|Year : 2018 | Volume
| Issue : 1 | Page : 53-58
Assessment of renal function and acute rejection using Cystatin C and kidney injury Molecule-1 in renal transplant recipients
Sailaja Kesiraju1, Lakshmi Kiran Chelluri2, Sumanlatha Gaddam3, Vijaya Lakshmi Valluri3, Uma Maheswara Rao Ch4, Sahariah Sarbeswar5
1 Transimmun-Transplantation Immunology and Research Centre; Bhagwan Mahavir Medical Research Center, Hyderabad, Telangana, India
2 Department of Transplant Immunology and Stem Cell Unit, Global Hospitals, Hyderabad, Telangana, India
3 Bhagwan Mahavir Medical Research Center, Hyderabad, Telangana, India
4 Department of Nephrology and Transplantation, Krishna Institute of Medical Sciences, Hyderabad, Telangana, India
5 Transimmun-Transplantation Immunology and Research Centre; Department of Nephrology and Transplantation, Krishna Institute of Medical Sciences, Hyderabad, Telangana, India
|Date of Web Publication||29-Mar-2018|
Dr. Sailaja Kesiraju
Transimmun-Transplantation Immunology and Research Centre, Somajiguda, Hyderabad, Telangana; Bhagwan Mahavir Medical Research Center, Bhagwan Mahavir Marg, A.C. Guards, Hyderabad, Telangana
Source of Support: None, Conflict of Interest: None
Purpose: Kidney injury molecule (KIM)-1, a transmembrane-tubular protein, is excreted in the urine within 12 h of the initial ischemic insult and before regeneration of the epithelium. Cystatin C (CysC) a low-molecular-weight nonglycosylated protein has been shown to be a good marker of kidney function. We aimed to evaluate the levels of KIM-1 and CysC immediately after transplantation as an early marker. Subjects and Methods: Urine and blood samples were collected from prospective renal transplant patients with chronic allograft dysfunction (CAD) at baseline and during the follow-up period at the interval of 12, 24 h, 7th postoperative day 15th and 6 m, and compared with healthy controls (HC) for KIM-1 CysC S.creatinine (SCr) and creatinine clearance. Results: Kidney transplant recipients showed significantly higher KIM-1and CysC values than the control group. Nonparametric receiver-operating characteristic (ROC) curve of the renal function with an area under the curve of 0.518 KIM-1, CysC was 0.841 and creatinine 0.74 indicating CysC at 12 h posttransplant (post-Tx) is a better biomarker among three. ROC of acute rejection (AR) of KIM-1 at 24 h post-Tx showed sensitivity of 0.938. ROC for distinguishing between graft survival and failure at 1 year showed a sensitivity of 0.763 for CysC, In CAD, both KIM-1and CysC were increased as compared to HC. Conclusion: Both KIM-1 and CysC are useful markers for predicting AR, renal function. Elevated urinary levels of KIM-1 independently predict AR. CysC is a valuable marker of predicting the long-term outcome.
Keywords: Biomarker, cystatin C, kidney injury molecule-1, rejection, renal function
|How to cite this article:|
Kesiraju S, Chelluri LK, Gaddam S, Valluri VL, Rao Ch UM, Sarbeswar S. Assessment of renal function and acute rejection using Cystatin C and kidney injury Molecule-1 in renal transplant recipients. Indian J Transplant 2018;12:53-8
|How to cite this URL:|
Kesiraju S, Chelluri LK, Gaddam S, Valluri VL, Rao Ch UM, Sarbeswar S. Assessment of renal function and acute rejection using Cystatin C and kidney injury Molecule-1 in renal transplant recipients. Indian J Transplant [serial online] 2018 [cited 2019 Jul 21];12:53-8. Available from: http://www.ijtonline.in/text.asp?2018/12/1/53/228924
| Introduction|| |
The rate of acute rejections (ARs) and chronic allograft dysfunction (CAD) are the major determinants of the long-term outcome in the solid organ transplantation. The underlying mechanisms are complex and include epithelial and endothelial damage due to tubular occlusion, impairment of microvascular flow, immunological responses, immune suppressive medication, and inflammation processes. It was found that endothelin-mediated vasospasm may be responsible for posttransplant (post-Tx) renal injury. It is also important to note that the role of proximal and distal tubules in acute kidney injury (AKI) is unclear. Early detection and intervention may help in improving the graft function. S.creatinine (SCr) – a commonly used “gold standard” marker for the assessment of renal function is affected by muscle mass, age, and gender. It has a long halflife and a large volume of distribution, preventing it from accurate measurement of renal injury and function. Many low-molecular-weight proteins, namely, retinol binding protein, β2 – microglobulin, alpha1-microglobulin, serum neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule (KIM)-1, and cystatin C (CysC) have been intensely investigated as possible biomarkers for predicting tubular injury., Many studies were conducted on the measurement of urinary tubular enzymes such as alanine aminopeptidase, N-acetyl-beta-glucosaminidase, alpha-glutathione s-transferase including proximal renal tubular epithelial antigen and found their incapability in distinguishing cause of injury.
