7.4; Thomson Reuters, Philadelphia, PA, USA).

Nomenclature

There have been sequential changes in nomenclature, which include the terms chronic rejection, chronic allograft nephropathy, chronic allograft dysfunction, chronic allograft damage, and chronic allograft injury to describe the clinical and histologic features leading to progressive deterioration of RT function and RTs loss. The concept of chronic rejection emerged gradually in the 1950s and 1960s. In 1955, Hume and associates reported a case in which rejection developed within 5.5 months after an RT, with obliteration of the arteries.⁴ Porter and associates and Jeannet and associates revealed that obliterative arterial intimal fibrosis was frequent and represented a reaction to immune injury, probably to allo­antibodies and coined the term chronic rejection.^5,6 These observations made in the presence of minimal immunosuppression, occurred within a few months after an RT, but are rarely seen currently because of immunosuppressive agents. The clinical course of CAI is slow and progressive. However, the Hume-Porter-Jeannet model remains the basis for understanding the current models of CAI. The term chronic rejection implies an ongoing immune response, which cannot, however, be proven in every case. It is now clear that there is involvement of both immune and non-immune factors in late RT loss with histologic changes described below; hence the term chronic rejection should be avoided.⁷

In 1991, Dr LC Paul from Netherlands coined the term chronic allograft nephropathy (CAN) to describe histologic changes such as interstitial fibrosis, tubular atrophy, glomerulosclerosis, and arteriolopathy.⁸ The term CAN was agreed on and promulgated by the Banff 1993 expert consensus, to include at least 4 entities that could not be distinguished by biopsy such as chronic rejection, chronic calcineurin inhibitor toxicity, hypertensive vascular disease, and chronic infection and/or reflux, to describe the pathology of tubular atrophy and chronic interstitial fibrosis of a chronically impaired renal allograft.⁹ When there was associated hyper-tension, proteinuria, and progressive deterioration of renal function, the condition was termed chronic allograft dysfunction and chronic allograft damage (CAD) to indicate the clinical function of the RT.¹⁰ Halloran and associates defined CAN as a state of an impaired graft function at least 3 months after an RT, but independent of acute rejection, drug toxicity, recurrence of de novo specific disease entities with typical histological features such as tubular atrophy (TA), interstitial fibrosis (IF), fibrous intimal thickening, and transplant glomerulopathy.¹¹

The 8th Banff Conference on allograft pathology held in Edmonton, Canada, July 15-21, 2005, introduced the term chronic allograft injury (CAI) to replace CAN, as the term CAN was being used as a generic term for all causes of CAD with fibrosis, which inhibited accurate diagnosis and appropriate therapy.¹² Histologically, the term CAN was replaced by interstitial fibrosis (IF) and tubular atrophy (TA). Currently, the term CAN is gradually being replaced by CAI, IF/TA, and CAD in the transplant literature to describe the clinical entities encompassed above.

Magnitude of Problem
Chronic allograft injury and death with functioning grafts are the 2 major causes of allograft loss 1 year after transplant.13 Schweitzer and associates from Minnesota in 1991 reported, in a cohort of 2396 patients over a period of 20 years (1970-1989), chronic rejection as the leading cause of graft loss after an RT amounting to 24%, followed by death with func­tioning graft (18%), infection (13%), and acute rejection (11%).¹⁴ Subsequently, Sijpkens and associates from The Netherlands reported that 54 of the 654 RTs (8%) performed between 1983 and 1997 had histologic evidence of CAN, and CAN accounted for 37% of graft loss after the first 6 months after an RT.¹⁵

The incidence of CAI is more accurately assessed if protocol biopsies are performed prospectively. Solez and associates reported the histopathological results of 2-year protocol biopsies from the patients enrolled in the US FK506 study (N = 144; seventy-nine in the tacrolimus and 65 in the cyclosporine group). Chronic allograft injury was found in 49 (62.0%) and 47 (72.3%) of tacrolimus- and cyclosporine-treated patients. The occurrence of CAI was significantly higher in patients who received a graft from an older donor, who experienced presumed cyclosporine or tacrolimus nephrotoxicity, who developed a Cytomegalovirus infection, or who experienced acute rejection in the first year after transplant. Multivariate analysis showed that nephrotoxicity and acute rejection were the most significant predictors for CAI.¹⁶

