Optimized immunosuppression to prevent graft failure in renal transplant recipients with HLA antibodies (OuTSMART): a randomised controlled trial

Background: 3% of kidney transplant recipients return to dialysis annually upon allograft failure. Development of antibodies (Ab) against human leukocyte antigens (HLA) is a validated prognostic biomarker of allograft failure. We tested whether screening for HLA Ab, combined with an intervention to improve adherence and optimization of immunosuppression could prevent allograft failure. Methods: Prospective, open-labelled randomised biomarker-based strategy (hybrid) trial in 13 UK transplant centres [EudraCT (2012-004308-36) and ISRCTN (46157828)]. Patients were randomly allocated (1:1) to unblinded or double-blinded arms and screened every 8 months. Unblinded HLA Ab+ patients were interviewed to encourage medication adherence and had tailored optimisation of Tacrolimus, Mycophenolate mofetil and Prednisolone. The primary outcome was time to graft failure in an intention to treat analysis. The trial had 80% power to detect a hazard ratio of 0.49 in donor specific antibody (DSA)+ patients. Findings: From 11/9/13 to 27/10/16, 5519 were screened for eligibility and 2037 randomised (1028 to unblinded care and 1009 to double blinded care). We identified 198 with DSA and 818 with non-DSA. Development of DSA, but not non-DSA was predictive of graft failure. HRs for graft failure in unblinded DSA+ and non-DSA+ groups were 1.54 (95% CI: 0.72 to 3.30) and 0.97 (0.54–1.74) respectively, providing no evidence of an intervention effect. Non-inferiority for the overall unblinded versus blinded comparison was not demonstrated as the upper confidence limit of the HR for graft failure exceeded 1.4 (1.02, 95% CI: 0.72 to 1.44). The only secondary endpoint reduced in the unblinded arm was biopsy-proven rejection. Interpretation: Intervention to improve adherence and optimize immunosuppression does not delay failure of renal transplants after development of DSA. Whilst DSA predicts increased risk of allograft failure, novel interventions are needed before screening can be used to direct therapy

Smad Mediated Regulation of Inhibitor of DNA Binding 2 and Its Role in Phenotypic Maintenance of Human Renal Proximal Tubule Epithelial Cells

<div><p>The basic-Helix-Loop-Helix family (bHLH) of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2) has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC), TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.</p> </div

BMP 7 induction of Id2 was prevented by combined Smad1/5 knock-down.

<p>HKC 8 cells were transfected with either Smad1 and Smad5 siRNAs or negative control siRNA for 24 h. After 24 h serum recovery and 24 h serum free period, the cells were treated with either vehicle (0.1% BSA) or BMP 7 (200 ng/ml) for a further 18 h. The cells were lysed and Id2, Smad1 and Smad5 expressions were assessed by immunoblotting. The representative immunoblots show the expression of Id2, Smad1 and Smad5. BMP 7 upregulation of Id2 was prevented by combined Smad1/5 knock-down. The data is expressed as mean±SD (n = 5, ns = P>0.05, ***<0.001).</p

Id2 was counter-regulated by TGFβ1 and BMP 7 in HKC 8 cells.

<p>HKC 8 cells were treated with either vehicle (0.1% BSA), TGFβ1 (5 ng/ml), BMP 7 (200 ng/ml) or both for 24 h and the expression of Id2 was determined by immunoblotting. The representative immunoblot shows the expression of Id2. TGFβ1 downregulated Id2 levels on the other hand BMP 7 upregulated Id2 levels and combined treatment of BMP 7 prevented TGFβ1 induced Id2 loss. The data is expressed as mean±SD (n = 3, ns = P>0.05, *<0.05).</p

BMP 7 inhibited TGFβ1 mediated α-SMA induction through Id2.

<p>HKC 8 cells were transfected with either Id2 siRNA or %GC content matched negative control siRNA for 24 h. After 24 h serum recovery and 24 h serum free period the cells were treated with either vehicle (0.1% BSA), TGFβ1 (5 ng/ml) or TGFβ1 (5 ng/ml) and BMP 7 (200 ng/ml) for 48 h. The expression of Id2 and α-SMA were determined by immunoblotting. The representative immunoblots shows the expression of Id2 and α-SMA (A). Id2 upregulation by BMP 7 was inhibited by siRNA (B). Id2 knock-down prevented BMP 7 inhibition of TGFβ1 mediated α-SMA induction (C). The data is expressed as mean±SD (n = 3, ns = P>0.05, *<0.05, **<0.01, ***<0.001).</p

Muscle insulin-like growth factor status, body composition, and functional capacity in hemodialysis patients

Hemodialysis (HD) patients typically have reduced muscle mass and diminished functional capacity. The role of the muscle insulin-like growth factors (IGFs), a principal anabolic system that is involved in protein synthesis and that has downregulation that is implicated in muscle loss in animal models of uremia, has previously not been assessed in vivo in HD patients. Seventeen HD patients were compared cross-sectionally with 17 age-, sex-, and body mass index-matched healthy controls. Body composition was assessed by dual energy x-ray absorptiometry and bioelectrical impedance spectrometry; functional capacity by hand grip strength, quadriceps strength, and 30-second sit-to-stand test; systemic inflammation by tumor necrosis factor-\u3b1 (TNF-\u3b1) and TNF receptor 1 (TNFR1); serum and muscle IGF-I and IGFBP-3 by radioimmunoassay; and fragmentation of serum IGFBP-3 by Western immunoblotting. Appendicular lean mass was significantly decreased in HD patients compared with controls (17.6 \ub1 0.9 versus 21.5 \ub1 1.5 kg, P &lt;. 05), as were all measures of functional capacity (P &lt;. 01 to. 001), and highly significant positive correlations between appendicular lean mass and functional capacity were evident (appendicular lean mass and hand-grip strength, quadriceps strength, 30-second sit-to-stand test, all P &lt;. 001). TNF-\u3b1 and TNFR1 were elevated in patients (P &lt;. 001). Although serum IGF-I and IGFBP-3 levels did not differ between the groups (P =. 295 and. 379 respectively), fragmented IGFBP-3 levels were increased (53.1 \ub1 16.0 versus 29.81 \ub1 15.3%, P &lt;. 005). In contrast, muscle IGF-I was substantially diminished in the patient group (n = 7) relative to control (n = 5) levels (0.84 \ub1 0.06 versus 2.78 \ub1 1.80 pg/\u3bcg, P &lt;. 05). We provide evidence of reduced IGF-I in HD patients' skeletal muscle that may be a causal factor in the muscle wasting characteristic of this population. Future research should determine the exact consequences and causes of alterations to the muscle IGF system in HD patients. \ua9 2004 by the National Kidney Foundation, Inc