Biomarkers of renal damage can be differentiated into markers of renal function and markers of renal injury. CysC a low-molecular-weight protein with a constant rate of production by nucleated cells and is not affected by age gender and muscle mass. It is freely filtered at the glomerulus and completely reabsorbed in the proximal tubule. Its presence in the urine reflects the impairment of protein uptake by the proximal tubules.. It has been observed that s CysC differentiates an abnormal glomerular filtration rate (GFR) more sensitively than SCr in stable transplant patients.. Earlier studies investigated potential biomarkers for the diagnosis of AKI and one among them was KIM., It is a transmembrane protein containing an Ig-like and mucin-like domain. It clears the debris from the damaged renal tubules playing a phagocytic process in activated epithelial renal tubules. It can be excreted in the urine immediately after the ischemic insult and before the regeneration of the epithelium. Urinary KIM-1 has been reported to be a noninvasive, rapid, sensitive, and reproducible biomarker of experimental nephrotoxic and ischemic AKI. It has been noted that urinary interleukin-18 and NGAL correlate well with the graft outcome, but in the immediate transplant period when intervention may not be helpful.
Thus, it is very important to identify a biomarker that predicts early graft dysfunction as well as the tubular injury which will help in the graft management and therapeutic interventions. We hypothesized that detecting early signs of rejection may enable early intervention leading to a better outcome and also biomarkers such as CysC and KIM-1 would help in distinguishing the renal tubular injury and predicting the long-term outcome. Hence, we aimed to assess whether sequential measurements of KIM-1, creatinine, and CysC in pretransplant and immediate post-Tx period could be used as an early biomarker in predicting the long-term outcome, AR episodes and also in detecting the renal tubular and epithelial cell injury when the morphological changes are minimal. In the present study, we enrolled 68 subjects between 2011 and 2012 and followed up them for 5 years.
| Subjects and Methods|| |
This prospective study was approved by the ethical committee. Obtained written informed consent from 25 renal transplant patients who received live related/unrelated donor kidney transplantation, 18 patients with CAD (characterized by renal function decline, elevated SCr and proteinuria), and 25 healthy controls (HC) during March 2011–January 2012. Clinical data collected included warm ischemia, cold ischemia time, episodes of ARs, and infections. Transplant patient group were recipients of renal transplant whose samples were collected from baseline to 4-year-post-Tx at regular intervals.
Compliance with ethical standards
The study protocol was approved by the Institute's Ethical Committee, written informed consent was obtained from all patients and study was conducted in compliance with the provisions of the Declaration of Helsinki and good clinical practice guidelines.
Urinary KIM-1 estimation – urine samples were collected before the operation, 12, 24 h, postoperative day (POD) 7, 15, and 6th month and at the time of discharge after transplantation. KIM-1 was measured quantitatively by sandwich ELISA (R and D Systems) according to the manufacturer's instructions. Samples were centrifuged, aliquoted, and stored at −80°C until processed. Serum CysC was measured using latex-enhanced immune turbid metric method (Dialab GmbH). Intensity of the turbidity was measured and calculated with multicalibrate graph.
Creatinine levels were measured using modified Jaffe reaction of fixed interval. Creatinine clearance (CrCl) was calculated from 24-h urinary creatinine excretion and serum creatinine. Estimated GFR was calculated with the modification of diet in renal disease formula. Creatinine reduction ratio was defined by the difference between baseline and the 7th day - SCr divided by baselines Cr.
All patients were on a triple-drug regime containing a calcineurin inhibitor (CNI) (either tacrolimus [TAC] or cyclosporine [CsA]/along with mycophenolate mofetil [MMF] and prednisolone. MMF 1000 mg twice daily and adjusted according to tolerability, TAC 6–8 mg/day adjusted accordingly based on the trough levels. The target TAC trough level was (10–12 ng/mL) ng/mL for the first 6 months and ≤5 ng/mL thereafter. For CsA, C0 was 200–300 ng/mL till month 3 and 150–250 ng/mL thereafter. Prednisolone was tapered to 5 mg/day. All the recipients received oral valganciclovir for 3 months and cotrimoxazole for 6 months post-Tx ation.