Clinical Manifestations
Chronic allograft injury is characterized by a relatively slow but variable rate of decline in renal function after the first 3 months of an RT, often in combination with proteinuria and hypertension.8 A definitive diagnosis of CAI requires the exclusion of other possible causes of deterioration of renal function and confirmation through biopsy. The decline in renal function as determined by the plot of reciprocal serum creatinine concentration over time, evident in more than 80% of patients with CAI, was found to be the best predictor of subsequent graft failure. The independent relative risk for graft failure attributable to Delta1/Cr less than - 40% was 5.91 (95% confidence interval, 3.25 to 10.8; P < .0001).¹⁷

The prevalence of proteinuria is variable. Twenty to 28% of patients with CAI have greater than 0.5 g proteinuria per 24 hours compared with 6 to 8% of patients free of CAI.¹⁸ Proteinuria in the nephrotic range post-transplant related to CAI was associated with poor allograft survival, irrespective of the time of onset of presentation, especially when renal function was reduced at the time of biopsy.¹⁹ Although hypertension is associated with CAI, its diagnostic significance remains limited as hyper­tension is prevalent among renal patients in as high as 50% of cases despite the use of modern immuno­suppressive regimens. Relationships between poor control of blood pressure and reduced RT survival have been clearly demonstrated and are analogous to the well-known acceleration of progressive renal disease by coexisting hypertension.²⁰

Pathology
The CAI kidney looks pale and fibrotic with a dense, thickened, adherent capsule, and sections show both the cortex and the medulla to appear smooth, indicating atrophy. Thickened obliterated arcuate and interlobar arteries often can be seen at the corticomedullary junction. The renal artery shows thickening and obliteration of its lumen up to the point of anastomosis with the recipient artery. The ureter is often involved in a similar process. The histology of a failing RT often shows collection of end pathway responses to injury within all 3 anatomic compartments of the kidney (microvascular, tubulointerstitial, and glomerular).²¹ Under light microscopy, the glomeruli appear ischemic, atubular with features of chronic transplant glomerulopathy (CTG). The ischemic glomeruli are characterized by wrinkling and collapse of glomerular capillary wall associated with extracapillary fibrotic material. In normal living or cadaveric donor kidneys, 1% to 2% of glomeruli are atubular, increasing to 17% to 18% with CAD and 29% in cyclosporine nephrotoxicity.²² Chronic transplant glomerulopathy comprises a spectrum of histologic abnormalities, which include thickening or duplication of the glomerular capillary basement membrane (double contour lesion) and mesangial expansion. Chronic transplant glomerulo­pathy implies chronic endothelial injury of glomerular capillary loops accompanied clinically by substantial or nephrotic range proteinuria, renal function impairment, and reduced RT survival (Figure 1A and 1C).

As a result of ischemia caused by the micro­vascular changes described above, the tubules undergo atrophy, which also may result from tubulitis. The interstitium shows fibrosis with variable mononuclear infiltrate with small lymphocytes, plasma cells, and mast cells. The fibrosis can have different patterns, such as dense and focal, diffuse and fine, striped, or subcapsular. The peritubular capillaries are depleted, leaving behind only traces of original basement membrane. The transplant arteriolopathy, characterized by severe intimal proliferation and luminal narrowing associated with sparse infiltration of T cells and macrophages, is seen in all arteries extending from main renal artery to the interlobar arteries (Figure 1, B and D).²³

