Kidney injury molecule-1 expression is closely associated with renal allograft damage

Th e aim of our study was to investigate the expression of kidney injury molecule- (KIM-) in renal allograft biopsy samples and assess the clinical signifi cance of its use as a biomarker for tissue damage. A total of  renal allograft biopsy samples from  patients with normal serum creatinine and  cases of increased serum creatinine were collected. Th ey were divided into diff erent groups according to the Banff  diagnostic criteria. KIM- expression was detected by immunohistochemical methods and the association of KIM- and blood biochemical indexes was analyzed. KIM- expression increased as Banff  classifi cation grade increased and was positively correlated with tubular infl ammation severity in the acute T-cell rejection group. Moreover, KIM- expression was strongly positive in the chronic active antibodymediated rejection group. Interestingly, KIM- was weakly positive in the normal group without obvious acute rejection and injury of immunosuppressant toxicity. In this group, . (/) of the cases with normal serum creatinine level showed weakly positive KIM- expression in their renal tissues. KIM- expression level is positively correlated with renal allograft damage and tubular cell injury. KIM- is expressed in tubular epithelial cells before blood biochemical indexes become elevated and morphological changes occur. KIM- expression is an early, sensitive, and specifi c biomarker to determine renal tubular epithelial cell injury in renal allograft tissue. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Kidney injury molecule- (KIM-) is a type I transmembrane protein that was fi rst identifi ed in renal cells after injury in  [].KIM- mRNA and protein are expressed at low levels in normal kidney but are specifi cally up regulated in dedifferentiated proximal tubular cells after ischemic or nephrotoxic acute kidney injury (AKI).An extracellular domain of KIM- is detectable in the urine soon after AKI [].KIM- represents a promising biomarker for the early diagnosis of AKI and its clinical outcomes [-].In hospitalized patients with established AKI, urinary KIM- levels predicted adverse clinical outcomes, such as dialysis requirement and mortality [].Preliminary studies have reported the potential utility of KIM- as a chronic kidney disease (CKD) biomarker.In a kidney biopsy study of  patients with CKD from various aetiologies, KIM- was primarily expressed at the luminal side of dediff erentiated proximal tubules, in areas with fi brosis and infl ammation [].KIM- staining in the kidney correlated positively with morphological damage and renal function decline.It appears to be a highly sensitive and specifi c biomarker for the diagnosis of acute or chronic renal injury [, ].In recent years, much attention has been paid to the role of KIM- in renal transplantation, although no consensus has been reached.Most researchers believe KIM- may become an early marker of kidney transplant rejection injury and an early biomarker of graft survival and prognosis.Our study was designed to explore KIM- expression in renal allograft biopsy and assess the clinical signifi cance of its use as a potential biomarker of renal tissue injury.The renal allograft biopsy tissues were collected, fixed in formalin and embedded with paraffin.All patients were given basiliximab before the transplantation and prednisone, mycophenolate mofetil, and calcineurin inhibitors after the operation.The clinical characteristics of the patients are listed in Table .The study was approved by the Ethics Committee of the First Hospital, Jilin University and informed consent was obtained from each patient.Th e renal allograft biopsy tissue samples were divided into six groups according to the Banff  standards [].The groups were acute T cell-mediated rejection group (group A,  cases), chronic active antibody-mediated rejection group (group B,  cases), chronic active T cells mediated rejection group (group C,  cases), borderline lesions group (group D,  cases), normal group without obvious acute rejection or injury of immunosuppressant toxicity (group E,  cases), and recurrence of allograft nephropathy group (group F,  cases).

Renal function
Serum creatinine was tested using the creatinine acid oxidase method and blood urea nitrogen (BUN) was tested by the ultraviolet glutamic acid deoxidizing enzymatic method (Hitachi - instrument).

