Bryan N. Becker

Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study.

Campath‐1H, an anti‐CD52 monoclonal antibody, was used as induction therapy (40 mg i.v. total dose) in 29 primary human renal transplants, and the patients were maintained on rapamycin monotherapy (levels 8–15 ng/mL) post‐transplant. Campath‐1H profoundly depletes lymphocytes long‐term and more transiently depletes B cells and monocytes. All patients are alive and well at 3–29 months of follow up. One graft was lost because of rejection. There have been no systemic infections and no malignancies. Eight of 29 patients have experienced rejection, which was successfully treated in seven of eight patients. Five of these patients had pathological evidence of a humoral component of their rejection. Seven of the 29 patients were converted to standard triple therapy on account of rejection. Rapamycin was generally well tolerated in that there were no significant wound‐healing problems; two lymphoceles required surgical drainage; and most patients were treated with a lipid‐lowering agent. Flow crossmatch testing post‐transplant revealed evidence of alloantibody in two patients tested with previous combined cellular and humoral rejection. Biopsies have shown no chronic allograft nephropathy to date. In view of the relatively high incidence of early humoral rejection, we plan to modify the immunosuppressive regimen in subsequent pilot studies. This clinical trial provides insight into the use of Campath‐1H induction in combination with rapamycin maintenance monotherapy.

Rituximab as Treatment for Refractory Kidney Transplant Rejection

Recent studies have shown that a high density of CD 20+ cells are seen in patients who have steroid‐resistant rejection episodes. Rituximab is a high‐affinity CD‐20 specific antibody that inhibits B‐cell proliferation while inducing cellular apoptosis. Thus, it is a rational choice for therapy in transplantation to abrogate B‐cell‐mediated events.

Prevalence of CKD and Comorbid Illness in Elderly Patients in the United States: Results From the Kidney Early Evaluation Program (KEEP)

BACKGROUND Elderly individuals with chronic kidney disease (CKD) have high rates of comorbid conditions, including cardiovascular disease and its risk factors, and CKD-related complications. In individuals aged > or = 65 years, we sought to describe the prevalence of CKD determined from laboratory test results in the Kidney Early Evaluation Program (KEEP; n = 27,017) and National Health and Nutrition Examination Survey (NHANES) 1999-2006 (n = 5,538) and the prevalence of diagnosed CKD determined from billing codes in the Medicare 5% sample (n = 1,236,946). In all 3 data sources, we also explored comorbid conditions and CKD-related complications. METHODS CKD was identified as decreased estimated glomerular filtration rate (<60 mL/min/1.73 m(2)) or increased albumin-creatinine ratio in KEEP and NHANES; CKD was identified using International Classification of Diseases, Ninth Revision, Clinical Modification codes in Medicare. Investigated comorbid conditions included diabetes, hypertension, high cholesterol level, coronary artery disease, congestive heart failure, cerebrovascular disease, peripheral vascular disease, and cancer, and CKD-related complications included anemia, hypocalcemia, hyperphosphatemia, and hyperparathyroidism. RESULTS The prevalence of CKD was approximately 44% in both KEEP and NHANES participants, and the prevalence of diagnosed CKD was 7% in Medicare beneficiaries. In all 3 data sets, the prevalence of CKD or diagnosed CKD was higher in participants aged > or = 80 years and those with comorbid conditions. For KEEP and NHANES participants, the prevalence of most comorbid conditions and CKD complications increased with decreasing estimated glomerular filtration rate. For participants with CKD stages 3-5, a total of 29.2% (95% CI, 27.8-30.6) in KEEP and 19.9% (95% CI, 17.0-23.1) in NHANES had anemia, 0.7% (95% CI, 0.4-0.9) and 0.6% (95% CI, 0.3-1.3) had hypocalcemia, 5.4% (95% CI, 4.7-6.1) and 6.4% (95% CI, 5.1-8.0) had hyperphosphatemia, and 52.0% (95% CI, 50.4-53.6) and 30.0% (95% CI, 25.9-34.3) had hyperparathyroidism, respectively. CONCLUSIONS CKD is common in the elderly population and is associated with high frequencies of concomitant comorbid conditions and biochemical abnormalities. Because CKD is not commonly diagnosed, greater emphasis on physician education may be beneficial.

Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule.