In immunofluorescence microscopy, CTG asso-ciated with chronic antibody-mediated rejection (C-AMR) often shows deposition of the complement degradation product C4d in the glomerular capillaries and peritubular capillaries basement membrane (Figure 2). Under electron microscopy, CTG is associated with deposition of flocculent or fibrillary material; mesangial cellular proliferation with matrix expansion; and multilamination or multilayering of the capillary basement membrane. Multilamination of capillary basement membrane as high as 7 or more layers indicates past or recent endothelial injury with subsequent repair, which was present in 38% of failed transplants ascribed to CAI (Figure 3).²⁴

Grading of Histology

Banff working schema
The most widely used classification system currently is the Banff working schema, introduced in 1993 by Soles and associates, to achieve consensus that would be useful for diagnosing and treating acute and chronic histologic changes in an RT.⁹ Although Banff is fully accepted as a scoring system, it is still undergoing revisions regularly based on available data.

Banff scores 3 elements for acute rejection: tubulitis (t), the extent of cortical mononuclear infiltrate (i), and vascular inflammation (v). The Banff grading of CAI is based on adding the scores of 3 components parts: tubulointerstitial, vascular, and glomerular. The grading and quantification of interstitial fibrosis (ci), tubular atrophy (ct), allograft glomerulopathy (cg), increase in mesangial matrix (mm), vascular fibrous intimal thickening (cv), and arteriolar hyalinosis (ah) are carried out.²⁵

Chronic allograft damage index
Similarly, Isoniemi and associates developed a Chronic Allograft Damage Index, which scores severity from 0 to 3 for six parameters such as interstitial inflammation, interstitial fibrosis, glomerulosclerosis, mesangial matrix expansion, vascular intimal proliferation, and tubular atrophy. The respective scores are added together to produce an overall Chronic Allograft Damage Index score.²⁶ The value of Chronic Allograft Damage Index in predicting long-term renal allograft outcome was examined by Inosiemi and associates, and it was observed that the posttransplant Chronic Allograft Damage Index score at 2 years significantly correlated with renal function at 6 years for 89 functioning renal allografts and significantly predicted which patient would later develop CAD.²⁷

Morphometry
Morphometry provides more precise quantification of interstitial fibrosis. The markers used for fibrosis include special stains (Masson/Mallory trichrome and Sirius/picrosirius red) and antibodies to collagen I and III. Digital images and computerized data analysis are used to calculate the cortical interstitial volume fraction (VIF). The VIF measured by morphometry in protocol biopsies correlates with time to graft failure.^28,29

Diagnostic markers
Urinary excretion of β2-microglobulin, tubular enzymes such as glutamine S-transferase, alanine aminopeptidase, γ-glutamyl transpeptidase and alkaline phosphatase, α1-microglobulin and N-acetyl-beta-d-glucosaminidase are useful indicators of transplant tubular injury but have not proven to be of diagnostic value.30 Urinary cell levels of TGF-ß mRNA levels measured using real-time PCR were significantly higher in patients with CAI compared with those with stable renal function independent of proteinuria, hence urinary cells may be a good resource for the noninvasive diagnosis of CAI.³¹

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemotactic protein-1, recruits monocytes, memory T cells, and dendritic cells to the sites of tissue injury, infections, and inflammation. Urinary CCL2 was measured and protocol biopsies performed prospectively in 111 RT recipients at 0, 6, and 24 months, which demonstrated urinary CCL2 at 6 months as an independent risk factor for subsequent development of interstitial fibrosis and tubular atrophy (IFTA)at 24 months, both in univariate and multivariate analyses.³²

Proteomic analysis of blood samples, using mass spectrometry, has identified several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAI, which can be used for proteogenomics classification of CAI based on peripheral blood profiling, although validity remains to be proven.³³ In 2003 Scherer and associates, using microarray technology, detected upregulation of several genes, which could predict the development of CAI. Those genes were acidic protein rich in interleukins, opiate-binding protein–cell adhesion molecule-like, the tumur suppressor gene (nitrogen permease regulator 2-like protein [NPRL2]), cytokeratin 15, homeobox gene B7, prolactin receptor, and guanine nucleotide-binding protein g7.³⁴