At each visit, patient and graft survival status; vital signs; hematology and biochemical parameters, dosage and levels of immunosuppressive agents were estimated. Findings of renal biopsies; and occurrence of rejection, infection, hospitalization, and other significant events were noted. Blood levels of Tac, CsA, and everolimus were measured by chemiluminescent microparticle immunoassay and liquid chromatography-mass spectrometry. All other variables were assessed by standard laboratory methodologies. Patients were followed up weekly for 1st month, fortnightly for the next 2 months, monthly for 6 months, and thereafter once in 6 months/1 year throughout the study period. All unexplained episodes of graft dysfunction were investigated by biopsy. AR was treated as per standard protocol.
For group comparison, Student's t-test was used if data was normally distributed. For analyzing related variables within group, paired t-test was used by two-tailed test wherein P #60; 0.05 was considered statistically significant. Nonparametric Spearman's correlations were used to determine the relationships between parameters. For multiple comparisons, Tukey's test was performed. Receiver operating characteristic (ROC) curve analysis to predict positive and negative predictive values was performed using SPSS version 18 software program IBM company, Chicago and Prism Graph Pad 5.
| Results|| |
Demographic characteristics age, gender, original disease, time on dialysis before transplant, human leukocyte antigen matching, and donor source are presented in [Table 1]. Biopsy-proven ARs were observed in two patients, and CNI toxicity was observed in two patients. Pretransplant values of CysC and KIM-1 were significantly high when compared to that of HC [Figure 1] and [Figure 2]. Mean CysC concentration at baseline was (6.41 ± 2.8 mg/L), at 12 h - (7.8 ± 3.2 mg/L), at 24 h - (4.4 ± 2.6 mg/L), at 7th POD-(3.4 ± 1.7 mg/L), at 15th POD-(2.8 ± 1.8 mg/L), at 6 m-(1.5 ± 0.7 mg/L) in CAD-(4.4 ± 2.0 mg/L), and in HC (0.65 ± 0.38 mg/L). KIM-1 levels were – at baseline (4.7 ± 2.1 ng/mL), at 12 h (2.5 ± 2.4 ng/mL), at 24 h (6.9 ± 3.2 ng/mL), at 7th POD-(2.0 ± 2.8 ng/mL), at 15th POD (2.35 ± 2.6 ng/mL), 6 m (1.04 ± 1.4 ng/mL), in CAD-(1.9 ± 1.6 ng/mL), and in HC (0.41 ± 0.3 ng/mL). Concentrations of CysC increased soon after transplantation and reached to peak (P ≤ 0.001) at 12 h posttransplantation and remained higher than the control up until day 15 of post-Tx. There is a significant increase of these levels observed in the CAD patients also. Urinary KIM-1 levels increased after transplantation and reached peak at 24 h (P ≤ 0.001) post-Tx and remained elevated up until day 15 of post-Tx as compared with HC. In 16 patients with uncomplicated post-Tx course, there was a drastic fall of both CysC and KIM-1 levels. Five patients had postoperative complications which did not interfere with the graft function where there was a change in the concentration of both CysC and KIM-1 and without statistical significance. Two patients had biopsy-proven AR [Figure 3]a and [Figure 3]b, reversed with the antirejection therapy. Both CysC and KIM-1 levels increased before the occurrence of the biopsy-proven AR; however, concentration of KIM-1 was significantly elevated and remained high in one patient. Two patients had CNI toxicity and were treated accordingly by dosage adjustment where CysC levels were altered.
|Table 1: Preoperative and demographic characteristics of patients and controls|
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|Figure 1: Concentration of cystatin C in serum. Samples were collected at pretransplantation, 12, 24 h, day 7, day 15, and month 6 after transplantation and compared with healthy controls and patients with chronic allograft dysfunction|
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|Figure 2: Concentration of kidney injury molecule-1 in whole urine pretransplant and posttransplant intervals|
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|Figure 3: (a and b) Pre- and post-transplant concentrations of S.creatinine, S cystatin C and urinary kidney injury molecule 1 in two patients biopsy-proven acute cellular rejection|
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The diagnostic accuracy of discharge levels of CysC, KIM-1, and SCr for predicting graft function at 4 years which was assessed by ROC curve and calculated by area under curve. CysC emerged as a better marker with an area under the curve (AUC) of 0.841 with 95% confidence interval (CI) of 0.906 with P ≤ 0.001 as depicted in [Figure 4]a. When we performed ROC in patients with AR KIM1 appeared as a better marker with an AUC of 0.938 with 95% CI of 1.0 and a P = 0.015 [Figure 4]b.