Immunohistochemistry
Paraffin-embedded tissue samples were observed by hematoxylin and eosin staining, PAS staining, and Masson staining.Samples were deparaffinized and rehydrated.After antigen retrieval, primary antibody against KIM- mAb (MAB; R&D; Minneapolis, MN, USA) was used at a dilution of :, and incubated overnight at °C.IgG conjugated horseradish peroxidase (HRP) and ,-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories; Bur-lingame, CA, USA) were used to visualize antibody binding.KIM- staining in renal tissues was evaluated by three pathologists who had no knowledge of patient clinicopathologic factors or outcomes.Immunoreactivity of samples was further graded as follows []: , no staining; +, weak fine granular staining focally present along the luminal surface of non-atrophic proximal tubules;  +, weak fine granular staining completely surrounding the luminal surface of non-atrophic proximal tubules; +, moderate granular staining completely surrounding the luminal surface of non-atrophic proximal tubules and extending into intercellular junctions; and  +, strong large granular staining completely surrounding the luminal surface of non-atrophic proximal tubules and extending into intercellular junctions.

Statistical analysis
Data are presented as mean ± SEM unless indicated otherwise.SPSS software package (versions ., SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses.Correlations of KIM- expression with clinicopathologic factors were examined by Chi-square tests.Correlation between KIM- staining scores and renal function indexes was assessed using simple regression analysis.Differences were considered signifi cant when the p values were less than ..

KIM- expression in renal allograft tissue KIM- was expressed in proximal tubular epithelial cells at various levels in the diff erent pathological diagnosis groups.
As shown in Table , the total positive KIM- expression rate was . (/).In the acute T cell mediated rejection group, . of the cases showed strongly positive expression, and the total positive expression rate was .Furthermore, . of the cases in the chronic active anti-  0  0 (0)  0 (0)  0 (0)  2 (15.4)  0 (0)  0 (0)  1+  1 (3.7)  1 (11.1)  0 (0)  2 (15.4)  1 (9.0)  0 (0)  2+  8 (29.6)  0 (0)  1 (20.0)  5 (38.5)  5 (45.5  body mediated rejection group showed strongly positive expression and the total positive expression rate was  as well.A preponderance of the cases () in the chronic active T cells mediated rejection group showed strongly positive expression and the total positive expression rate was .In contrast, only . of the cases in the borderline lesions group showed strongly positive expression and the positive expression rate was ..In the normal group without obvious acute rejection and injury of immunosuppressant toxicity, . of the cases also showed weakly positive expression and . of the cases showed strongly positive expression.All four cases in the recurrence of allograft nephropathy group had strongly positive expression.

KIM- expression correlated with declines in renal function in renal transplant patients
The association of KIM- expression with creatinine and BUN was analyzed.KIM- expression was signifi cantly correlated with creatinine level (p = .,Table ).BUN level is easily aff ected by many factors and analysis did not show KIM- expression was correlated with BUN level.The results indicated that KIM- expression in renal allograft tissue was positively correlated with declines in kidney function.

KIM- expression contributed to acute T cells mediated rejection
Acute T cells mediated rejection was graded as IA, IB, IIA, IIB, and III [].KIM- expression was detected in  (.) grade I tissues and in  (.) grade II tissues (Table ).KIM- expression increased as the Banff  classification grade increased and was positively correlated with the acute T-cell rejection group.KIM- expression is related to severity of tubular infl ammation In the acute T cell mediated rejection group, four cases that showed strongly positive expression of KIM- although no tubular infl ammation was observed.Th is indicated that renal tubular epithelial cells had been damaged before the morphological change of tubular infl ammation had occurred.Th e number of cases of KIM- expression increased as tubular inflammation grade rose; KIM- expression was strongly positive when tubular infl ammation grade was  + (Table ).

KIM- expression in cases with normal serum creatinine levels
In the present study we also found that . (/) of the cases showed weakly positive KIM- expression and . (/) of the cases showed relatively strongly positive expression although they had normal levels of serum creatinine (data not shown).In the normal group without obvious acute rejection and injury of immunosuppressant toxicity, . (/) of the cases had normal serum creatinine levels, but they showed weakly positive KIM- expression in their renal tissues.

