Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis.

Introduction: Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. Methods: We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Results and Conclusion: Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.

A large-scale retrospective study enabled deep-learning based pathological assessment of frozen procurement kidney biopsies to predict graft loss and guide organ utilization.

Lesion scores on procurement donor biopsies are commonly used to guide organ utilization for deceased-donor kidneys. However, frozen sections present challenges for histological scoring, leading to inter- and intra-observer variability and inappropriate discard. Therefore, we constructed deep-learning based models to recognize kidney tissue compartments in hematoxylin & eosin-stained sections from procurement needle biopsies performed nationwide in years 2011-2020. To do this, we extracted whole-slide abnormality features from 2431 kidneys and correlated with pathologists' scores and transplant outcomes. A Kidney Donor Quality Score (KDQS) was derived and used in combination with recipient demographic and peri-transplant characteristics to predict graft loss or assist organ utilization. The performance on wedge biopsies was additionally evaluated. Our model identified 96% and 91% of normal/sclerotic glomeruli respectively; 94% of arteries/arterial intimal fibrosis; 90% of tubules. Whole-slide features of Sclerotic Glomeruli (GS)%, Arterial Intimal Fibrosis (AIF)%, and Interstitial Space Abnormality (ISA)% demonstrated strong correlations with corresponding pathologists' scores of all 2431 kidneys, but had superior associations with post-transplant estimated glomerular filtration rates in 2033 and graft loss in 1560 kidneys. The combination of KDQS and other factors predicted one- and four-year graft loss in a discovery set of 520 kidneys and a validation set of 1040 kidneys. By using the composite KDQS of 398 discarded kidneys due to "biopsy findings", we suggest that if transplanted, 110 discarded kidneys could have had similar survival to that of other transplanted kidneys. Thus, our composite KDQS and survival prediction models may facilitate risk stratification and organ utilization while potentially reducing unnecessary organ discard.

HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy.

Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.

Allograft tissue under the microscope: only the beginning.

PURPOSE OF REVIEW: To review novel modalities for interrogating a kidney allograft biopsy to complement the current Banff schema. RECENT FINDINGS: Newer approaches of Artificial Intelligence (AI), Machine Learning (ML), digital pathology including Ex Vivo Microscopy, evaluation of the biopsy gene expression using bulk, single cell, and spatial transcriptomics and spatial proteomics are now available for tissue interrogation. SUMMARY: Banff Schema of classification of allograft histology has standardized reporting of tissue pathology internationally greatly impacting clinical care and research. Inherent sampling error of biopsies, and lack of automated morphometric analysis with ordinal outputs limit its performance in prognostication of allograft health. Over the last decade, there has been an explosion of newer methods of evaluation of allograft tissue under the microscope. Digital pathology along with the application of AI and ML algorithms could revolutionize histopathological analyses. Novel molecular diagnostics such as spatially resolved single cell transcriptomics are identifying newer mechanisms underlying the pathologic diagnosis to delineate pathways of immunological activation, tissue injury, repair, and regeneration in allograft tissues. While these techniques are the future of tissue analysis, costs and complex logistics currently limit their clinical use.

Infliximab Induction Lacks Efficacy and Increases BK Virus Infection in Deceased Donor Kidney Transplant Recipients: Results of the CTOT-19 Trial.