More recently, Enicke and associates analyzed gene expression measured by microarray in RT biopsy specimens and showed that genes associated with graft failure were related to tissue injury, epithelial dedifferentiation, matrix remodelling, and TGF-β. The molecular risk score correlated with interstitial fibrosis, tubular atrophy, tubulitis, inter­stitial inflammation, proteinuria, and glomerular filtration rate and were predictors of graft loss.³⁵

Risk factors implicated in chronic allograft Damage index
Both antigen-dependent (immunologic) and antigen-independent (non-immunologic) factors, usually in combinations, are implicated in the cause of CAI (Table 1). Recurrent episodes of acute tubular-interstitial rejection can explain the IF and TA observed in some cases. Cytokines released during episodes of rejection including interleukin-1, fibroblast growth factor, and platelet derived growth factor likely play a role in promoting the fibroblast and smooth muscle proliferation seen in allograft vessels. In cases with previously documented intimal arteritis, vessel thickening can be explained as a direct result of immunologic vascular injury. Graft atherosclerosis leads to ischemic glomerulopathy.³⁶

Once glomerulosclerosis occurs, the remaining glomeruli undergo compensatory hypertrophy, in­creased glomerular capillary hydraulic pressure, and increased glomerular filtration. These hemo­dynamic forces damage the glomerular capillary endothelium, cause mesangial expansion, and accentuate the evolution of chronic transplant glomerulopathy.³⁶ In support of this hypothesis, it has been shown experimentally that if the increase in glomerular filtration rate is prevented by putting animals on a severely protein restricted diet, the rate of progression of glomerular sclerosis in allograft kidneys is retarded.³⁷ Arteriolosclerosis and interstitial fibrosis in the allograft also may occur as a result of hyper­tension, recurrent pyelonephritis, and chronic cyclosporine or tacrolimus toxicity. The relative contribution of these various processes to the ultimate loss of any given allograft may be difficult to determine by pathological evaluation alone. The etiologically noncommittal term chronic allograft nephropathy was in fact coined to accommodate this difficulty.³⁸

Theories of Pathogenesis of CAI
Chronic allograft injury represents the summated effects of tissue injury resulting from several pathogenic insults, and the healing response of the kidney to injury, in addition to the influence of alloimmunity and immunosuppression. The follow­ing hypotheses have been proposed to explain the pathogenesis of CAI.

Chronic antibody-mediated rejection
In the modern era with potent immunosuppressive agents, C-AMR remains the leading cause of CAI, which occurs in immunologically competent, nonadherent recipients or after reduction of immuno­suppressive agents for treatment of cancer or infection. Persistent low level of alloimmune activity manifests by cellular interstitial inflammation and fibrointimal hyperplasia; or with transplant glomerulopathy asso­ciated with circulating donor-specific antibody (DSA) and C4d in the peritubular capillaries.18 Antibody-mediated rejection is responsible for up to half of the acute rejection episodes in kidney transplant patients and more than half of late graft failures.

Despite a complete HLA match between the donor and recipient such as in HLA-identical siblings RT, acute and chronic rejection can occur, which are due to the presence of immune response to non-HLA antigens.39 Besides the major HLA, there are small endogenous peptides, known as the minor histo­compatibility antigen, which occupy the antigen-binding sites of major histocompatibility complex (MHC) molecules and are recognized by CD8+ T cells in the context of self -MHC leading to graft rejection. The H-Y minor histocompatibility antigen is encoded by the Y-chromosomes in men and can induce alloimmune response when a male organ is transplanted into a female recipient. MHC class 1 related chain A and B (MICA and MICB) are expressed in endothelial cells. Antibody-mediated rejections occurring in the absence of DSA directed against HLA on many occasions are due to antibodies directed against MICA and/or MICB leading to transplant loss.⁴⁰