Angiotensin II (Ang II) is an important regulator of proximal tubule salt and water reabsorption. Recent studies indicate that rabbit proximal tubule angiotensin II receptors are the type-1 (AT1R) subtype. We studied the effect of Ang II on proximal tubule receptor expression. Rabbits were treated with either angiotensin converting enzyme inhibitors or a low salt diet to modulate endogenous Ang II levels. In captopril-treated rabbits, liver and glomerular AT1R mRNA levels increased 242 +/- 125 and 141 +/- 60%, respectively (n = 6-7; P < 0.05), as determined by quantitative PCR. In contrast, proximal tubule AT1R mRNA levels decreased 40 +/- 11% (n = 6; P < 0.05). Binding of 125I Ang II to renal cortical basolateral membranes of captopril-treated rabbits decreased from 2.9 +/- 0.55 to 1.4 +/- 0.17 fmol/mg protein (n = 6; P < 0.025). In rabbits fed a sodium chloride-deficient diet for 4 wk, AT1R mRNA levels decreased 52 +/- 11% in liver and 43 +/- 7% in glomeruli (n = 4-5; P < 0.05), whereas they increased 141 +/- 85% (n = 5; P < 0.05) in proximal tubule. In basolateral membranes from rabbits on the sodium chloride-deficient diet, specific binding of 125I Ang II increased from 2.1 +/- 0.2 to 4.3 +/- 1.1 fmol/mg protein (n = 7; P < 0.05). To determine whether Ang II directly regulates expression of proximal tubule AT1 receptors, further studies were performed in cultured proximal tubule cells grown from microdissected S1 segments of rabbit proximal tubules and immortalized by transfection with a replication-defective SV40 vector. Incubation of these cells with Ang II (10(-11) to 10(-7) M) led to concentration-dependent increases in both AT1R mRNA levels and specific 125I Ang II binding. Pretreatment with pertussis toxin inhibited Ang II stimulation of AT1R mRNA. AT1R mRNA expression was decreased by either forskolin or a nonhydrolyzable cAMP analogue (dibutryl cAMP). Simultaneous Ang II administration overcame the inhibitory effect of forskolin but not dibutryl cAMP. These results indicate that proximal tubule AT1R expression is regulated by ambient Ang II levels, and Ang II increases AT1R mRNA at least in part by decreasing proximal tubule cAMP generation through a pertussis toxin-sensitive mechanism. Upregulation of proximal tubule AT1R by Ang II may be important in mediating enhanced proximal tubule sodium reabsorption in states of elevated systemic or intrarenal Ang II.

Functionally significant renal allograft rejection is defined by transcriptional criteria

Renal allograft acute cellular rejection (ACR) is a T‐cell mediated disease that is diagnosed histologically. However, many normally functioning allografts have T‐cell infiltrates and histological ACR, and many nonimmune processes cause allograft dysfunction. Thus, neither histological nor functional criteria are sufficient to establish a significant rejection, and the fundamental features of clinical rejection remain undefined. To differentiate allograft lymphocyte infiltration from clinically significant ACR, we compared renal biopsies from patients with ACR to patients with: sub‐clinical rejection (SCR, stable function with histological rejection); no rejection; and nontransplanted kidneys. Biopsies were compared histologically and transcriptionally by RT‐PCR for 72 relevant immune function genes. Neither the degree nor the composition of the infiltrate defined ACR. However, transcripts up‐regulated during effector TH1 T‐cell activation, most significantly the transcription factor T‐bet, the effector receptor Fas ligand and the costimulation molecule CD152 clearly (p = 0.001) distinguished the patient categories. Transcripts from other genes were equivalently elevated in SCR and ACR, indicating their association with infiltration, not dysfunction. Clinically significant ACR is not defined solely by the magnitude nor composition of the infiltrate, but rather by the transcriptional activity of the infiltrating cells. Quantitative analysis of selected gene transcripts may enhance the clinical assessment of allografts.