BACKGROUND: Ischemia-reperfusion (IR) of a kidney transplant (KTx) upregulates TNF α production that amplifies allograft inflammation and may negatively affect transplant outcomes. METHODS: We tested the effects of blocking TNF peri-KTx via a randomized, double-blind, placebo-controlled, 15-center, phase 2 clinical trial. A total of 225 primary transplant recipients of deceased-donor kidneys (KTx; 38.2% Black/African American, 44% White) were randomized to receive intravenous infliximab (IFX) 3 mg/kg or saline placebo (PLBO) initiated before kidney reperfusion. All patients received rabbit anti-thymocyte globulin induction and maintenance immunosuppression (IS) with tacrolimus, mycophenolate mofetil, and prednisone. The primary end point was the difference between groups in mean 24-month eGFR. RESULTS: There was no difference in the primary end point of 24-month eGFR between IFX (52.45 ml/min per 1.73 m 2 ; 95% CI, 48.38 to 56.52) versus PLBO (57.35 ml/min per 1.73 m 2 ; 95% CI, 53.18 to 61.52; P =0.1). There were no significant differences between groups in rates of delayed graft function, biopsy-proven acute rejection (BPAR), development of de novo donor-specific antibodies, or graft loss/death. Immunosuppression did not differ, and day 7 post-KTx plasma analyses showed approximately ten-fold lower TNF ( P <0.001) in IFX versus PLBO. BK viremia requiring IS change occurred more frequently in IFX (28.9%) versus PLBO (13.4%; P =0.004), with a strong trend toward higher rates of BKV nephropathy in IFX (13.3%) versus PLBO (4.9%; P =0.06). CONCLUSIONS: IFX induction therapy does not benefit recipients of kidney transplants from deceased donors on this IS regimen. Because the intervention unexpectedly increased rates of BK virus infections, our findings underscore the complexities of targeting peritransplant inflammation as a strategy to improve KTx outcomes.Clinical Trial registry name and registration number:clinicaltrials.gov (NCT02495077).

Role of time from transplantation to biopsy in histologic ABMR: A single center report.

BACKGROUND: Allograft biopsies with lesions of Antibody-Mediated Rejection (ABMR) with Microvascular Inflammation (MVI) have shown heterogeneous etiologies and outcomes. METHODS: To examine factors associated with outcomes in biopsies that meet histologic ABMR criteria, we retrospectively evaluated for-cause biopsies at our center between 2011 and 2017. We included biopsies that met the diagnosis of ABMR by histology, along with simultaneous evaluation for anti-Human Leukocyte Antigen (HLA) donor-specific antibodies (DSA). We evaluated death-censored graft loss (DCGL) and used a principal component analysis (PCA) approach to identify key predictors of outcomes. RESULTS: Out of the histologic ABMR cohort (n = 118), 70 were DSA-positive ABMR, while 48 had no DSA. DSA(+)ABMR were younger and more often female recipients. DSA(+)ABMR occurred significantly later post-transplant than DSA(-)ABMR suggesting time-dependence. DSA(+)ABMR had higher inflammatory scores (i,t), chronicity scores (ci, ct) and tended to have higher MVI scores. Immunodominance of DQ-DSA in DSA(+)ABMR was associated with higher i+t scores. Clinical/histologic factors significantly associated with DCGL after biopsy were inputted into the PCA. Principal component-1 (PC-1), which contributed 34.8% of the variance, significantly correlated with time from transplantation to biopsy, ci/ct scores and DCGL. In the PCA analyses, i, t scores, DQ-DSA, and creatinine at biopsy retained significant correlations with GL-associated PCs. CONCLUSIONS: Time from transplantation to biopsy plays a major role in the prognosis of biopsies with histologic ABMR and MVI, likely due to ongoing chronic allograft injury over time.

Deep learning identified pathological abnormalities predictive of graft loss in kidney transplant biopsies.

Interstitial fibrosis, tubular atrophy, and inflammation are major contributors to kidney allograft failure. Here we sought an objective, quantitative pathological assessment of these lesions to improve predictive utility and constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte infiltrates. Periodic acid- Schiff stained slides of transplant biopsies (60 training and 33 testing) were used to quantify pathological lesions specific for interstitium, tubules and mononuclear leukocyte infiltration. The pipeline was applied to the whole slide images from 789 transplant biopsies (478 baseline [pre-implantation] and 311 post-transplant 12-month protocol biopsies) in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss. Our model accurately recognized kidney tissue compartments and mononuclear leukocytes. The digital features significantly correlated with revised Banff 2007 scores but were more sensitive to subtle pathological changes below the thresholds in the Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of one-year graft loss, while a Composite Damage Score in 12-month post-transplant protocol biopsies predicted later graft loss. ITASs and Composite Damage Scores outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITASs or Composite Damage Scores also demonstrated significantly higher incidence of estimated glomerular filtration rate decline and subsequent graft damage. Thus, our deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies and demonstrated superior ability for prediction of post-transplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

AMPK mediates regulation of glomerular volume and podocyte survival.