DISCUSSION
Renal tubular epithelial cells are very sensitive to toxic injury and anoxic damage, and a series of changes occur after injury.Tubular cells lose polarity and integrity of the cytoskeleton, apoptosis, and necrosis occur, and surviving cells start to proliferate and diff erentiate.KIM- is expressed in the damaged renal tubular epithelial cells after toxic and anoxic injury [].
It is a marker with high sensitivity and specifi city in the diagnosis of renal tubular damage.In our study, KIM- expression was assessed in proximal tubular epithelial cells from several groups representing patients with varying degrees of renal damage.KIM- expression was detected in . of renal tissues in the acute T cell mediated rejection group, . of renal tissues in the chronic rejection group, and . of renal tissues in the borderline lesions group.In the normal group without obvious acute rejection and injury of immunosuppressant toxicity, . of renal tissues showed had weakly positive KIM- expression, while . of renal tissues showed strongly positive KIM- expression.KIM- was highly expressed in the recurrence of allograft nephropathy group.

Grade (case number)
KIM-1 expression (case number) 0 (0) 1+ ( 1) 2+ ( 7) 3+ ( 12) 4+ ( 7) 0 ( 4 Sensitive biological indexes are needed to identify renal tubular epithelial injury and to evaluate renal tubular damage when morphological changes have not yet occurred in the allograft.In this study, we detected KIM- expression by immunohistological methods in renal allograft tissue to judge renal tubular epithelial injury and acute cellular rejection damage, and found that KIM- expression was positively related with kidney function decline [].Th e results show that KIM- expression in renal allograft tissue is more sensitive than rises in serum creatinine or morphological changes in the assessment of renal tubular epithelial injury.Chronic renal allograft function loss is the main cause of graft failure.Th erefore, the early evaluation of renal allograft function is benefi cial for early anti-rejection treatment.In a prospective study of  kidney transplant patients followed for an average of four years, elevated urinary KIM- levels were associated with a .-fold increased risk of graft loss [].Prediction of graft loss by KIM- was independent of donor age, creatinine clearance, and proteinuria.In a retrospective study of non-diabetic proteinuric subjects, anti-proteinuric therapies reduced the urinary excretion of KIM-, suggesting its use as an effi cacy marker [].KIM- has proven to be an excellent marker of nephrotoxicity in preclinical studies []. In our study, . (/) of cases showed strongly positive KIM- expression in IA and IB acute T cells mediated rejection subgroup; in the grade II subgroup, . (/) of the cases showed strongly positive KIM- expression.KIM- expression gradually increased as the Banff  classifi cation level rose in acute cellular rejection.In the  cases of acute T cell mediated rejection, KIM- was highly expressed in four cases with no tubular infl ammation, which suggests that damage to renal tubular epithelial cells occurred before the morphological change appeared.Th e number of cases of strongly positive KIM- expression increased when the classifi cation grade of tubular infl ammation rose.All renal tissues showed strongly positive KIM- expression in grade +, which suggests that renal tubular epithelium is significantly damaged.In the chronic active antibody mediated rejection group, . of the cases showed strongly positive expression and total positive expression rate was .In the chronic active T cell mediated rejection group,  of the cases exhibited strongly positive expression.All these data strongly suggest that active rejection caused kidney injury and KIM- positive expression helps to identify early renal tubular epithelial injury.We also found KIM- positive expression in patients with normal biochemical indexes, including . (/) of cases with weak expression and . (/) with strong expression.Therefore, increased KIM- expression in kidney tissue occurs earlier than changes in blood biochemical indexes and better reflects renal tubular epithelial cell damage.Th ere was weak KIM- expression in three cases when there was no rejection and immunosuppressant toxic injury confirmed by morphology, which suggests increases in KIM- expression, can be an earlier marker than the visible morphological change in identifying renal tubular damage.KIM- expression was weakly positive in . of cases (/) with normal serum creatinine, which is consistent with the result that KIM- expression was detected in  of the cases with procedural renal biopsy [].
Our study also had some limitations.First, . (/) of patients in our study had abnormal renal function, and renal biopsy indicated that KIM- expression was positive in most of the total patient pool.Second, we did not compare other related clinical data and did not follow up patients.Th ird, we did not investigate the eff ect of treatment on the relationship between KIM- and renal function.We also did not assess the effect on renal function after blocking KIM- expression.Finally, the sample number may limit the reliability of the research results and increased sample numbers in future studies may be helpful to further defi ne the relationship between KIM- expression and renal function.
The molecular biological function of KIM- is not fully understood, although evidence indicates a role for its involvement in renal tubular cells at early stages of damage and repair, in adhesion and immune reaction processes, and in renal fi brosis [, , , ].Most patients had varying levels of renal dysfunction, and a renal biopsy was performed.Most renal tissues exhibited high levels of KIM- expression and rejection injury was the primary cause.Thus, KIM- may be involved in the renal allograft rejection process.The role of KIM- in graft immune injury or downstream molecular events needs further research.It has been shown that KIM- is mainly expressed and increased in tubular interstitial inflammation and fibrosis induced atrophy.The extracellular domain of KIM- is short, which includes a conserved tyrosine phosphorylation site used for signal transduction mediated by receptor and ligand interactions.The extracellular domain of KIM- can be cleaved into soluble portions by matrix metalloproteases and released into the urine [].Additionally, urinary KIM- expression is also related to declines in allograft function [, ].Therefore, while our study supports the conclusion that urinary KIM- amount may be an early, non-invasive, specific, sensitive biochemical indicator to identify renal tubular epithelial injury, further investigation is needed to better understand the function of KIM- and more completely validate its use as a biomarker.

