Joseph P. Grande

Evidence for antibody-mediated injury as a Major determinant of late kidney allograft failure

Background. Late graft failure (LGF) is believed to be the consequence of immunologic and nonimmunologic insults leading to progressive deterioration in kidney function. We studied recipients with new onset late kidney graft dysfunction (n=173) to determine the importance of C4d staining and circulating donor-specific antibody (DSA) in subsequent LGF. Methods. One hundred seventy-three subjects transplanted before October 1, 2005 (mean time after transplant 7.3±6.0 years) had a baseline serum creatinine level of 1.4±0.3 mg/dL before January 1, 2006 and underwent biopsy for new onset graft dysfunction after that date (mean creatinine at biopsy 2.7±1.6 mg/dL). Statistical analysis was based on central DSA and blinded pathology determinations. Results. Subjects were divided into four groups based on C4d and DSA: no C4d, no DSA (group A; n=74); only DSA (group B; n=31); only C4d (group C; n=28); and both C4d and DSA (group D; n=40). Among DSA+ recipients (groups B and D), group D had broader reactivity and a stronger DSA response. After 2 years, groups C and D (C4d+) were at significantly greater risk for LGF than groups A and B. Adjusting for inflammation (Banff i, t, g, and ptc scores) did not change the outcome. Local diagnosis of calcineurin inhibitor nephrotoxicity was spread across all four subgroups and did not impact risk of LGF. Conclusions. Evidence of antibody-mediated injury (DSA or C4d) is common (57%) in patients with new onset late kidney allograft dysfunction. The risk of subsequent graft failure is significantly worse in the presence of C4d+ staining.

Predicting subsequent decline in kidney allograft function from early surveillance biopsies

Identifying factors that are predictive of allograft loss might be an important step toward prolonging kidney allograft survival. In this study we sought to determine the association between histologic changes on 1‐year surveillance biopsies, changes in graft function and survival. This analysis included 292 adults, recipients of kidneys from living donors (69%) or deceased donors (31%), transplanted between 1998 and 2001 and followed up for 46 ± 14 months. The primary end point was death‐censored graft loss or a >50% reduction in GFR beyond 1 year. One‐year biopsies were classified as: (i) Normal (N = 87, 30%), (ii) inflammation (N = 6, 2%), (iii) fibrosis (N = 131, 45%), (iv) fibrosis and inflammation (N = 53, 18%) and (v) transplant glomerulopathy (N = 15, 5%). By multivariate Cox analysis, survival related to biopsy classification (HR = 4.2, p = 0.001), graft function (HR = 0.97, p = 0.001) and HLA mismatches (HR = 1.003, p = 0.004). Using normal histology as a reference, fibrosis and inflammation (HR = 8.5, p < 0.0001) and glomerulopathy (HR = 10, p < 0.0001) related to poorer survival but mild fibrosis alone did not. Importantly, the degree of inflammation associated with fibrosis generally did not qualify for the diagnosis of borderline rejection. In conclusion, inflammation and glomerulopathy 1 year post‐transplant predict loss of graft function and graft failure independently of function and other variables.

The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo

Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, and its isozyme, HO-1, may protect against tissue injury. One posited mechanism is the degradation of heme released from destabilized heme proteins. We demonstrate that HO-1 is a critical protectant against acute heme protein-induced toxicity in vivo. In the glycerol model of heme protein toxicity-one characterized by myolysis, hemolysis, and kidney damage-HO-1 is rapidly induced in the kidney of HO-1 +/+ mice as the latter sustain mild, reversible renal insufficiency without mortality. In stark contrast, after this insult, HO-1 -/- mice exhibit fulminant, irreversible renal failure and 100% mortality; HO-1 -/- mice do not express HO-1, and evince an eightfold increment in kidney heme content as compared to HO-1 +/+ mice. We also demonstrate directly the critical dependency on HO-1 in protecting against a specific heme protein, namely, hemoglobin: doses of hemoglobin which exert no nephrotoxicity or mortality in HO-1 +/+ mice, however, precipitate rapidly developing, acute renal failure and marked mortality in HO-1 -/- mice. We conclude that the induction of HO-1 is an indispensable response in protecting against acute heme protein toxicity in vivo.