Other antibodies such as antiangiotensin-2 receptor, antiglutathione S-transferase T1, and antiendothelial antibodies are identified to be involved in causing AMR. Antiendothelial antibody can be detected by using donor monocyte for crossmatch. Some minor transplant antigens may come from mitochondrial proteins and enzymes. In the future, several allo- and autoreactive antibodies are likely to be identified with the advancement of transplant immunology. ^41,42

Despite use of intensive treatment for C-AMR, outcomes have not always been promising. Recently, prevention, rather than treatment, of C-AMR has been attempted, and this approach appears to be a more effective option for reducing the incidence of C-AMR and, ultimately, improving long-term survival.⁴³

Input-stress model
The input-stress model is a composite model that describes the interaction between the starting input of the transplanted kidney and the subsequent posttransplant stress, which have been postulated to drive the cells from a normal state into a senescent phenotype, exhaust the repair process, and deplete the finite nephron supply leading to graft failure. The input variable includes the overall quality or condition of the organ, early events including pro­curement, preservation, and reimplantation injury. The posttransplant stresses include immune (cellular and antibody-mediated injuries) and non-immune stresses such as hypertension, hyperlipidemia, drug nephrotoxicity, infection, proteinuria, and hyper­filtration.¹¹

Cumulative damage hypothesis
This hypothesis is based on sequential observational pathology and assumes that CAI is the end result of a series of time-dependent immune and non-immune insults inflicted on the transplanted kidney, resulting in incremental loss of individual nephrons, combined with additional internal structural damage leading to transplant failure.²¹

Oxidative stress
Oxidative stress in the renal allograft is induced by ischemia-reperfusion injury, immunosuppressive drugs, and comorbid conditions such as diabetes, hypertension, proteinuria, anemia and dyslipidemia. Oxidative stress leads to production of reactive oxygen species (ROS), which include superoxide anion (O₂^•-), hydrogen peroxide, hydroxyl radicals, and peroxynitrite from mitochondrial electron transport system, peroxisomes, cytochrome p-450 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes. Oxidative stress causes cellular injury, apoptosis, epithelial-mesenchymal transition EMT and expression of a senescent phenotype. Studies have shown that interstitial inducible nitric oxide synthase protein expression, nitrotyrosine, and ex vivo ROS production are increased in CAI.^44,45 The balance between the ROS production and antioxidant defenses defines the degree of OS in any given tissue.⁴⁵

Injury to the tubular epithelium causes endo­plasmic reticulum (ER) stress and unfolded protein response, thereby releasing ROS leading to ischemia-reperfusion injury. The unfolded protein response initially serves as an adaptive response, but also will induce apoptosis in cells under severe or prolonged ER stress.⁴⁶

Cytokine excess theory
This theory postulates that CAI is due to acute and repeated tissue injury caused by excessive cytokine production, leading to interstitial and vascular fibrosis by transforming growth factor-β1 (TGF-β1). A role of other mediators, such as vascular endothelial growth factors, endothelin-1, plasminogen activating factor-1, monocyte chemoattractant protein-1, platelet-derived growth factor A and b, RANTES (regulated on activation, T-cell expressed), and advanced glycation end products are supported by their altered expression in experimental and human chronic transplant fibrosis.^47,48

Epithelial-mesenchymal transition induced fibrosis
Tubular epithelial cells of the kidney, with the exception of the distal collecting duct are derived from the fetal mesenchyme, which undergo tran­sition to an epithelial phenotype during embryonic development. Epithelial-mesenchymal transition is a mechanism for generating primitive mesenchymal cells during gastrulation or mobile tumour cells during cancer metastasis and is a principal stimulus for renal fibrosis.⁴⁹ Exposure of mature tubular epithelial cells to hypoxic injury, TGF-β1, or interleukin-1 is followed by a series of genetically programmed and orchestrated processes, including loss of cell-cell adhesion, E-cadherin expression, de novo α-smooth actin (α-SMA) expression, actin reorganization, tubular basement membrane disruption, cell migration, and myofibroblasts invasion with production of collagen type I and III, and fibronectin.⁵⁰