CONCLUSION
KIM- expression level is positively correlated with renal allograft damage and tubular cell injury.Increased KIM- expression in allograft tissue occurs earlier than changes in blood biochemical indexes and better refl ects renal tubular epithelial cell damage.KIM- expression is an early, sensitive, and specific biomarker to determine renal tubular epithelial cell injury in renal allograft tissue.


A total of  patients ( women,  men) who underwent kidney transplantation between May,  and  Bosn J Basic Med Sci 2013; 13 (3): 171-174 LIANLIAN SONG ET AL.: KIDNEY INJURY MOLECULE1 EXPRESSION IS CLOSELY ASSOCIATED WITH RENAL ALLOGRAFT DAMAGE June,  in the Urology and Nephrology Center of the First Hospital, Jilin University were enrolled in this study.
Bosn J Basic Med Sci 2013; 13 (3): 174-174 LIANLIAN SONG ET AL.: KIDNEY INJURY MOLECULE1 EXPRESSION IS CLOSELY ASSOCIATED WITH RENAL ALLOGRAFT DAMAGE KIM- expression in renal allograft tissue is more sensitive than rises in serum creatinine or morphological changes in the assessment of renal tubular epithelial injury.

TABLE 1 .
Basic clinical characteristics of patients and KIM-1 expression in diff erent groups.Legend: group A, acute T cell-mediated rejection group (27 cases); group B, chronic active antibody-mediated rejection group (9 cases); group C, chronic active T cells mediated rejection group (5 cases); group D, borderline lesions group (13 cases); group E, normal group without obvious acute rejection or injury of immunosuppressant toxicity (11 cases); group F, recurrence of allograft nephropathy group (4 cases).The numbers in parentheses are the percentage of cases with positive KIM-1 expression in each group.

TABLE 2 .
KIM-1 expression correlation with decreased renal function in renal transplant patients.

TABLE 3 .
Correlation between KIM-1 expression and acute T-cell mediated rejection.