Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection [BORTEJECT] Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib (=21) or placebo (=23). The 0.5-ml/min per 1.73 m per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant (=0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m; =0.31), 2-year graft survival (81% versus 96%;=0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR.

Rationale: Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS) including the balanced synthesis of its main effector peptides angiotensin (Ang) II and Ang-(1-7). The condition of enzymatic overproduction of Ang II relative to Ang- (1-7) is termed RAS dysregulation, and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. Angiotensin-converting enzyme 2 (ACE2) and neprilysin (NEP) induce the 'alternative', and potentially reno-protective axis by enhancing Ang-(1-7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD), has not yet been elucidated. Objective: To examine whether NEP-mediated Ang-(1-7) generation exceeds Ang II formation in the healthy kidney compared to diseased kidney. Methods and Results: In this exploratory study, we used mass spectrometry (LC-MS/MS) to measure Ang II and Ang-(1-7) synthesis rates of ACE, chymase and NEP, ACE2, prolyl-endopeptidase (PEP), prolyl-carboxypeptidase (PCP) in kidney biopsy homogenates in 11 healthy living kidney donors and 12 patients with CKD. Spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang-(1-7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity. Conclusions: Ang-(1-7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.

Abbreviations Ang Angiotensin CKD Chronic kidney disease LLOQ Lower limit of quantification RAS Renin-angiotensin system SGLT-2 Sodium-glucose cotransporter 2 TGF Tubuloglomerular feedback To the Editor: Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are a highly promising class of glucose-lowering therapies and have been found to reduce cardiovascular and kidney disease risk in patients with type 2 diabetes [1-3]. The mechanisms underlying these cardio-and renoprotective effects of SGLT-2 inhibition are, however, incompletely understood and it is not known if these beneficial effects are also evident in people with type 1 diabetes.

Aim: Sodium glucose co-transporter-2 inhibitors (SGLT-2i) improve cardiorenal outcomes in patients with chronic kidney disease (CKD), with and without type 2 diabetes. The molecular mechanisms underlying these pleiotropic effects remain unclear, yet it is speculated that SGLT-2i elicit a neurohormonal modulation resulting in reninangiotensin system (RAS) activation. We hypothesized that combined SGLT-2 and angiotensin-converting enzyme inhibition (ACEi) favours RAS regulation towards the beneficial angiotensin-(1-7)-driven axis. Materials and Methods: This randomized controlled prospective study investigated the effect of 12 weeks treatment with the SGLT-2i empagliflozin on top of ACEi on the molecular RAS dynamics in 24 diabetic and 24 non-diabetic patients with CKD. Systemic RAS peptides were quantified by mass spectrometry. Results: In patients with type 2 diabetes, combined SGLT-2i and ACEi significantly upregulated plasma renin activity [pre-treatment median and interquartile range 298.0 (43.0-672.0) pmol/L versus post-treatment 577.0 (95.0-1543.0) pmol/L; p = .037] and angiotensin I levels [pre-treatment 289.0 (42.0-668.0) pmol/L versus post-treatment 573.0 (93.0-1522.0) pmol/L; p = .037], together with a significant increase of angiotensin-(1-7) levels [pre-treatment 14.0 (2.1-19.0) pmol/L versus post-treatment 32.0 (5.7-99.0) pmol/L; p = .012]. Empagliflozin treatment resulted in a 1.5 to 2-fold increase in main RAS peptides in patients with diabetes compared with placebo. No significant effect of empagliflozin on top of ACEi on RAS peptides was found in patients with CKD without diabetes.

Treating hyperglycemia in previously non-diabetic individuals with exogenous insulin immediately after kidney transplantation reduced the odds of developing Posttransplantation Diabetes Mellitus (PTDM) in our previous proof-of-concept clinical trial. We hypothesized that insulin-pump therapy with maximal insulin dosage during the afternoon would improve glycemic control compared to basal insulin and standard-of-care. In a multi-center, randomized, controlled trial testing insulin isophane for PTDM prevention, we added a third study arm applying continuous subcutaneous insulin lispro infusion (CSII) treatment. CSII was initiated in 24 patients aged 55±12 years, without diabetes history, receiving tacrolimus. The mean daily insulin lispro dose was 9.2±5.2 IU. 2.3±1.1% of the total insulin dose were administered between 00:00 and 6:00, 19.5±11.6% between 6:00 and 12:00, 62.3±15.6% between 12:00 and 18:00 and 15.9±9.1% between 18:00 and 24:00. Additional bolus injections were necessary in five patients. Mild hypoglycemia (52–60 mg/dL) occurred in two patients. During the first post-operative week glucose control in CSII patients was overall superior compared to standard-of-care as well as once-daily insulin isophane for fasting and post-supper glucose. We present an algorithm for CSII treatment in kidney transplant recipients, demonstrating similar safety and superior short-term efficacy compared to standard-of-care and once-daily insulin isophane.