Therapeutic apheresis in kidney transplantation: An updated review

Maurizio Salvadori,Aris Tsalouchos

Abstract

Key words: Kidney transplantation； Therapeutic plasma exchange； Double-filtration plasmapheresis； Immunoadsorption； Extracorporeal photopheresis； Desensitization；Antibody-mediated rejection； Focal segmental glomerulosclerosis

INTRODUCTION

Therapeutic apheresis (TA),from the Greek αφαιρ?,i.e.,remove,is a therapeutic method by which pathogenic blood components such as cells,harmful antibodies and inflammatory mediators causing morbidity,are separated and selectively removed,exchanged or modified by an extracorporeal apheresis system.The clinical applications of TA include renal diseases in native kidneys,metabolic diseases,autoimmune and rheumatic diseases,hematological diseases,neurological disorders,overdose and poisoning,and cover the field of solid organ transplantation[1].

TA techniques widely used in transplantation medicine,as an adjunctive therapeutic option include therapeutic plasma exchange (TPE) and selective TA techniques such as double-filtration plasmapheresis (DFPP),immunoadsorption (IA),and extracorporeal photopheresis (ECP)[1](Table 1).In the specific field of kidney transplantation (KT),TA is principally employed as an adjunctive therapeutic option to immunosuppressive agents in protocols both for preoperative procedures and during the posttransplant period in the clinical conditions reported in Table 2.

The objectives of this review are the description of technical characteristics,mechanisms of action,advantages,disadvantages,and complications of the TA techniques used in KT,and the rationale examination and evidence supporting the application of TA in treating clinical conditions in KT through the presentation of the current therapeutic protocols.

THERAPEUTIC PLASMA EXCHANGE

Mechanisms of action

TPE,through the removal and replacement of plasma,removes high-molecular-mass pathological substances (＞ 15000 Da) such as pathogenic antibodies,immune complexes,paraproteins,cytokines and adhesion molecules,and exogenous poisons[2].In some clinical conditions such as in thrombotic thrombocytopenic purpura (TTP),replacement with normal plasma is indicated to supply the deficient or missing plasma components[2].

However,evidence suggests that TPE also has immunomodulatory effects also.TPE has been associated with a variety of autoimmune diseases with a decline in B cells and natural killer (NK) cells,an increase in T cells,an increase in T suppressor cell function,and an increase in regulatory T cells (Tregs)[3-6].The immunomodulatory effects of TPE determine an increased susceptibility of cell-mediated and humoral immunity to immunosuppressive agents,and numerous therapeutic protocols integrate the administration of these agents with TPE to enhance their immunosuppressive effects.

The influence of TPE on the Th1/Th2 cytokine-producing-cell balance is controversial.Some studies suggest that TPE induces a shift of the Th1/Th2 balance in favor of Th2 differentiation and the suppression of the Th1 cytokines (IFN-γ and IL-2)[7,8]which evoke cell-mediated immunity and phagocyte-dependent inflammation[9].Conversely,other studies indicate that TPE is associated with a shift in cytokineproducing peripheral blood lymphocytes from a Th2 dominant pattern (IL-4,IL-6,IL-10),primarily involved in the humoral immune response,to a Th1 predominance[10,11].Accordingly,further studies are required to elucidate whether TPE contributes to the shift of Th1/Th2 balance and in what way.

Techniques of plasma removal: Centrifugation- vs filtration-based devices

TPE can be achieved by employing centrifugation- or filtration-based devices.Centrifugal TPE (cTPE) is an automated system designed to separate plasma from whole blood utilizing centrifugal force as the basis of operation[2,12].During treatment,blood is withdrawn from the patient and pumped through an extracorporeal circuit into a rapidly rotating centrifuge chamber,enabling a nonselective plasma separation and removal based on the density of the individual blood substances.The rest of the blood elements returns to the patient by intermittent or continuous flow mixed with a replacement fluid (RF),typically albumin or fresh frozen plasma (FFP),which is required to avoid hypotension[2,12].

Conventional membrane TPE (mTPE) uses highly permeable membranes,with pore sizes of 0.2-0.6 μm diameter,sufficient to separate plasma notselectively from the cellular blood components based on molecular size[13].The choice of RF depends essentially on the indication for TPE and patient clinical parameters,and does not differ between cTPE and mTPE[13].A head-to-head comparison of cTPE and mTPE provides a comparable treatment quality[14].However,mTPE devices are less effective at removing higher-molecular-mass proteins such as IgM and immune complexes[15].

Plasma removal efficiency (PRE； the percentage of plasma removedvsplasma processed) is much higher with cTPE than with mTPE.For each 1-1.5 plasma volume exchanged or 2.5-4.0 L,during a session,almost 60%-70% of the original plasma components will be removed with a cTPE device[16].When the procedure is extended beyond 1.5 plasma volumes,the amount of the removed plasma components decreases as large-molecular-mass substances are slowly equilibrated between their extra vascular and intravascular distribution[16].In mTPE,to avoid filter clotting and to prevent hemolysis due to high transmembrane pressure (TMP),the PRE is limited to 30%-35%[13].A consequence of this disparity in PRE is that mTPE devices need to process three or four times the patient's blood volume to obtain an equivalent reduction in the target molecule[17].As a result,procedure times lead to be longer and/or require higher blood flow rates (BFRs) on mTPE devices.

Choice of vascular access:To achieve higher BFRs,mTPE devices are almost all in need of a central venous catheter (CVC) that is able to maintain BFRs typically in the 150-200 mL/min range,while the lower BFR needed for a cTPE device (50 mL/min)can often be achieved through 17 gauge peripheral vein needles[17,18].Recently,an update of the World Apheresis Association (WAA) registry data showed more severe adverse events (AEs) in the procedures performed with a CVC[19].Common severe AEs of CVCs include central-line-associated bloodstream infections (CLABSIs),deep vein thrombosis (DVT),and arterial or venous bleeding[13,19].Nevertheless,mTPE with a CVC vascular access is the preferred technique in patients with renal failure who require hemodialysis and TPE as they can receive both treatments sequentially using the same dialysis machine.