In a Fisher-to-Lewis rat transplant model, oxidative stress has been shown to be involved in the pathogenesis of EMT-associated CAI as evidenced by increased expression of superoxide ion (O₂^-), inducible nitric oxide synthase, and endothelial nitric oxide synthase.⁵¹ Likewise, phagocytic NADPH oxidase 2 and electron transfer subunits of NADPH oxidase (Nox) colocalized with α-SMA in areas of injury in the tubulointerstitium, suggesting EMT.⁵²

Features of EMT demonstrated in failing transplants with IFTA. Relative to implantation biopsies, IFTA kidneys showed loss of epithelial markers (E-cadherin, cytokeratin), expression of mesenchymal markers (vimentin, S100A4, α-SMA), and collagen synthesis marker (heat shock protein-47).⁵³

Replicative senescence
Cellular replicative senescence is the aging process occurring in normal cells, which leads to cellular exhaustion as seen in RTs from old donor kidneys. The “mitotic clock,” which limits cell division, is controlled in humans by telomeres, which are DNA repeats at the end of chromosomes that shorten with each mitotic division. Progressive shortening of the telomeres leads to senescent phenotype and mitotic arrest in its G1 phase. There is little evidence of shortening of telomeres on human CAI, although this is seen in native and transplanted older kidneys driven by oxidative stress and aging^54,55

Insertion/deletion polymorphism of angiotensin-converting enzyme
Angiotensin-converting enzyme is a major enzyme of the renin-angiotensin-aldosterone system, which exhibits genetic polymorphisms affecting its concentration in blood.⁵⁶ The insertion/deletion (I/D) polymorphism, located in intron 16, has been intensively studied in cardiovascular, native renal, and renal transplant dysfunction.⁵⁷ It confirmed that the deletion/deletion (DD) genotype is related to increase in angiotensin-converting enzyme con­centration, elevated enzyme activity, and increased angiotensin II concentration when compared with insertion/insertion (II) genotype heterozygotes. This has an important bearing on angiotensin-converting enzyme inhibitor therapy in patients with CAI. Renal transplant recipients who develop CAI are shown to be associated with DD genotype more frequently compared with RT recipients with normal renal function. Hence, presence of DD genotype can be considered as an indication for angiotensin-con­verting enzyme inhibitor in the prevention of CAI.⁵⁸

Acetylcholine and chronic allograft injury
Winczynska and associates have demonstrated in the Fisher-to Lewis rat model of CAI, a significant accumulation of leukocytes in the allograft blood vessels during acute rejection, which produce acetylcholine-inducing changes in the blood vessels, which are present in CAI. They demonstrated significantly increased expression of high-affinity choline transporter-1 and choline acetyltransferase in the leucocytes from allografts compared to the isografts. Treatment with rivastigmine, an acetyl­choline-esterase inhibitor, significantly exacerbated CAI compared with placebo, thereby indicating the contribution of acetylcholine to the pathogenesis of CAI and identifying a potential target for therapeutic intervention.⁵⁹

Management Strategies

Chronic allograft injury, once established, is irreversible.^8,21 Delaying the progression of renal fibrosis and preservation of allograft function should be the goal, which is being achieved through sub­stitution with less nephrotoxic immuno­suppressive agents and modification of risk factors, such as adequate control of hypertension, diabetes, hyper­lipidemia, proteinuria (angiotensin blockade), infections (Cytomegalovirus, BK virus, and urinary tract infections). Early diagnosis of CAI through protocol biopsies and institution of appropriate immunosuppressive regimens and treatment of subclinical rejection are essential for preventing late diagnosis of CAI.⁶⁰