Herein, we report that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without focal segmental glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease (MCD), where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral and 5/6th nephrectomy models in Shroom3-KD mice. Knockdown mice exhibited less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying that Fyn was downstream of Shroom3. Using SHROOM3 or FYN knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMPK, a negative regulator of anabolism. AMPK activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMPK abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMPK activation. In agreement with this, treatment of glomerular injury models with AMPK activators restricted glomerular volume, podocytopenia, and progression to FSGS. Glomerular transcriptomes from MCD biopsies also showed significant enrichment of Fyn inactivation and Ampk activation versus FSGS glomeruli. In summary, we demonstrated the important role of AMPK in glomerular volume regulation and podocyte survival. Our data suggest that AMPK activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

Influence of patient characteristics and immunosuppressant management on mortality in kidney transplant recipients hospitalized with coronavirus disease 2019 (COVID-19).

The influence of patient characteristics and immunosuppression management on COVID-19 outcomes in kidney transplant recipients (KTRs) remains uncertain. We performed a single-center, retrospective review of all adult KTRs admitted to the hospital with confirmed COVID-19 between 03/15/2020 and 05/15/2020. Patients were followed from the date of admission up to 1 month following hospital discharge or study conclusion (06/15/2020). Baseline characteristics, laboratory parameters, and immunosuppression were compared between survivors and patients who died to identify predictors of mortality. 38 KTRs with a mean baseline eGFR of 52.5 ml/min/1.73 m2 were hospitalized during the review period. Maintenance immunosuppression included tacrolimus (84.2%), mycophenolate (89.5%), and corticosteroids (81.6%) in the majority of patients. Eleven patients (28.9%) died during the hospitalization. Older age (OR = 2.05; 1.04-4.04), peak D-dimer (OR = 1.20; 1.04-1.39), and peak white blood cell count (OR = 1.11; 1.02-1.21) were all associated with mortality among KTRs hospitalized for COVID-19. Increased mortality was also observed among KTRs with concomitant HIV infection (87.5% vs. 36.1%; p < .01). Conversely, immunosuppression intensity and degree of reduction following COVID-19 diagnosis were not associated with either survival or acute allograft rejection. Our findings potentially support a strategy of individualization of immunosuppression targets based on patient-specific risk factors, rather than universal immunosuppression reduction for KTRs at risk from COVID-19.

4D flow MRI for the assessment of renal transplant dysfunction: initial results.

OBJECTIVES: (1) Determine inter-observer reproducibility and test-retest repeatability of 4D flow parameters in renal allograft vessels; (2) determine if 4D flow measurements in the renal artery (RA) and renal vein (RV) can distinguish between functional and dysfunctional allografts; (3) correlate haemodynamic parameters with estimated glomerular filtration rate (eGFR), perfusion measured with dynamic contrast-enhanced MRI (DCE-MRI) and histopathology. METHODS: Twenty-five prospectively recruited renal transplant patients (stable function/chronic renal allograft dysfunction, 12/13) underwent 4D flow MRI at 1.5 T. 4D flow coronal oblique acquisitions were performed in the transplant renal artery (RA) (velocity encoding parameter, VENC = 120 cm/s) and renal vein (RV) (VENC = 45 cm/s). Test-retest repeatability (n = 3) and inter-observer reproducibility (n = 10) were assessed by Cohen's kappa, coefficient of variation (CoV) and Bland-Altman statistics. Haemodynamic parameters were compared between patients and correlated to the estimated glomerular filtration rate, DCE-MRI parameters (n = 10) and histopathology from allograft biopsies (n = 15). RESULTS: For inter-observer reproducibility, kappa was > 0.99 and 0.62 and CoV of flow was 12.6% and 7.8% for RA and RV, respectively. For test-retest repeatability, kappa was > 0.99 and 0.5 and CoV of flow was 27.3% and 59.4%, for RA and RV, respectively. RA (p = 0.039) and RV (p = 0.019) flow were both significantly reduced in dysfunctional allografts. Both identified chronic allograft dysfunction with good diagnostic performance (RA: AUC = 0.76, p = 0.036; RV: AUC = 0.8, p = 0.018). RA flow correlated negatively with histopathologic interstitial fibrosis score ci (ρ = - 0.6, p = 0.03). CONCLUSIONS: 4D flow parameters had better repeatability in the RA than in the RV. RA and RV flow can identify chronic renal allograft dysfunction, with RA flow correlating with histopathologic interstitial fibrosis score. KEY POINTS: • Inter-observer reproducibility of 4D flow measurements was acceptable in both the transplant renal artery and vein, but test-retest repeatability was better in the renal artery than in the renal vein. • Blood flow measurements obtained with 4D flow MRI in the renal artery and renal vein are significantly reduced in dysfunctional renal transplants. • Renal transplant artery flow correlated negatively with histopathologic interstitial fibrosis score.