Choice of anticoagulation:cTPE commonly uses regional citrate anticoagulation(RCA),which binds ionized calcium,a necessary cofactor in the coagulation cascade,to prevent clotting.Bleeding disorders are not common with RCA.However,citrate utilization is often complicated with systemic hypocalcemia (60%-70% of the overall complications during cTPE) resulting from intravascular citrate accumulation potentially leading to severe complications ranging from perioral and/or acral paresthesias to frank tetany and a QT prolongation of the electrocardiogram (ECG)with life-threatening arrhythmia requiring intravenous calcium replacement,often continuous infusion,with the return fluid[19-21].Hypocalcemia can be further exacerbated if the replacement fluid is FFP,which contains up to 14% citrate byvolume[12,13].

In mTPE,systemic anticoagulation with unfractionated heparin (UFH) is routinely used to maintain circuit patency,while citrate is not preferred because the higher BFRs,as well as the lower PRE,lead to a greater fraction of citrate being returned to the patient[13].During TPE,antithrombin III (AT III) levels decrease significantly,and heparin itself is filtered with a sieving coefficient (SC) of 1.As a consequence,in comparison to hemodialysis,in mTPE,higher doses of heparin may be required to achieve a clot-free circuit that in association with the bulk removal of plasma,which also involves the nonselective removal of clotting factors,results in a higher risk for bleeding[13].The risk of heparin-induced thrombocytopenia (HIT) type II is less frequent with low molecular weight heparin (LMWH) in comparison to UFH[18].

Additional differences between cTPE and mTPE are the increased risk of platelet(PLT) loss in centrifugal devices and the potential activation of complement and leukocytes on the artificial membrane described for mTPE[22,23].

SELECTIVE TA TECHNIQUES

Over time,selective TA techniques have been developed to avoid the removal of key plasma constituents that occur with conventional TPE by targeting a specific molecule,antibody,or cellular element[24].Below,we focus on selective TA techniques that find application in transplantation medicine.

Double filtration plasmapheresis

Double-filtration plasmapheresis (DFPP),or cascade filtration plasmapheresis,is a variation of mTPE,introduced in Japan by Agishiet al[25]in the 1980s,for desensitization in ABO-i KT,and over time it has been used for other indications.The circuit contains two plasma filters with different pore sizes,a primary membrane plasma separator to isolate the plasma,and then the plasma fractionator (PF),which is a high molecular-mass filter that removes target macromolecules based on molecular size and mass,primarily immunoglobulins (Ig)[23-25].The advantage of DFPP is that the PF allows smaller molecules,such as albumin,to pass through the membrane and return to the patient.This results to minimize,or potentially eliminate,the need for an RF and the associated complications,including allergic reaction and infection[23-25].A disadvantage of DFPP is that the performance of the PF is not sufficient to remove small-molecular-mass IgG and substances smaller than albumin[23-25].

Immunoadsorption

Immunoadsorption (IA) is a TA technique that enables the selective removal of humoral factors from separated plasma through a secondary device with high-affinity absorbers.The adsorption columns contain a specific ligand for the substance to be removed,and the depleted plasma is then returned to the patient[24].An advantage of IA is that RF is not required because the plasma volume remains the same and albumin is not adsorbed.Over time,different IA devices have been developed.

IA using immobilized antibodies:IA columns containing immobilized antibodies selectively bind a circulating molecule and remove it from the plasma[24].A Thera-SorbTM-Ig adsorber column,containing polyclonal sheep anti-human IgG antibodies immobilized on sepharose,has been shown to be effective in depleting all subclasses of IgG and has been used in ABO-i KT[26].IA using bound antibodies can also be applied for the depletion of preformed or newly synthesized cytotoxic antibodies in the rejection of allogeneic organ transplants[24].These columns are utilized in pairs,one working while the other is being regenerated with washing fluids,shifting periodically during the procedure.The online regeneration of the columns enables large volumes of plasma to be treated so that IgG extraction more efficient[24].Usually,up to two plasma volumes are processed during an Ig apheresis treatment.

IA using immobilized staphylococcal protein A:IA columns containing immobilized staphylococcal protein A (SPA),which has a high avidity for the Fc portions of IgG1,IgG2,and IgG4,have been used to deplete IgG auto antibodies or circulating immune complexes that contain IgG[24].Furthermore,SPA has been shown to be a B-cell super antigen[27].The interaction of SPA with peripheral B cells,expressing B cell receptors(BCRs) with VH regions capable of binding SPA,induces B cell apoptosis through the dissipation of mitochondrial membrane potential,the induction of the caspase pathway,and DNA fragmentation[27].Thus,the exposure of the patient's blood to SPA during IA may also trigger a beneficial immunosuppressive effect.The Immunosorba column,containing SPA bound to sepharose,has been used in acute AMR in KT and in highly sensitized patients waiting for KT[28-31].During a treatment,two absorbers work alternately.While one is adsorbing,the other is regenerated through the elution of bound antibodies,and vice versa.

IA using immobilized antigens and synthetic epitopes:IA columns containing immobilized antigens and synthetic epitopes are the most specific way to remove Ig as these columns are developed to extract only the antibodies that are reactive with that specific antigen,leaving untouched all other plasma components[24].

The Globaffin column is a regenerative twin adsorber system that utilizes the synthetic peptide GAM which covalently binds to an insoluble sepharose carrier matrix.Peptide GAM has a strong binding affinity,especially to the constant (Fc)section of subclass 1,2 and 4 IgG antibodies,and finds clinical application in different conditions,including acute AMR and perioperative Ig depletion,in sensitized renal transplant recipients[32].

