Alkesh Jani

Urine NGAL and IL-18 are Predictive Biomarkers for Delayed Graft Function Following Kidney Transplantation

Delayed graft function (DGF) due to tubule cell injury frequently complicates deceased donor kidney transplants. We tested whether urinary neutrophil gelatinase‐associated lipocalin (NGAL) and interleukin‐18 (IL‐18) represent early biomarkers for DGF (defined as dialysis requirement within the first week after transplantation). Urine samples collected on day 0 from recipients of living donor kidneys (n = 23), deceased donor kidneys with prompt graft function (n = 20) and deceased donor kidneys with DGF (n = 10) were analyzed in a double blind fashion by ELISA for NGAL and IL‐18. In patients with DGF, peak postoperative serum creatinine requiring dialysis typically occurred 2–4 days after transplant. Urine NGAL and IL‐18 values were significantly different in the three groups on day 0, with maximally elevated levels noted in the DGF group (p < 0.0001). The receiver–operating characteristic curve for prediction of DGF based on urine NGAL or IL‐18 at day 0 showed an area under the curve of 0.9 for both biomarkers. By multivariate analysis, both urine NGAL and IL‐18 on day 0 predicted the trend in serum creatinine in the posttransplant period after adjusting for effects of age, gender, race, urine output and cold ischemia time (p < 0.01). Our results indicate that urine NGAL and IL‐18 represent early, predictive biomarkers of DGF.

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation.

Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

IL-33 Exacerbates Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury (AKI). IL-33 is a proinflammatory cytokine, but its role in AKI is unknown. Here we observed increased protein expression of full-length IL-33 in the kidney following induction of AKI with cisplatin. To determine whether IL-33 promotes injury, we administered soluble ST2 (sST2), a fusion protein that neutralizes IL-33 activity by acting as a decoy receptor. Compared with cisplatin-induced AKI in untreated mice, mice treated with sST2 had fewer CD4 T cells infiltrate the kidney, lower serum creatinine, and reduced acute tubular necrosis (ATN) and apoptosis. In contrast, administration of recombinant IL-33 (rIL-33) exacerbated cisplatin-induced AKI, measured by an increase in CD4 T cell infiltration, serum creatinine, ATN, and apoptosis; this did not occur in CD4-deficient mice, suggesting that CD4 T cells mediate the injurious effect of IL-33. Wildtype mice that received cisplatin and rIL-33 also had higher levels of the proinflammatory chemokine CXCL1, which CD T cells produce, in the kidney compared with CD4-deficient mice. Mice deficient in the CXCL1 receptor also had lower serum creatinine, ATN, and apoptosis than wildtype mice following cisplatin-induced AKI. Taken together, IL-33 promotes AKI through CD4 T cell-mediated production of CXCL1. These data suggest that inhibiting IL-33 or CXCL1 may have therapeutic potential in AKI.

Caspase Inhibition Prevents the Increase in Caspase‐3, ‐2, ‐8 and ‐9 Activity and Apoptosis in the Cold Ischemic Mouse Kidney

Prolonged cold ischemic time is a risk factor for the development of delayed graft function. The adverse impact of cold ischemia may be associated with tubular cell death in the kidney. Caspase‐3 is a major mediator of apoptotic cell death. We hypothesized that caspase inhibition would reduce apoptosis and other features of cold ischemia. Kidneys of C57BL/6 mice were perfused with cold University of Wisconsin solution containing a pancaspase inhibitor or vehicle via the left ventricle. The contralateral right kidney was used as a control. The left kidney was stored for 48 h at 4°C to produce cold ischemia. Caspase‐3 activity was massively (100‐fold) increased in cold ischemic kidneys compared with controls. On immunoblot analysis, the processed form of caspase‐3 was increased in cold ischemic kidneys compared with controls. The increase in caspase‐3 was associated with significantly more renal tubular apoptosis and brush‐border injury. In addition, caspase‐2, ‐8 and ‐9 activities were increased in cold ischemic kidneys. The pancaspase inhibitor prevented the formation of the processed form of caspase‐3 and the increase in caspase activity, and reduced apoptosis and brush‐border injury. Caspase inhibition may prove useful in kidney preservation.

Interleukin-18 binding protein transgenic mice are protected against ischemic acute kidney injury

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

NLRP3 Inflammasome Knockout Mice Are Protected against Ischemic but Not Cisplatin-Induced Acute Kidney Injury