KIDNEY AND PANCREAS TRANSPLANTATION IN THE UNITED STATES, 1999-2008: THE CHANGING FACE OF LIVING DONATION

The waiting list for kidney transplantation continued to grow between 1999 and 2008, from 41 177 to 76 089 candidates. However, active candidates represented the minority of this increase (36 951–50 624, a 37% change), while inactive candidates increased over 500% (4226–25 465). There were 5966 living donor (LD) and 10 551 deceased donor (DD) kidney transplants performed in 2008. The total number of pancreas transplants peaked at 1484 in 2004 and has declined to 1273. Although the number of LD transplants increased by 26% from 1999 to 2008, the total number peaked in 2004 at 6647 before declining 10% by 2008. The rate of LD transplantation continues to vary significantly as a function of demographic and geographic factors, including waiting time for DD transplant. Posttransplant survival remains excellent, and there appears to be greater use of induction agents and reduced use of corticosteroids in LD recipients. Significant changes occurred in the pediatric population, with a dramatic reduction in the use of LD organs after passage of the Share 35 rule. Many strategies have been adopted to reverse the decline in LD transplant rates for all age groups, including expansion of kidney paired donation, adoption of laparoscopic donor nephrectomy and use of incompatible LD.

Plasma F2-isoprostane levels are elevated in chronic hemodialysis patients.

AIMS Cardiovascular mortality has been reported to be 10- to 20-fold higher in chronic dialysis patients than in the age-matched general population. It has been suggested that increased oxidant stress and resulting vascular wall injury due to uremia and the hemodialysis procedure may be one of the mechanisms predisposing to these cardiovascular complications. Further, hemodialysis membrane bioincompatibility can contribute to increased oxidative stress and prevalence of inflammation. MATERIALS We studied 18 chronic hemodialysis (CHD) patients (age 62.8 +/- 14.7 years, 39% male, 61% African-American, 44% insulin-dependent diabetic, 61% smokers, 61% with documented coronary artery disease) during hemodialysis with 2 membranes with different flux and complement activating properties. METHODS We have measured free and phospholipid-bound F2-isoprostane (F2-IsoP) levels, a sensitive marker of oxidative stress, in CHD patients and compared them to levels in healthy subjects. We have also examined the acute effects of the hemodialysis procedure using both biocompatible and bioincompatible membranes on F2-IsoP levels. RESULTS The results indicated that, compared to controls, both free (96.2 +/- 48.8 pg/ml versus 37.6 +/- 17.2 pg/ml) and bound F2-IsoP (220.4 +/- 154.8 pg/ml versus 146.8 +/- 58.4 pg/ml) levels were significantly higher (p < 0.05 for both). There was a statistically significant decrease in free F2-IsoP concentrations at 15 and 30 minutes of HD, which rebounded to baseline levels at the completion of the procedure. There were no significant differences in F2-IsoP concentrations between the 2 study dialyzers at any time point. Age, smoking status, diabetes mellitus and presence of cardiovascular disease were also not correlated with F2-IsoP levels in this patient population. There was a significant association between predialysis F2-IsoP and C-reactive protein concentrations. CONCLUSION Using a sensitive and specific assay for the measurement of F2-IsoP, we demonstrated that CHD patients are under increased oxidative stress. During a single hemodialysis treatment, the hemodialysis membrane appears to have no discernable effect on oxidative stress status. Measurement of F2-isoprostanes may be a useful biomarker of oxidative stress status as well as in developing new therapeutic strategies to ameliorate inflammatory and oxidative injury in this patient population.

Epithelial-to-mesenchymal transition and oxidative stress in chronic allograft nephropathy.

Epithelial‐to‐mesenchymal transition (EMT) and oxidative stress contribute to kidney tissue fibrosis in various forms of native kidney disease. However, their role in chronic allograft nephropathy (CAN) remains somewhat uncertain. To address this question, kidney transplants were performed in 3‐month‐old rats, using the Fisher 344 → Lewis model of CAN. Six‐month posttransplant, kidney allografts displayed significant tubular atrophy, interstitial fibrosis and vascular wall thickening. Allograft recipients had significantly higher levels of serum creatinine (4.7 ± 1.3 versus 0.59 ± 0.08 mg/dL, p = 0.03) and proteinuria (380 ± 102 versus 30.2 ± 8 mg/dL, p = 0.04) compared to syngeneic grafts. Semiquantitative PCR, immunoblot and immunohistochemical analyses demonstrated increased α‐smooth muscle actin (α‐SMA) mRNA and protein levels coupled with reduced E‐cadherin mRNA and protein immunoreactivity, confirming the presence of CAN‐associated EMT. Allograft α‐SMA levels were increased as early as 1–2 weeks posttransplant. Immunohistochemical studies for collagen type I and III, superoxide anion (O2−), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) confirmed that tubular O2−, eNOS and iNOS, and interstitial collagen I, III and O2− levels were significantly increased in CAN‐associated EMT. In conclusion, these observations suggest that CAN‐associated EMT may be a link between oxidative stress and allograft fibrosis.