The glycosorb ABO column contains synthetic terminal trisaccharide A/B blood group antigens covalently linked to a sepharose matrix and has been developed to remove A or anti-B antibodies in recipients of organ transplants from ABO-i donors[33].However,in a minority of patients,antibody elimination has been demonstrated to be incomplete with the glycosorb ABO column[34].The inadequate adsorption of corechain-dependent A/B antibodies may explain this finding[35],but further studies are needed.

ECP

ECP is a cell therapy procedure that begins with the separation of peripheral white blood cells (WBCs) and nonnucleated cells from plasma by centrifugation.Then,the isolated suspension of WBCs undergoes extracorporeal treatment with 8-methoxypsoralen (8-MOP) followed by exposure to ultraviolet A (UVA) light prior to reinfusion in the patient[36].The combination of 8-MOP and UVA results in the crosslinking of pyrimidine bases in DNA,leading to the apoptosis of lymphoid cells,largely T-cells and natural killer (NK) cells[37].Upon reinfusion,the phagocytosis of apoptotic lymphoid cells is performed by immature dendritic antigen-presenting cells(iDCs),which subsequently undergo maturation and present self-antigens in a protolerant signaling environment[38].The activated T cells differentiate into several cell lineages,particularly Tregs,which mediate a specific immunological tolerance by inducing anergy or apoptosis in self-reactive lymphocytes[38].

ECP was initially used in patients with cutaneous T-cell lymphoma (CTCL)[39].However,over the years,the indications for ECP have increased as it promotes antiinflammatory and tolerogenic responses without causing global immunosuppression[40].In solid organ transplantation,ECP has been successfully used to treat acute heart allograft rejection and chronic allograft dysfunction after lung transplantation[41,42].In addition,ECP was also used as a part of calcineurin inhibitor(CNI) sparing protocols to reduce drug side effects such as nephrotoxicity,and neurological or infectious complications[43].

In KT,there are only a few reports available on the use of ECP in recurrent or refractory acute rejection after the failure of standard immunosuppression and in antibody-mediated chronic rejection (AMCR),but they have encouraging preliminary results[44].Finally,ECP was also employed as a preventive treatment in a small case series with a favorable outcome: Rejection did not occur in any of the treated patients,and the authors described a notable increase in circulating Tregs[45].

INDICATIONS FOR TA IN KT

Desensitization in ABO-incompatible KT

ABO blood group incompatibility is the first and most significant immunological barrier to a successful transplantation and for a long time has been a contraindication to KT.Hyper acute rejection or AMR in nondesensitized ABO-incompatible (ABO-i)KTs occurs due to the presence of circulating preformed antibodies against the blood group antigens A and B (isohemagglutinins),which are strongly exposed on the surface of endothelial cells and kidney parenchymal cells[46].However,ABO-i KT was first attempted in the 1970s using A2 donors for recipients of blood groups O and B with only regular immunosuppression[47].This was possible because,compared to blood group A1 and blood group B individuals,the A2 antigen is less reactive with isohemagglutinins and is expressed in lower amounts on the surface of red blood cells and tissue cells[48].As experience increased,it became clear that low initial anti-A2 antibody titers in the recipient (IgG ≤ 1:2) were a requirement for the transplantations to be successful from an ABO A2 donor,significantly restricting the number of possible candidates[48-51].

To overcome the ABO barrier in KT and to increase donor pools,specific desensitization protocols have been refined to achieve a depletion of preformed antiA and/or antiB antibodies and the modulation of Bcell immunity[52].In this context,the use of TA techniques represents a cornerstone of current desensitization protocols.

In the early days of ABO-i KT,Alexandreet al[53]introduced an effective desensitization protocol based on plasmapheresis and splenectomy.Subsequently,splenectomy was progressively replaced by the anti-CD20 antibody rituximab (RTX)due to the surgical risk and increased risk of sepsis.Initially,RTX has been used in combination with DFPP and splenectomy in 2002[54],while the first report of the use of RTX instead splenectomy came from Karolinska University Hospital in 2003[55].In this protocol,in combination with RTX and conventional immunosuppression(tacrolimus,mycophenolate mofetil,and prednisolone),antigen-specific IA with a Glycosorb ABO column on pretransplant days - 6,- 5,- 4,and - 1[55].After transplantation,three more IA sessions were performed every third day.Moreover,if there was a significant increase in the antibody titers,more sessions were added[55].

In contrast to the Swedish protocol[55],Wilpertet al[56]adopted an on-demand strategy for postoperative IA.Instead of scheduling pre-emptive posttransplant IA,they submitted patients to IA if their antibody titers were higher than 1: 8 in the first postoperative week and higher than 1: 16 in the second postoperative week,without any additional risk for the patients[56].

Ishidaet al[57],in a retrospective cohort of 191 ABO-i KT recipients without postoperative administration of any prophylactic treatment for rejection,found no correlation between levels of antibody rebound and the incidence of AMR,even with antibody titers higher than 1: 64.The authors concluded that no treatment is necessary for rebounded anti-A/B antibodies as there is an immunological accommodation for elevated titers[57].In fact,immunological accommodation is established early (2 wk)after successful KT and could explain the resistance to AMR despite the rebound of anti-A/B antibodies in the recipient[58].The exact mechanisms of accommodation remain to be elucidated,although several have been proposed[59].Similar results have been reported by previous studies[34].

In contrast,a group from Johns Hopkins reported that the incidence of AMR was significantly higher in recipients with high postoperative titers (≥ 1:64),but the clinical significance was variable,as there was no consistent clinical correlation for AMR[60].The authors hypothesized that postoperative TPE could be helpful in preventing the rebound of anti-A/B titers until tolerance or accommodation occurs[60].

Consequently,the utility of postoperative antibody monitoring and prophylactic apheresis appears unclear and controversial.The transplant community should conduct larger studies with sufficient statistical power and with uniform and validated antibody titer measurements to find appropriate answers to this delicate issue.