To reduce drug toxicity, CNI minimization and steroid-sparing regimens have been shown to reduce the progression of CAI. Substitution of CNIs with sirolimus and mycophenolate mofetil, both in clinical and experimental settings, leads to improvement and preservation of renal function in CAI cases.^61,62 In the Efficacy Limiting Toxicity Elimination Symphony study, a regimen of daclizumab, mycophenolate mofetil, and corticosteroids in combination with low-dose tacrolimus was superior to regimens involving daclizumab induction plus either low-dose cyclo-sporine, low-dose sirolimus, or standard-dose cyclosporine without induction, in improving renal function, allograft survival, and acute rejection rates.^63,64

Calcineurin inhibitors reducing or sparing strategies have been shown to reduce premature graft loss. The Sirolimus Renal Conversion Trial (CONVERT), which examined the effects of converting from CNI to sirolimus as maintenance therapy in renal transplant recipients, revealed (particularly in the subgroup with a baseline GFR of > 40 mL/min and a urinary protein-to creatinine ratio of ≤ 0.11) superior renal function in patients treated with sirolimus for 12 to 24 months.⁶¹

The Phase III study Belatacept Evaluation of Nephroprotection and Efficacy as First line Immunosuppression Trial (BENEFIT) assessed a more intensive or less intensive regimen of belatacept, a costimulation blocker, versus cyclosporine in adults receiving a kidney transplant from living or standard criteria deceased donors. At 1 year, the belatacept group had superior graft function, but was associated with higher rate of early acute rejection and posttransplant lymphoproliferative disorders.65 At 3 and 5 years (BENEFIT-EXT trial), there was superior graft function in the belatacept group but with no difference in the rate of acute rejection, infections, malignancies, graft loss, and patient survival between the groups.^66,67

The role of alemtuzumab (a potent lymphocyte-depleting antibody) as an induction treatment followed by an early reduction in CNI and myco­phenolate exposure and steroid avoidance (3-C Trial in UK) after kidney transplant was examined by comparing with basiliximab-based induction treatment. The patients were randomly assigned to either alemtuzumab-based induction treatment (ie, alemtuzumab followed by low-dose tacrolimus and mycophenolate without steroids) or basiliximab-based induction treatment (basiliximab followed by standard-dose tacrolimus, mycophenolate, and prednisolone). Compared with standard basiliximab-based treatment, alemtuzumab-based induction therapy, followed by reduced CNI and myco­phenolate exposure and steroid avoidance, reduced the risk of biopsy-proven acute rejection in patients receiving a kidney transplant (7% vs 17%). There was no difference in the incidence of infections and graft loss.⁶⁸

In antibody-mediated rejection, to prevent C-AMR, removal of antibodies by plasmapheresis or immunoadsorption, inactivation of antibodies with intravenous immunoglobulins, and prevention of antibody production with anti-CD20 monoclonal antibody (Rituximab) has proven to be the most effective form of current desensitization protocols.⁶⁹ In refractory cases unresponsive to standard desen­sitization treatments, bortezomib, a proteasome inhibitor to eliminate plasma cells, and eculizumab, a monoclonal antibody against C5, have been successful in preventing and treating earlier stages of C-AMR.^70,71

Several interventional strategies have been examined to block the intracellular and extracellular cascades of events at molecular levels in both clinical and experimental settings to prevent CAI, but limited success has been achieved.⁷² Preventive and treatment strategies targeting TGF-ß1 signaling pathway are reasonable antifibrotic options in RT, but TGF-ß expression in RT is being considered to be beneficial because of its effect in gaining tolerance.⁷² More specifically pirfenidone and therapies targeting bone morphogenetic protein-7, hepatocyte growth factor and connective tissue growth factor, although having shown promising results, still are in the experimental phase.⁷³ Exploration of alternative pathways and downstream molecules are critical for developing new strategies to ameliorate graft fibrosis and atrophy. Clinical trials must examine their long-term effects in RT.^74,75

In summary, this paper has consolidated the body of knowledge on the evolution of the concepts and pathogenesis related to the development of CAI and highlighted the ongoing research in the prevention and treatment of CAI. Further research must gain insight into the pathogenesis and molecular biology of CAI for its successful prevention, progression, and treatment purposes.

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