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

BackgroundEpithelial to Mesenchymal Transition (EMT) induced by Transforming Growth Factor-β (TGF-β) is an important cellular event in organogenesis, cancer, and organ fibrosis. The process to reverse EMT is not well established. Our purpose is to define signaling pathways and transcription factors that maintain the TGF-β-induced mesenchymal state.ResultsInhibitors of five kinases implicated in EMT, TGF-β Type I receptor kinase (TβRI), p38 mitogen-activated protein kinase (p38 MAPK), MAP kinase kinase/extracellular signal-regulated kinase activator kinase (MEK1), c-Jun NH-terminal kinase (JNK), and Rho kinase (ROCK), were evaluated for reversal of the mesenchymal state induced in renal tubular epithelial cells. Single agents did not fully reverse EMT as determined by cellular morphology and gene expression. However, exposure to the TβRI inhibitor SB431542, combined with the ROCK inhibitor Y27632, eliminated detectable actin stress fibers and mesenchymal gene expression while restoring epithelial E-cadherin and Kidney-specific cadherin (Ksp-cadherin) expression. A second combination, the TβRI inhibitor SB431542 together with the p38 MAPK inhibitor SB203580, was partially effective in reversing EMT. Furthermore, JNK inhibitor SP600125 inhibits the effectiveness of the TβRI inhibitor SB431542 to reverse EMT. To explore the molecular basis underlying EMT reversal, we also targeted the transcriptional repressors ZEB1 and ZEB2/SIP1. Decreasing ZEB1 and ZEB2 expression in mouse mammary gland cells with shRNAs was sufficient to up-regulate expression of epithelial proteins such as E-cadherin and to re-establish epithelial features. However, complete restoration of cortical F-actin required incubation with the ROCK inhibitor Y27632 in combination with ZEB1/2 knockdown.ConclusionsWe demonstrate that reversal of EMT requires re-establishing both epithelial transcription and structural components by sustained and independent signaling through TβRI and ROCK. These findings indicate that combination small molecule therapy targeting multiple kinases may be necessary to reverse disease conditions.

Posttransplant infection in enteric versus bladder-drained simultaneous pancreas-kidney transplant recipients

BACKGROUND Although bladder drainage of the pancreas remains the most common site for drainage of exocrine secretions, enteric drainage is becoming more common in the United States. The most common cause of morbidity after pancreas transplantation is infection, particularly recurrent urinary tract infection. METHODS We examined the incidence of infectious complications for enteric-drained (ED) versus bladder-drained (BD) simultaneous pancreas-kidney transplants (PTx) to determine the incidence of post-transplant infection. The patient cohort included simultaneous pancreas-kidney PTx recipients from June 1995 through August 1997 using a similar induction protocol with antithymocyte globulin, mycophenolate mofetil, prednisone, and Neoral. During this time period, 48 BD PTx and 78 ED PTx were performed. Demographic data including age of transplant, gender, race, and duration of initial hospital stay were similar. However, mean follow-up for the BD PTx was 1.9 years vs. 0.9 years for ED PTx. Rejection, infection, and graft and patient survival rates were estimated by the method of Kaplan and Meier. RESULTS For the entire cohort, 1-year patient survival was 98%, kidney survival 94%, and pancreas survival 93%. There was no difference in survival between ED or BD PTx. At 6 months, kidney transplant rejection had occurred in 38% of BD PTx vs. 30% of ED PTx. Steroid resistant rejection was similar (BD 19%, ED 17%). Postoperative pancreatic leak occurred in 12% BD PTx and 5% ED PTx (P=0.06). There was no significant difference in time to first infection or first abdominal infection between groups. Opportunistic infections were much less likely to occur in ED recipients by 1 year (12% vs. 31%, P=0.002). Both cytomegalovirus infection rates (BD 21% vs. ED 8%, P=0.04) and fungal infection rates (BD 17% vs. ED 4%, P=0.04) were lower in ED PTx. The rate of first urinary tract infection was dramatically decreased with ED. At 1 year, only 20% of ED PTx developed a urinary tract infection vs. 63% of BD PTx (P=0.0001). CONCLUSION Enteric drainage of the pancreas is more physiologic, has similar results to bladder drainage, but has less infectious complications, particularly urinary tract infections.