Currently,cTPE is the preferred antibody removal strategy in the United States；membrane separation use is widespread in Japan,while IA is frequently practiced in Europe because of its safety and efficacy[58].

In many protocols,the number of pretransplant apheresis sessions is scheduled according to baseline antiA/B antibody titers[61,62].Typically,on the day of transplantation,the target for an antibody level is ≤ 1:8 regardless of the applied TA because higher levels have been correlated with a higher incidence of AMR[63].However,the choice of TA technique could also be scheduled according to baseline antibody titers.In fact,the Guy's Hospital ABO-i desensitization regimen introduced such a desensitization scheme tailored to initial antibody titers[64].In patients with baseline titers of ≤ 1:8,apheresis treatment was omitted,while RTX was not applied in patients with titers ＜ 1:16[64].DFPP was used in those with titers between 1:16 and 1:64 and antigen-specific IA (glycosorb-ABO IA columns) was used in those with titers above 1:64[64].The justification for the use of IA only for those patients with titers ＞1:64 was that these patients were expected to require the highest number of sessions,and DFPP is notably correlated with a higher risk of bleeding[65].Instead,DFPP was preferred in patients with titers between 1:16 and 1:64 because it is a less-expensive technique,and fewer cycles of antibody removal should not significantly alter coagulation parameters[64].The exact number of apheresis sessions depended on the course of the titers[64].In conclusion,tailoring the intensity of desensitization treatment according to individual immunological risk should be the recommended strategy.

Desensitization in patients with preformed HLA-antibodies

Preformed anti-HLA antibodies represent another major immunological barrier to a successful KT.Sensitization occurs when the transplant candidate develops immunological memory to the donor's antigens from prior transplants,blood transfusions,and pregnancies[66,67].Approximately 30% of the KT candidates have detectable anti-HLA antibodies and approximately half of them are “highly”sensitized with HLA antibody reactivity to over 80% of potential donors (panel reactivity antibody ≥ 80%)[68].

KT with donor-specific anti-HLA antibodies (DSAs) at pretransplant is known as HLA-incompatible transplantation.After transplantation,DSAs in high amounts cause hyperacute rejection,while in small amounts they reduce the survival of the graft by causing acute AMR and/or chronic humoral rejection[69,70].As such,highly sensitized candidates present difficulties in finding a cross-match-negative kidney,and waiting on the list for an acceptable match may be exhausted.According to Fuggleet al[71],sensitized candidates remain on the waiting list for a compatible donor kidney two to three times longer than nonsensitized KT candidates.The possibilities for the highly sensitized candidate that is waiting on the deceased-donor transplant list are higher after a desensitization protocol and even better in those with an available living donor.In this context,TA has a central role as an anti-humoral therapeutic strategy.

Desensitization of deceased donor kidney transplant recipients:Current desensitization protocols commonly use a combination of high-dose intravenous immunoglobulin (IVIG) and RTX to lower the titers of preformed HLA-antibodies in candidates on the waiting list and increase the chances of finding an acceptable deceased-donor[72].Moreover,TA (TPE or IA),if performed while on the waiting list,has historically been shown to reduce the long waiting times in highly sensitized candidates[29,73,74].Such strategies,however,are not always effective and may produce risks correlated with extended immunosuppression on dialysis.

Regarding the efficacy of HLA antibody reduction,in preventing hyperacute rejection,acute AMR and later transplant glomerulopathy,by peri-pretransplant TPE in deceased-donor KT (DDKT),the available data are limited[75-77].Beimleret al[75]reported for the first time a successful DDKT in two cross-match-positive recipients with a single peri-pretransplant TPE session and RTX.Cold ischemic time (CIT) due to the therapeutic protocol was not prolonged because TPE was performed during the transport of the kidneys from the donor center to the transplant center.After desensitization,the cross match turned negative,and TPE sessions were extended during the posttransplant period until stable allograft function was achieved to avoid an early rebound of DSAs[75].Both patients showed good graft outcomes two years after KT[75].Using the same desensitization protocol,the same group reported excellent short- and medium-term outcomes in a larger cohort of 12 DDKTs with positive cross matches,which turned negative after desensitization[76].Recently,a retrospective cohort study of DSA-positive recipients who received DDKT showed that a single peri-pretransplant TPE session,in combination with anti-human thymocyte globulin (ATG) as induction immunosuppression,did not result in a lower incidence of acute AMR within 6 mo in comparison with the DSA-positive recipients who did not receive a TPE session[77].Posttransplant TPE was not performed because the protocol included 3 to 5 d of ATG induction[77].

Loupyet al[78],from the Paris group,reported the results of a combined posttransplant prophylactic IVIg/RTX/TPE treatment in DDKT with preformed DSAs but a negative cross match on the day of transplant.The patients received 9 TPE sessions on an alternate-day basis at posttransplant plus IVIg 2 g/kg at days 0,2,42,and 63 and RTX on days 2 and 22.At 1-year posttransplant,patient and graft survival rates and the rate of acute AMR were comparable between the patients who received only IVIg and those who also received RTX and TPE.However,the estimated glomerular filtration rate (e-GFR) was significantly worse,and proteinuria was significantly higher in the IVIg group,as well as the rate of chronic AMR[78].These differences in long-term function were characterized by a significant decrease in the DSA mean intensity of fluorescence (MFI),as detected with the Luminex solid phase immunoassay,in the group of patients receiving the more intensive post transplant prophylactic regimen in comparison with the IVIg group[78].Recently,the Paris group reported the long-term results of a high immunological risk program including patients with high peak DSA levels (MFI ＞ 3000) and a negative cross match at transplantation day who received a posttransplant desensitization protocol with highdose IVIg,TPE and RTX.The results were compared to a control group including patients with a lower immunological risk (MFI between 500 and 3000) on transplantation day and in whom posttransplant desensitization was based on IVIg alone[79].Patient survival was the same between the two groups.However,there were significantly more cases of acute T-cell rejection and AMR in the group with MFI ＞3000,which clinically translated into significantly lower graft survival[79].