Key driver genes as potential therapeutic targets in renal allograft rejection.

Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. Molecules with regulatory control among immune pathways of AR that are inadequately suppressed, despite standard-of-care immunosuppression, could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative, network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators - the key driver genes (KDGs) - within dysregulated AR pathways. A 982-meta-gene signature with differential expression in AR versus non-AR was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across 7 data sets. Fourteen KDGs were derived from this signature. Interrogation of 2 publicly available databases identified compounds with predicted efficacy against individual KDGs or a key driver-based gene set, respectively, which could be repurposed for AR prevention. Minocycline, a tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline attenuated the inflammatory profile of AR compared with controls and when coadministered with immunosuppression prolonged graft survival. This study demonstrates that a network-based strategy, using expression and genotype data to predict KDGs, assists target prioritization for therapeutics in renal allograft rejection.

Multiparametric magnetic resonance imaging shows promising results to assess renal transplant dysfunction with fibrosis.

Here we assessed the diagnostic value of a quantitative multiparametric magnetic resonance imaging (mpMRI) protocol for evaluation of renal allograft dysfunction with fibrosis. Twenty-seven renal transplant patients, including 15 with stable functional allografts (eGFR mean 71.5 ml/min/1.73m2), and 12 with chronic dysfunction/established fibrosis (eGFR mean 30.1 ml/min/1.73m2), were enrolled in this prospective single-center study. Sixteen of the patients had renal biopsy (mean 150 days) before the MRI. All patients underwent mpMRI at 1.5T including intravoxel-incoherent motion diffusion-weighted imaging, diffusion tensor imaging, blood oxygen level dependent (BOLD R2*) and T1 quantification. True diffusion D, pseudodiffusion D*, perfusion fraction PF, apparent diffusion coefficient (ADC), fractional anisotropy (FA), R2* and T1 were calculated for cortex and medulla. ΔT1 was calculated as (100x(T1 Cortex-T1 Medulla)/T1 Cortex). Test-retest repeatability and inter-observer reproducibility were assessed in four and ten patients, respectively. mpMRI parameters had substantial test-retest and interobserver repeatability (coefficient of variation under 15%), except for medullary PF and D* (coefficient of variation over 25%). Cortical ADC, D, medullary ADC and ΔT1 were all significantly decreased, while cortical T1 was significantly elevated in fibrotic allografts. Cortical T1 showed positive correlation to the Banff fibrosis and tubular atrophy scores. The combination of ΔT1 and cortical ADC had excellent cross-validated diagnostic performance for detection of chronic dysfunction with fibrosis. Cortical ADC and T1 had good performance for predicting eGFR decline at 18 months (4 or more ml/min/1.73m2/year). Thus, the combination of cortical ADC and T1 measurements shows promising results for the non-invasive assessment of renal allograft histology and outcomes.

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection.

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

Disruption of MAGI2-RapGEF2-Rap1 signaling contributes to podocyte dysfunction in congenital nephrotic syndrome caused by mutations in MAGI2.