Distinct Renal Injury in Early Atherosclerosis and Renovascular Disease

Background—Atherosclerotic renovascular disease may augment deterioration of renal function and ischemic nephropathy compared with other causes of renal artery stenosis (RAS), but the underlying mechanisms remain unclear. This study was designed to test the hypothesis that concurrent early atherosclerosis and hypoperfusion might have greater early deleterious effects on the function and structure of the stenotic kidney. Methods and Results—Regional renal hemodynamics and function at baseline and during vasoactive challenge (acetylcholine or sodium nitroprusside) were quantified in vivo in pigs by electron-beam computed tomography after a 12-week normal (n=7) or hypercholesterolemic (HC, n=7) diet, RAS (n=6), or concurrent HC and a similar degree of RAS (HC+RAS, n=7). Flash-frozen renal tissue was studied ex vivo. Basal cortical perfusion and single-kidney glomerular filtration rate (GFR) were decreased similarly in the stenotic RAS and HC+RAS kidneys, but tubular fluid reabsorption was markedly impaired only in HC+RAS. Perfusion responses to challenge were similarly blunted in the experimental groups. Stimulated GFR increased in normal, HC, and RAS (38.3±3.6%, 36.4±7.6%, and 60.4±9.3%, respectively, P <0.05), but not in HC+RAS (6.5±15.1%). These functional abnormalities in HC+RAS were accompanied by augmented perivascular, tubulointerstitial, and glomerular fibrosclerosis, inflammation, systemic and tissue oxidative stress, and tubular expression of nuclear factor-&kgr;B and inducible nitric oxide synthase. Conclusions—Early chronic HC+RAS imposes distinct detrimental effects on renal function and structure in vivo and in vitro, evident primarily in the tubular and glomerular compartments. Increased oxidative stress may be involved in the proinflammatory and progrowth changes observed in the stenotic HC+RAS kidney, which might potentially facilitate the clinically observed progression to end-stage renal disease.

Accommodation in ABO-incompatible kidney allografts, a novel mechanism of self-protection against antibody-mediated injury.

To elucidate the mechanism of self‐protection against anti‐donor blood‐group antibody known as accommodation, we studied 16 human ABO‐incompatible living‐donor kidney transplant recipients at 3 and 12 months post transplantation. Both circulating anti‐blood‐group antibody and the target blood‐group antigen in the graft were demonstrable in all patients after transplantation. Thirteen of 16 grafts had normal renal function and histology, while three grafts with prior humoral rejection demonstrated significant glomerulopathy and thus did not meet the criterion for accommodation. Using microarrays, we compared five 1‐year protocol ABO‐compatible renal graft biopsies to four accommodated ABO‐incompatible graft biopsies. Significant alterations in gene expression in 440 probe sets, including SMADs, protein tyrosine kinases, TNF‐α and Mucin 1 were identified. We verified these changes in gene expression using RT‐PCR and immunohistochemistry. Heme oxygenase‐1, Bcl‐2 and Bcl‐xl were not increased in ABO‐incompatible grafts at any time‐point. We conclude that accommodation is always present in well‐functioning, long‐surviving ABO‐incompatible kidney transplants. This self‐protection against antibody‐mediated damage may involve several novel mechanisms including the disruption of normal signal transduction, attenuation of cellular adhesion and the prevention of apoptosis.

Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation.

Recent protocols have allowed successful positive crossmatch (+XM) and ABO incompatible (ABOI) kidney transplantation, although their long‐term outcome is not clear. To begin to assess this issue we compared protocol biopsies performed 12 months posttransplant in 37 +XM, 24 ABOI and 198 conventional allografts. Although the majority in all three groups had only minimal histologic changes, transplant glomerulopathy (TG) was significantly increased in +XM (22% vs. 13% ABOI vs. 8% conventional, p = 0.015), and correlated with prior humoral rejection (HR) by multivariate analysis (odds ratio 17.5, p ≤ 0.0001). Patients with a prior history of HR also had a significant increase in interstitial fibrosis (No HR 54% vs. HR 86%, p = 0.045). In the absence of HR no difference in histologic changes was seen between groups, although all three groups had a demonstrable mild increase in interstitial fibrosis from biopsies performed at the time of transplant. Thus, although HR is associated with an increase in TG, in its absence allograft histology is similar in +XM, ABOI and conventional allografts 1 year posttransplant.

ABO-incompatible kidney transplantation using both A2 and non-A2 living donors

Background. Given the scarcity of cadaveric organs, efforts are intensifying to increase the availability of living donors. The current study assessed the feasibility of using ABO-incompatible living-donor kidneys to expand the donor pool. Methods. The authors performed 18 ABO-incompatible living-donor kidney transplants between May 1999 and April 2001. Ten patients received living-donor kidneys from A2 and eight patients received kidneys from non-A2 blood group donors. Immunosuppression consisted of Thymoglobulin antibody induction, tacrolimus, mycophenolate mofetil, and prednisone. Eight non-A2 and two A2 kidney recipients also received a pretransplant conditioning regimen of four plasmapheresis treatments followed by intravenous immunoglobulin and splenectomy at the time of transplantation. Antidonor blood group antibody titer was measured at baseline, pretransplant, at 1- to 3-month and 1-year follow-up, and at the time of diagnosis of antibody-mediated rejection. Results. No hyperacute rejection episodes occurred. One-year graft and patient survival rates in the 18 ABO-incompatible recipients were only slightly lower than those of 81 patients who received ABO-compatible kidney transplants during the same period (89% vs. 96% and 94% vs. 99%, respectively). Glomerular filtration rate and serum creatinine levels did not differ between the groups. Antibody-mediated rejection occurred in 28% of ABO-incompatible recipients, and was reversible with plasmapheresis, intravenous immunoglobulin, and increasing immunosuppression in all patients except one. Conclusions. ABO-incompatible living donor kidney transplants can achieve an acceptable 1-year graft survival rate using an immunosuppressive regimen consisting of Thymoglobulin induction, tacrolimus, mycophenolate mofetil, and prednisone combined with pretransplant plasmapheresis, intravenous immunoglobulin, and splenectomy.