IA,aimed at preventing humoral graft injury,has also been used with mixed results.The Vienna transplantation center reported a favorable allograft outcome in a series of highly sensitized kidney transplant recipients after a peri-pretransplant IA session with a staphylococcal protein A column supplemented by repeat posttransplant treatment[80].Subsequently,the same group described that a single peripretransplant IA,in addition to pre-emptive ATG,can turn a positive cross match into a negative cross match,enabling a successful DDKT supported by a favorable longterm graft survival at 3 years[81].The authors confirmed these data by extending their initial experience in a later paper[82].Repeated posttransplant IA sessions have been performed in this protocol to prevent a potentially harmful rebound of DSAs[81,82].In line with the Vienna group,Higginset al[83],in a previous study,reported a cohort with a successful cross-match conversion and prevention of hyper-acute rejection by peri-pretransplant IA treatment.However,in this case,a considerably high graft loss rate was observed during follow-up,with only 54% of transplants surviving after a median follow-up of 26 mo[83].The difference in the outcome between these studies could be explained by the significant differences between the desensitization protocols.Unlike the Vienna group[81,82],Higginset al[83]did not repeat post-transplant IA sessions.In addition,the Vienna group[81,82],to obviate an exaggerated increase of CIT,excluded transplantation for patients in whom a negative cross match could not be obtained by treatment with 6 L of plasma,while Higginset al[83]in some patients prescribed more than 30 L plasma volume to convert a positive cross match,which resulted in significant increases in CIT (up to 62 h).However,the Vienna group recently reported that one-third of 101 DSA-positive recipients of DDKT underwent intense IA-based desensitization and experienced acute AMR and that DSA MFI levels were significantly associated with acute rejection (20vs71% AMR rates at ＜5000vs＞ 15000 peak DSA MFI)[84].The 3-year graft-survival rate in DSA-positive recipients was significantly lower than that of the DSA-negative recipients (79%vs88%；P= 0.008)[84].

These data highlight that MFI levels have significant prognostic value and suggest that the intensification of TA treatment in posttransplant desensitization protocols must be personalized according to MFI levels.

Desensitization of living-donor kidney transplant recipients:For sensitized candidates with an available but incompatible living donor,paired donor exchange(PDE) is the best alternative option.However,for most highly sensitized candidates,the chance of finding a match in the relatively small pools of donors in PDE programs is reduced,and desensitization alone or desensitization in combination with PDE present almost the only viable option for transplantation[85].HLA-incompatible desensitized living-donor KT (LDKT)vsHLA-compatible LDKT has significantly lower graft survival[86].Multicenter study results indicate,however,that it is worth desensitizing HLA-incompatible patients who have a potential living donor,as after KT these patients have significantly better long-term survival than highly sensitized candidates on a KT waiting list who did not receive a kidney from a deceased donor[87-89].

TA has a central role in current desensitization protocols.The most commonly used protocol is a combination of alternate-day TPE followed by low-dose IVIg (100-150 mg/kg) prior to transplantation[87-91].Most transplant centers also initiate antirejection medications,tacrolimus,and mycophenolate mofetil (MMF),up to 2 wk prior to surgery[92].Montgomeryet al[87],in the largest series of HLA desensitization based on TPE plus low-dose IVIg,at the 5-year follow-up,showed a significantly greater survival in patients who received LDKT (90.6%) than in those who remained on dialysis (51.5%) or in those placed on a DDKT wait list with or without KT (65.6%).On average,patients received 4 ± 4 TPE treatments before LDKT and 5 ± 4 TPE treatments after LDKT[87].More recently,Orandiet al[88],in a larger multicenter (n= 22)United States study that involved 1025 patients,validated the results from the Baltimore group[87].Glooret al[93],to overcome a positive cross match in 14 LDKT recipients added RTX and splenectomy to the protocol TPE/low-dose IVIg in an attempt to decrease the high AMR rate.

However,a 43% AMR rate was detected,while the patient and graft survival rates were 86% and 78%,respectively at 15 mo.Mageeet al[94]reported their experience with TPE/low-dose IVIG plus RTX in 28 cross-match-positive patients.The AMR rate was high (39%),but within a mean follow-up of 22 mo,the mean serum creatinine level was good (1.5 mg/dL),and only 3 grafts were lost.Similar results,applying TPE/low-dose IVIg plus RTX,have been reported by the University of Illinois in 51 transplanted patients[95].The acute rejection rate was 33%,with optimal graft survival at 2 years (93%).

Morathet al[96]examined the effect of adding one dose of RTX (375 mg/m2) just prior to KT with IA performed before and after transplantation.After a median of 10 IA treatments,all ten patients were desensitized successfully and transplanted.The recipients also received a median of 7 posttransplant IA treatments.After a median follow-up of 19 mo,the reversible AMR rate was 30%,and the patient and allograft survival rates were 100% and 90%,respectively,with a mean serum creatinine level of 1.6 mg/dL[96].Similar results with RTX plus IA have been reported recently by Kaukeet al[97]on a small series of 8 LDKT recipients.Kleinet al[98],on a series of 23 sensitized patients,performed pretransplant IA sessions plus tacrolimus,MMF,and steroids,with the goal of achieving an MFI ＜ 1000 on transplantation day.On days 0 and 1,recipients also received one dose of RTX.The induction therapy was based on either ATG or basiliximab,and IA sessions were maintained posttransplantation until serum creatinine became ＜ 2 mg/dL and MFI was stable at ＜ 1000.This desensitization protocol showed excellent results at the 2-year follow-up,with a graft survival rate of 100% and a median serum creatinine level of 1.42 mg/dL[98].To allow LDKT in 6 highly sensitized patients,Rostainget al[99]performed an IA-based desensitization protocol plus IVIg,RTX,and ATG as induction therapy.This protocol effectively reduced or eliminated DSAs in 71% of recipients at the time of transplant.Three recipients manifested an AMR,but long-term renal function was good.