The essential role of membrane associated guanylate kinase 2 (MAGI2) in podocytes is indicated by the phenotypes of severe glomerulosclerosis of both MAGI2 knockout mice and in patients with congenital nephrotic syndrome (CNS) caused by mutations in MAGI2. Here, we show that MAGI2 forms a complex with the Rap1 guanine nucleotide exchange factor, RapGEF2, and that this complex is lost when expressing MAGI2 CNS variants. Co-expression of RapGEF2 with wild-type MAGI2, but not MAGI2 CNS variants, enhanced activation of the small GTPase Rap1, a central signaling node in podocytes. In mice, podocyte-specific RapGEF2 deletion resulted in spontaneous glomerulosclerosis, with qualitative glomerular features comparable to MAGI2 knockout mice. Knockdown of RapGEF2 or MAGI2 in human podocytes caused similar reductions in levels of Rap1 activation and Rap1-mediated downstream signaling. Furthermore, human podocytes expressing MAGI2 CNS variants show severe abnormalities of cellular morphology and dramatic loss of actin cytoskeletal organization, features completely rescued by pharmacological activation of Rap1 via a non-MAGI2 dependent upstream pathway. Finally, immunostaining of kidney sections from patients with congenital nephrotic syndrome and MAGI2 mutations showed reduced podocyte Rap1-mediated signaling. Thus, MAGI2-RapGEF2-Rap1 signaling is essential for normal podocyte function. Hence, disruption of this pathway is an important cause of the renal phenotype induced by MAGI2 CNS mutations.

SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts.

BACKGROUND: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. RESULTS: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. CONCLUSIONS: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

Pre-liver transplant renal dysfunction and association with post-transplant end-stage renal disease: A single-center examination of updated UNOS recommendations.

Simultaneous liver-kidney allocation protocols allocate dual organs based on a sustained eGFR of 30 mL/min or less. A 2017-UNOS update includes CKD3 as dual organ candidates but only when the listing eGFR is <30 mL/min while recommending a "safety net" for prioritized kidney listing post-LT. We retrospectively reviewed adult LTs examine whether the UNOS proposal captured the LT population at highest risk for developing post-LT ESRD. Among 290 LT recipients, 67 had pre-LT CKD3, 141 had AKI, of whom 47 required dialysis (<4 weeks). During follow-up, 25 (8.62%) developed ESRD, while 70 (24.1%) died. In adjusted Cox models, CKD3 had an independent association with post-LT ESRD (adjusted HR 4.8; P = 0.001), independent of AKI. Interestingly, CKD3 with listing GFR >30 mL/min was still significantly associated with post-LT ESRD. AKI was associated with reduced post-LT survival (adjusted HR 1.9; P = 0.02), albeit only in the first-year post-LT. Severe AKI-D was associated with post-LT ESRD and mortality. The safety net would have captured only 60% of all post-LT ESRD cases in our cohort. Pre-LT CKD3 was associated with increased risk of post-LT ESRD above the recommended cutoff for listing GFR. These findings, if generalizable in larger cohorts have important implications for dual organ allocation.

Early conversion to belatacept after renal transplantation.

Belatacept is a non-nephrotoxic immunosuppressive agent, which may make it the ideal agent for patients with delayed or slow graft function on calcineurin inhibitors. There are limited data on conversion of patients to belatacept within 6 months of transplantation. Between January 2012 and December 2015, 16 patients were converted to belatacept for delayed or poor graft function (eGFR<30 mL/min/1.73 m2 , MDRD); three were HIV positive. Conversion protocols were analyzed in patients ≤4 months and 4-6 months post-transplantation. Mean serum creatinine levels after belatacept conversion were compared with preconversion levels. Patient survival was 100%, and graft survival was 88%. The mean creatinine fell from 3.9±1.82 mg/dL prebelatacept conversion to 2.1±1.1 mg/dL at 6 months and 1.9±0.47 mg/dL (median 1.8 mg/dL) at 12 months postconversion. There was no significant increased risk of rejection, infection, or malignancy. HIV parameters remained largely stable. Early conversion to belatacept in patients with DGF or slow graft function is safe and efficacious, in a single-center nonrandomized retrospective analysis.