We have demonstrated that caspase-1 is a mediator of both cisplatin-induced acute kidney injury (AKI) and ischemic AKI. As caspase-1 is activated in the inflammasome, we investigated the inflammasome in cisplatin-induced and ischemic AKI. Mice were injected with cisplatin or subjected to bilateral renal pedicle clamping. Immunoblot analysis of whole kidney after cisplatin-induced AKI revealed: 1) an increase in apoptosis-associated Speck-like protein containing a caspase recruitment domain (ASC), the major protein that complexes with nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing proteins (NLRP) 1 or 3 to form the inflammasome; 2) an increase in caspase-1 activity, caspase-5, and NLRP1, components of the NLRP1 inflammasome; and 3) a trend toward increased NLRP3. To determine whether the NLRP3 inflammasome plays an injurious role in cisplatin-induced AKI, we studied NLRP knockout (NLRP3−/−) mice. In cisplatin-induced AKI, the blood urea nitrogen, serum creatinine, acute tubular necrosis score, and tubular apoptosis score were not significantly decreased in NALP3−/− mice compared with wild-type mice. We have previously demonstrated the injurious role of caspase-1 in ischemic AKI. NLRP3, but not ASC or NLRP1, is increased in ischemic AKI. NLRP3−/− mice with ischemic AKI had significantly lower blood urea nitrogen, serum creatinine, and acute tubular necrosis and apoptosis scores than the wild-type controls. The difference in protection against cisplatin-induced AKI compared with ischemic AKI in NLRP3−/− mice was not explained by the differences in proinflammatory cytokines interleukin (IL)-1β, IL-6, chemokine (C-X-C motif) ligand 1, or tumor necrosis factor α. NLRP3 inflammasome is a mediator of ischemic AKI but not cisplatin-induced AKI, and further investigation of the NLRP1 inflammasome in cisplatin-induced AKI should prove interesting.

Kidney Transplantation for Systemic Sclerosis Improves Survival and may Modulate Disease Activity

Systemic sclerosis (SS) may lead to sclerodema renal crisis, an unusual cause of end‐stage renal disease (ESRD) with historically poor hemodialysis outcomes. Little information is available on outcomes after kidney transplantation. Information from the UNOS registry was obtained on SS patients in the United States, listed for kidney transplants between 1985–2002. We compared survival at 1 and 3 years in patients who received cadaveric transplants with patients who remained on the waiting list. Graft survival, cause of graft loss, frequency of early graft loss and pre‐ and post‐transplant skin scores were analyzed. Two hundred and fifty‐eight patients with SS were listed for transplantation. Survival was significantly prolonged in patients receiving transplants (p = 0.005). Graft survival at 1 and 3 years was 68% and 60%. Early graft loss was common. Skin scores improved in all four subjects at our center, with an average decline of 60.7% (p = 0.024). Kidney transplantation confers a survival benefit in ESRD due to SS. Transplantation may be associated with an improvement in systemic manifestations of disease. Despite suboptimal graft survival, kidney transplant should be considered the treatment of choice in ESRD due to SS.

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.

Long-Term Renal Outcomes After Delayed Graft Function

Delayed graft function (DGF) describes dysfunction of the kidney allograft immediately after transplantation and is the most common complication in the immediate posttransplantation period. Although a standardized definition for DGF is lacking, it is most commonly defined as the need for dialysis within the first week after transplant. DGF is caused by a variety of factors related to the donor and recipient as well as organ procurement techniques. The occurrence of DGF affects both allograft and patient outcomes. In addition to prolonging hospital stay and increasing the costs associated with transplantation, DGF is associated with an increased incidence of acute rejection after transplantation and is associated with poorer long-term graft outcomes. Both immunologic and nonimmunologic mechanisms contribute to DGF. The risk factors for DGF that have been identified are reviewed as well as the impact of DGF on long-term outcomes.

Urine IL-18, NGAL, IL-8 and serum IL-8 are biomarkers of acute kidney injury following liver transplantation

BackgroundAKI is common following liver transplantation and is associated with significant morbidity and mortality. Biomarkers of AKI have not been well established in this setting but are needed to help guide patient care and facilitate development of novel therapeutics.MethodsSerum creatinine, cystatin C, IL-6, and IL-8 and urine IL-18, NGAL, IL-6, and IL-8 were measured before and within 24 hours after liver transplantation in 40 patients. AKI was defined as a ≥50% sustained increase in creatinine above pre-operative values occurring within 24 hours of transplantation and persisting for at least 24 hours.ResultsSeven patients met criteria for AKI (17.5%), with mean creatinines of 0.81 mg/dL pre-operatively and 1.75 mg/dL post-operatively. While pre-operative biomarker levels in patients with AKI were similar to those in patients without AKI, differences were seen between the groups with regard to median post-operative serum IL-8 (pg/mL) (242.48 vs. 82.37, p = 0.0463) and urine NGAL (ng/mL) (386.86 vs. 24.31, p = 0.0039), IL-6 (pg/mL) (52 vs. 7.29, p=0.0532), IL-8 (pg/mL) (14.3 vs. 0, p = 0.0224), and IL-18 (pg/mL) (883.09 vs. 0, p = 0.0449). The areas under receiver operating characteristic (ROC) curves were 0.749 for urine IL-18, 0.833 for urine NGAL, 0.745 for urine IL-6, 0.682 for serum IL-6, 0.773 for urine IL-8, and 0.742 for serum IL-8. Post-operative cystatin C was not significantly different between AKI and no AKI groups.ConclusionSerum IL-8 and urine IL-18, NGAL, IL-6, and IL-8 are elevated in AKI within the first 24 hours following liver transplantation.