Woodleet al[100]in an alternative protocol incorporating TPE,the proteasome inhibitor bortezomib,and RTX,showed a significant decrease in DSAs in both LDKT and DDKT with successful transplantation in 19 of 44 highly sensitized patients and low acute rejection rates (18.8%) at 6 mo.

In a recent review,Malvezziet al[101]proposed an algorithm based on MFI pretransplant levels for the use of the various TA techniques in desensitization protocols.In their experience,the authors suggest that the use of TPE should be restricted in cases where the highest pretransplant MFI is ≤ 9000.In such circumstances,TPE should be delivered on a daily basis until MFI becomes ≤ 3000.MFI must be assessed after every 5 sessions.If the MFI of the DSA is ＞ 9000 but below 13000,DFPP can be implemented on a daily basis.When the target of MFI ＜ 9000 is reached,DFPP can be converted to TPE.In the event that MFI is ＞ 12000 before starting desensitization,IA has to be applied on a daily basis.When the MFI is reduced (i.e.,＜ 6000),IA can be replaced by DFPP or TPE to obtain an MFI threshold of about 3000.The authors conclude that in all of these scenarios,as soon as MFI is reduced to ＜ 3000,KT can be performed as in this case DSA strength is low.In our opinion,based on current studies,the best strategy is to apply TA,preferably IA,plus RTX until MFI becomes ＜ 3000.The addition of IVIG might also be relevant in this setting.

Antibody-mediated rejection

Antibody-mediated rejection (AMR) is a severe complication after KT with potentially deleterious effects on graft survival.Currently,AMR is widely recognized as a continuous process with varying degrees of activity and damage,clinically and histologically,expressed with multiple phenotypes,now identified as acute AMR,subclinical AMR,and chronic AMR[102,103].

Despite the use of desensitization protocols,up to one-third of highly sensitized recipients may develop AMR following transplantation[104,105].Hence,the ability to successfully deliver incompatible transplants and optimize long-term results is contingent on the ability to successfully approach and manage an AMR.AMR is also of significant burden in non-sensitized individuals,asde novoDSA (dnDSA) can emerge early or late following KT[106].

Early acute AMR can be severe and result in graft loss,but it is also potentially responsive to current treatments[103].Instead,late acute AMR (more than 6 months posttransplant),can be a mixed cellular and humoral rejection,and it is often nonresponsive to current treatments,such as chronic AMR and,in some cases,subclinical AMR.Late acute and chronic AMR may result from dnDSA formation,the incomplete elimination of DSA following an earlier acute AMR episode,or the persistence of preformed DSA after desensitization[103].TA,as an adjunctive therapeutic option,has a central role in the treatment of AMR.

TA and IVIG:When acute AMR occurs,TPE or IA plus IVIG and increased immunosuppression is considered the current standard of care (SOC) treatment,as it can be used to decrease antibody levels and arrest the rejection process in the majority of patients[103].

In a recent meta-analysis,Wanet al[107],regarding graft survival after antibody removal with TPE or IA,based on 5 RCTs,showed no benefit in the trials with a shorter follow-up (1-7 mo)[108,109],while those with a longer follow-up (2-5 years)showed a trend towards a benefit[28,110,111].

In a recent retrospective cohort study investigating TPE plus IVIG in late AMR,with approximately 50% of patients having chronic histology lesions,Leeet al[112]showed an improvement of graft survival in the intervention group compared to the control group who did not receive any therapy,in a mean follow-up of 7 years.In contrast,Eineckeet al[113]observed no effect on graft survival after treatment with TPE plus IVIG in late AMR,with approximately 63% of patients having chronic histology lesions.

In conclusion,based on current data,the basis of establishing TPE plus IVIG as SOC treatment in AMR is lacking strong evidence,and a high-quality RCT with sufficient power to evaluate the efficacy of this treatment would provide reassurance on this delicate topic.However,it is extremely improbable that such a trial will be conducted due to the ethical perplexity of enrolling patients to a no-treatment group,which is historically related to high risks of graft failure.

Add-on treatments to TA and IVIG:Different add-on treatments in the current SOC treatment have been proposed over time per transplant center preference[103,107].

The use of RTX in acute AMR showed promising results in several small retrospective series[114,115].In the first controlled trial using RTX plus TPE/IVIGvsIVIG alone,Lefaucheuret al[116]concluded that high-dose IVIG is inferior to combination therapy.However,in this trial,it was impracticable to determine which of RTX or TPE led to the improvement[116].

In addition,2 retrospective cohort studies compared RTX plus TPE/IVIG to TPE/IVIG or IVIG alone,and both showed an improvement in graft survival in the RTX group[117,118].The patients in the RTX group,however,received a higher dose of TPE and IVIG,limiting the ability to make a direct comparison between groups.

In a small multicenter double-blind RCT comparing RTX plus TPE/IVIG to placebo plus TPE/IVIG for the treatment of acute AMR,Sautenetet al[119]showed no additional benefit from RTX in graft survival after 1 year.However,the 1-year followup period may not have been long enough to identify a difference in graft survival.Recently,Oblaket al[120],with the limitations that a retrospective cohort study can provide,confirmed no evidence of any benefit in adding RTX to SOC treatment for AMR in a longer follow-up period (2 years).

Bortezomib,a proteasome inhibitor,in several nonrandomized retrospective studies and case reports,showed benefit to treat acute AMR in combination with TPE and IVIG[121,122]or TPE and RTX[123],while other studies have shown no improvement in e-GFR after bortezomib when used as add-on therapy with TPE and IVIG for late AMR[124].

The single RCT comparing the use of bortezomib,in patients with mixed AMR and acute cellular rejection,in conjunction with TPE and ATGvsTPE,RTX,and ATG or TPE and ATG alone,showed no difference in graft survival between the 3 groups[125].