Oxidative Stress and Induction of Heme Oxygenase-1 in the Kidney in Sickle Cell Disease

Chronic nephropathy is a recognized complication of sickle cell disease. Using a transgenic sickle mouse, we examined whether oxidative stress occurs in the sickle kidney, the origins and functional significance of such oxidant stress, and the expression of the oxidant-inducible, potentially protective gene, heme oxygenase-1 (HO-1); we also examined the expression of HO-1 in the kidney and in circulating endothelial cells in sickle patients. We demonstrate that this transgenic sickle mouse exhibits renal enlargement, medullary congestion, and a reduced plasma creatinine concentration. Oxidative stress is present in the kidney as indicated by increased amounts of lipid peroxidation; heme content is markedly increased in the kidney. Exacerbation of oxidative stress by inhibiting glutathione synthesis with buthionine-sulfoximine dramatically increased red blood cell sickling in the sickle kidney: in buthionine-sulfoximine-treated sickle mice, red blood cell sickling extended from the medulla into the cortical capillaries and glomeruli. HO activity is increased in the sickle mouse kidney, and is due to induction of HO-1. In the human sickle kidney, HO-1 is induced in renal tubules, interstitial cells, and in the vasculature. Expression of HO-1 is increased in circulating endothelial cells in patients with sickle cell disease. These results provide the novel demonstration that oxidative stress occurs in the sickle kidney, and that acute exacerbation of oxidative stress in the sickle mouse precipitates acute vaso-occlusive disease. Additionally, the oxidant-inducible, heme-degrading enzyme, HO-1, is induced regionally in the murine and human sickle kidney, and systemically, in circulating endothelial cells in sickle patients.

Genetically obese MMTV-TGF-α/Lepob Lepob female mice do not develop mammary tumors

Elevated body weight is a risk factor for postmenopausal breast cancer and is associated with increased incidence of spontaneous and chemically induced mammary tumors (MTs) in rodents. In this study, genetically obese LepobLepob female mice that overexpress human TGF-α (transforming growth factor-alpha) were used to assess the role of body weight on oncogene-induced MT development in comparison to lean counterparts. MMTV (mouse mammary tumor virus)-TGF-α and Lep strain mice were crossed to produce TGF-α/Lep+Lep+ (homozygous lean), TGF-α/Lep+Lepob (heterozygous lean) and TGF-α/LepobLepob (homozygous obese) genotypes. Body weights were determined weekly and mice palpated for the presence of MTs until 104 weeks of age. Despite their significantly higher body weight, obese TGF-α/LepobLepob mice failed to develop MTs. MTs were detected between 48 and 104 weeks of age for 26/39 TGF-α/Lep+Lepob mice and for 19/38 TGF-α/Lep+Lep+ mice between 67 and 104 weeks of age. Although MT incidence was not statistically different between the lean groups, age of MT detection tended to be younger for TGF-α/Lep+Lepob mice (p < 0.09). There were significant effects of both genotype and MTs on final body weight, that is, TGF-α/Lep+Lepob mice weighed more than homozygous lean mice, and mice with MTs weighed more than those without MTs. TGF-α/LepobLepob mice are not a good model to evaluate the effect of body weight on MT development possibly due to leptin deficiency. However, the finding that increased body weight is associated with increased oncogene-induced MT development within the normal weight range provides experimental support for the role of body weight in breast cancer.

Mechanisms of Renal Structural Alterations in Combined Hypercholesterolemia and Renal Artery Stenosis

Objective—Atherosclerotic renovascular disease (ARVD) aggravates renal scarring more than other causes of renal artery stenosis (RAS), but the underlying pathogenic mechanisms of this potential profibrotic effect remain unclear. We tested the hypothesis that coexistence of atherosclerosis and RAS interferes with renal tissue remodeling. Methods and Results—Single-kidney hemodynamics and function were quantified in vivo with electron-beam computed tomography in 3 groups of pigs (n=7 each): normal pigs, pigs 12 weeks after induction of unilateral RAS (RAS group), and pigs with similar-degree RAS fed a 12-week 2% hypercholesterolemic diet (HC+RAS, simulating early ARVD). Kidneys were studied ex vivo by Western blotting and immunohistochemistry. Renal volume, renal blood flow, and glomerular filtration rate were similarly decreased in RAS and HC+RAS ischemic kidneys, accompanied by similar increased expression of profibrotic factors like transforming growth factor-&bgr;, tissue inhibitor of metalloproteinase-1, and plasminogen activator inhibitor-1. Nevertheless, HC+RAS kidneys showed increased intrarenal fibrosis compared with RAS-only kidneys. Furthermore, expression of nuclear factor-&kgr;B was increased, expression of extracellular (matrix metalloproteinase-2) and intracellular (ubiquitin) protein degradation systems was decreased, and apoptosis was blunted. Conclusions—Diet-induced HC superimposed on RAS accelerates the development of fibrosis in the stenotic kidney by amplifying profibrotic mechanisms and disrupting tissue remodeling. These alterations might contribute to renal disease progression in ARVD and might account for the increased propensity for end-stage renal disease.