|Figure 4: (a) Receiver-operating characteristic curve of graft function. Nonparametric receiver-operating characteristic plot for diagnostic accuracy of S.creatinine, cystatin C and kidney injury molecule 1 in predicting graft function showed AUC 0.740, 0.841 and 0.581 respectively. (b) Receiver-operating characteristic curve of acute rejection. Nonparametric receiver-operating characteristic curve of kidney injury molecule 1, cystatin C and S.creatinine, in two patients with acute rejection with the area under curve 0.938, 0.781 and 0.875 respectively|
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When we performed Tukey's multiple comparison correlation studies [Table 2] of discharge creatinine levels, CysC levels and KIM-1 levels with 6 months CrCl only discharge CysC showed statistical significance with P = 0.01. Discharge levels of KIM-1 correlated with discharge SCr while discharge CysC correlated with discharge CrCl and 4th year CrCl.
KIM1 levels elevated in rejection patients while CysC changed in response to the changes in GFR induced by either AR or CNI toxicity/infections. In CAD, both KIM-1 and CysC elevated.
| Discussion|| |
In the present study, we evaluated the ability of CysC and KIM1 as biomarkers of renal function, renal tubular and cellular injury in pre- and post-transplant subjects and in CAD patients. We observed that both CysC and KIM-1 did not show correlation with age, gender, and body mass index of patient. Both KIM-1 and CysC levels elevated after transplantation CysC peaked at 12 h post-Tx whereas KIM-1 peaked at 24 h post-Tx. In uncomplicated and complicated post-Tx, clinical courses CysC correlated well with CrCl than KIM-1 and SCr showing it as a better marker of glomerular function. It is evident that there is a leak back of creatinine into the circulation through the damaged proximal tubules whenever there is low GFR consequently underestimating GFR. It was also observed that there was a fall in the levels of CysC in three patients despite rise in the creatinine levels with the alteration in the urine output, which would help the clinician to reverse adverse insults beforehand.
When we compared the levels of CysC, KIM-1 and S. Cr levels at the time of discharge with that of CrCl at the time of discharge and 4 years posttransplantation only CysC was significantly correlated with CrCl at both the time points. Both KIM-1 and SCr were just above the upper range (2.4 ng/mL and1.75 mg/dL) of the normal whereas CysC (3.3 mg/L) was almost 3-fold above the upper range of the normal range correlating well with the discharge CrCl (45.3 mL/mt). The elevated levels of CysC may reflect proximal tubular injury.. All the three parameters CysC, KIM-1, and SCr levels fell to the baseline in 16 patients with uncomplicated post-Tx course. However, CysC seems to adjust rapidly than the other two in response to changes in GFR. Two patients with biopsy-proven CNI toxicity, it has been noticed that both CysC and KIM-1 levels were on rise, but CysC levels were more prominent reflecting that both may provide an earlier indication of renal damage.
Concentrations of CysC and KIM-1were increased before the occurrence of biopsy-proven AR. Cytatin C concentration decreased with the antirejection treatment whereas it took a while for KIM-1 concentrations to come down owing to the tubular injury. Patient survival was 92% at the end of 4th year and it was interesting note that the CysC levels at the time of discharge of these two patients were high (>7 mg/L) while SCr and KIM-1 levels were normal.
In CAD patients, all the three parameters increased. Both KIM-1 and CysC did not perform well in predicting CAD.
Earlier studies noticed a correlation between CycC and GFR in renal transplantation , but none has established either prognostic value or the long-term outcome. This study is novel by determining the role of these two biomarkers in terms of predicting rejection episodes as well a long-term outcome. The possible limitation of the study is that of smaller sample size. However, preliminary findings were encouraging and warrants for the study in larger cohort.
| Conclusion|| |
Noninvasive measurements of urinary tubular biomarkers can provide information of the microenvironment of the allograft in transplant recipients. Monitoring their response to host immune system may reveal early state of injury and thus allow the clinician to provide timely intervention. Future advancements in modulating the expression of these biomarkers on tubular cells may also potentially aid in identifying new therapeutic targets.
In summary, both KIM-1 and CysC are useful markers for predicting AR, renal function, and graft survival. Elevated urinary levels of KIM-1 independently predict AR, while CysC is a valuable marker of GFR and a useful tool in predicting graft survival in renal transplant recipients. Further studies with large sample size are required to clarify the specific roles.
The authors would like to thank the Bhagwan Mahavir Hospital and Research Centre and laboratory technicians of Transimmun for their support in collecting the samples. The authors thank UGC for funding.
Financial support and sponsorship
The study was funded by UGC, PDF for Women (Sailaja Kesiraju) – F-15-31/11 (SA-II). Co-Authors have no proprietary interest in products discussed.
Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]