The complement inhibitors eculizumab,a humanized monoclonal IgG antibody that binds to complement protein C5 and inhibits the formation of MAC,and C1-INH,a serine protease inhibitor that inactivates both C1r and C1s,inhibiting in this way the first step of the complement cascade,have also been evaluated in combination with TPE and IVIG in the treatment of AMR.

Lockeet al[126]reported the first case report on the use of eculizumab in combination with TPE and IVIG to treat severe AMR,demonstrating a reversal of the AMR episode.In a study of 24 patients who developed severe oliguric AMR after HLAincompatible LDKT,Orandiet al[127]showed that a combination of splenectomy plus eculizumab and RTX as an add-on therapy to TPE/IVIG resulted in an effective intervention for rescuing and preserving allograft function in comparison with splenectomy alone or eculizumab alone as an add-on therapy.

In an RCT in which 18 patients with acute AMR were assigned to C1-INH (Cinryze)plus TPE/IVIG or placebo plus TPE/IVIG,Montgomeryet al[128]showed less transplant glomerulopathy at 6 months in the C1-INH group.A multicenter phase III RCT (NCT02547220) evaluating C1-INH as an add-on therapy to TPE/IVIG or IA/IVIG has just concluded,and we are waiting for the results to be published.

In conclusion,various add-on treatment options are employed for the current SOC treatment based on their targets in the steps of AMR pathogenesis with different results.Future RCTs should assess definitive endpoints,and until then,the regimen to be used should be considered on a case-by-case basis.

ECP: There are only a few reports available on the use of ECP in chronic AMR.Sunder-Plassmanet al[129]employing intensive and long term ECP treatments (2 consecutive procedures every 2 wk for 17 cycles),showed a benefit in treating a single patient with chronic rejection.Dall'Amicoet al[130]reported progressive improvement in renal function and consecutive biopsy specimens during the course of ECP in treating one patient with chronic rejection.In contrast,Horinaet al[131]showed no response in treating two patients with two consecutive ECP procedures per month for 3 mo.Further experience on the usefulness of ECP in AMR is required.

Recurrence of primary focal segmental glomerulosclerosis

Approximately 30% of cases of primary focal segmental glomerulosclerosis (FSGS)recur after first KT and are associated with early graft loss in up to 50% of patients[132].The prediction of recurrence is even higher than 75% in subsequent grafts when the first graft has been lost because of recurrence[133].

Primary FSGS seems to be induced by a circulating factor that targets podocytes.Several candidates have been suggested,although until now,the specific factor(s)involved remain unknown[134].Recently,Delvilleet al[135]identified a panel of seven antibodies (CD40,PTPRO,CGB5,FAS,P2RY11,SNRPB2,and APOL2) that predict posttransplant FSGS recurrence with 92% accuracy.The pretransplant elevation of anti-CD40 antibody alone had the best correlation (78% accuracy) with recurrence of FSGS after transplantation[135].In addition,anti-CD40 antibodies purified from patients with FSGS recurrence have been proven to be particularly pathogenic in human podocyte cultures[135].

TPE or IA with either a protein A or anti-IgG column have been used with benefit,alone or in combination with cyclophosphamide,with the scope to remove the putative circulating permeability factor[136-140].Dantalet al[140]showed that the administration to rats of material eluted from protein A columns from patients with disease recurrence after KT increased the urinary albumin excretion.

In a literature review,Ponticelli[141]reported that approximately 70% of children and 63% of adults with recurrent FSGS receiving TPE or IA achieved complete or partial remission of proteinuria.Similar data have been reported in two recent metaanalyses[142,143].

The duration and frequency of TPE sessions are not yet unanimously agreed upon.A typical TPE regimen is 1.5 plasma volume exchanges for three consecutive days and then every other day for a total of two weeks[132].

TPE has also been used as an adjunctive treatment to other immunosuppressive agents.Canaudet al[144],in a series of 10 patients,reported good results by combining intravenous cyclosporine with high-dose steroids,mycophenolate,and frequent TPE sessions slowly tapered down for nine months.

In the last ten years,the use of RTX in recurrent FSGS has rapidly expanded with beneficial effects[145,146].In addition to being a selective depleting agent of Blymphocytes,RTX seems to have a direct protective effect on podocytes.RTX is able to protect sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) and acid sphingomyelinase (ASMase) by binding to SMPDL-3b,a protein exposed in podocyte lipid rafts that may be the target of the permeability factor of FSGS and that displays a sequence identified by RTX[147,148].RTX,in combination with TPE,seems to have better efficacy,as suggested by case reports[149,150].Other immunosuppressive agents,such as abatacept and antiTNFα agents,have shown prominent results in recurrent FSGS[151,152],but the experience of these agents in combination with TPE is inexistent.

Other indications of TA in KT

Complement-mediated atypical hemolytic uremic syndrome:Complementmediated atypical hemolytic uremic syndrome (aHUS) is a rare disease that results from genetically determined complement deregulation with an alternative pathway of activity secondary to either loss-of-function mutations in regulators [factor H,factor I,and membrane cofactor protein (MCP)] or gain-of-function mutations in activators(C3 and factor B) of the alternative pathway[153].In addition,complement-mediated aHUS may result from autoimmune mechanisms,including the development of auto antibodies to complement proteins[153].Mutations in factors H,factor I,factor B,and C3 have a high risk of recurrence (75%),and more than 90% of those with recurrence are strongly associated with graft failure,typically within the first year,because the altered proteins persist in the blood after KT[154].In contrast,mutations of MCP are associated with a recurrence rate of only 20% and considerably more favorable graft survival rates because kidney transplants express normal proteins[155].

TPE can remove auto-antibodies against complement proteins or mutated circulating complement regulators while replacing absent or defective complement regulators and has been used in regimens for the prevention of recurrence,prior KT,and the recurrence of complement-mediated aHUS posttransplantation with relatively poor response to treatment[156].The introduction of eculizumab,an anti-C5 monoclonal antibody,has favorably changed the outcomes and challenged the role of TPE in the treatment of aHUS.

The added therapeutic benefits ofTPE in a pre-emptive prophylactic protocol with eculizumab prior to KT,used by some centers[157],remain unclear and questionable.TPE remains an alternative therapeutic option only when eculizumab is not available in patients with anti-complement factor H antibodies and when thrombocytopenia is still present during the first days of eculizumab administration[158,159].

De novo thrombotic microangiopathy:De novothrombotic microangiopathy (TMA)after KT may be due to any of the etiologies that induce TMA in the general population.However,the most common causes of TMA among kidney transplant recipients include drug-induced TMA due to calcineurin inhibitors and mammalian target of rapamycin (mTOR) inhibitors,ischemia reperfusion injury,AMR,and viral infections[160].

If switching to a different immunosuppressive regimen or if the treatment of underlying infection does not lead to a resolution of signs and symptoms of TMA and there is a clinical deterioration,TPE can be attempted to improve the course of the disease and subsequent graft damage[161],although the level of evidence is low.If available,eculizumab is the treatment of choice in these cases[162,163].

In AMR-associated TMA,improved outcomes have been reported with TPE and IVIG therapy[164].Eculizumab is the recommended treatment in AMR-associated TMA if hemolysis persists despite maximal management with TPE and in those with TPE dependency[160].

Antiphospholipid syndrome and systemic lupus erythematosus:The antiphospholipid syndrome (APS) is a multisystem autoimmune disorder characterized clinically by thrombotic episodes in the arterial or venous circulation,and serologically by the persistent evidence of antiphospholipid antibodies (aPL).APS occurs either as a primary condition or secondary in the setting of an underlying systemic autoimmune disease,mainly systemic lupus erythematosus (SLE)[165].The kidney is one of the organs that can be compromised by occlusion of a broad spectrum of renal blood vessels,ranging from glomerular capillaries to the main renal artery and vein[165].

Early graft arterial or venous thrombosis,or TMA,remains the most frequent cause of renal graft failure in patients with APS[166].In addition,several studies have found that patients on maintenance hemodialysis,and consequently a substantial number of renal transplant recipients have a high prevalence of circulating aPL,which can damage the allograft[167,168].Treatment of APS with long-term warfarin for arterial or venous thrombosis is recommended after renal transplantation and most transplant nephrologists prefer to inhibit the coagulation system in all patients with aPL and a history of coagulation events during the peritransplant period[169,170].However,anticoagulation therapy increases the risk of bleeding complications,which may lead to early graft loss,and graft thrombosis takes place in 40% of the APS population despite anticoagulant therapy[171].

Prophylaxis with TPE for antibody removal,in addition to full anticoagulation therapy,before living-donor KT has been reported effective in one patient with primary APS[172]and in one patient with secondary APS in the setting of SLE[173].However,in case of catastrophic APS (CAPS),which is characterized by diffuse TMA(vascular occlusions involving three or more organ systems)[174],prophylactic administration of eculizumab to prevent recurrence of CAPS after KT should be considered the preferred therapeutic option as have been used with success in one patient together with continuous systemic anticoagulation and standard immunosuppression[175].

Barbouret al[166]reported a case of acute recurrence of TMA after KT,in a patient with APS and lupus nephritis successfully treated with TPE albeit with some irreversible graft damage and renal impairment.These results suggest that further studies are warranted.

Recurrent and de novo anti-glomerular basement membrane disease:The histological recurrence of anti-glomerular basement membrane disease (GBM) may be as high as 50% in patients who receive a transplant while circulating anti-GBM antibodies persist[176,177].However,there are only a limited number of documented cases of symptomatically recurrent anti-GBM disease,as most patients are asymptomatic[176].

De novoanti-GBM disease is seen in up to 15% of transplant recipients with Alport syndrome who develop anti-GBM antibodies to a collagen component [alpha5 (IV)NC1] carried by the transplanted kidney that is lacking in Alport patients[178].The approach to the treatment is the same as in the native kidneys.TA should be used promptly to remove the causative antibody plus glucocorticoids and cyclophosphamide to inhibit further autoantibody production[177].IA and TPE have comparable outcomes[179,180]

Recurrence of antineutrophil cytoplasmic antibody-associated vasculitis:The relapse of antineutrophil cytoplasmic antibody-associated vasculitis in KT patients is a rare event.In a recent review of 11 studies,including 441 patients,the relapse rate was 10%[181].

In the case of a recurrence,the treatment options for remission induction are similar to those of nontransplanted patients.Both cyclophosphamide- and RTX- based induction regimens have shown effectiveness in the treatment of posttransplant relapses[182].

TPE is recommended,in conjunction with glucocorticoids and either cyclophosphamide or RTX in the setting of relapse manifesting as alveolar hemorrhage,severe segmental necrotizing glomerulonephritis with serum creatinine above 4.0 mg/dL,and concurrent anti-GBM disease[182-184].

CONCLUSION

The application of TA in KT is currently a cornerstone of therapy for several clinical conditions,such as in desensitization protocols for ABO-i KT and in patients with preformed HLA-antibodies,in the treatment of AMR,and with the recurrence of different glomerulopathies after KT as in recurrent primary FSGS.However,strong evidence is scarce,and more clinical researches,with a high standard of quality RCTs,are demanded to establish the use of each TA method for the clinical problems that occur in KT.

In addition,in the era of new and emerging biological immunosuppressive therapies with an increasing number of specific actions and immune targets directed against cell-surface antigens or plasma-soluble molecules,the use of TA,and the optimal timing and dose,as an adjunctive therapeutic option becomes challenging in the study of future therapeutic protocols,which will best address open issues for better clinical outcomes.