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.

Multiscale genetic architecture of donor-recipient differences reveals intronic LIMS1 mismatches associated with kidney transplant survival.

Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLAs) contribute to kidney allograft loss, but the mechanisms remain unclear, specifically for intronic mismatches. We quantified non-HLA mismatches at variant-, gene-, and genome-wide scales from single nucleotide polymorphism (SNP) data of D-Rs from 2 well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n = 385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n = 146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major-allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T cell transcriptome analyses associated the GCC2 gene and LIMS1 SNPs with the TGF-ß1/SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGF-ß1 and downstream signaling in T cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGF-ß1-dependent effects on T cells.

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli.

Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time-consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic- and paraffin-embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2-profile and Weibel-Gomez (WG) methods and a novel 3-profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10-glomerular sampling versus 20-glomerular sampling using the Cav method, while 5-glomerular sampling was less precise. In plastic tissue, 2- or 3-profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2- or 3-profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3-profile method offered incremental benefit to the 2-profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin-embedded versus plastic-embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3-profile method offers slightly improved concordance with reduced bias versus 2-profile. Our findings have implications for future studies using glomerular morphometry.

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.

Blood Transcriptomes of SARS-CoV-2-Infected Kidney Transplant Recipients Associated with Immune Insufficiency Proportionate to Severity.

BACKGROUND: Among patients with COVID-19, kidney transplant recipients (KTRs) have poor outcomes compared with non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort. METHODS: We ascertained clinical data by chart review. A single sample of blood was collected for transcriptome analysis. Total RNA was poly-A selected and RNA was sequenced to evaluate transcriptome changes. We also measured cytokines and chemokines of serum samples collected during acute infection. RESULTS: A total of 64 patients with COVID-19 in KTRs were enrolled, including 31 with acute COVID-19 (<4 weeks from diagnosis) and 33 with post-acute COVID-19 (>4 weeks postdiagnosis). In the blood transcriptome of acute cases, we identified genes in positive or negative association with COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways but downregulation of T cell and adaptive immune activation pathways. This finding was independent of lymphocyte count, despite reduced immunosuppressant use in most KTRs. Compared with acute cases, post-acute cases showed "normalization" of these enriched pathways after 4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of immunosuppression. Analysis of the non-KTR cohort with COVID-19 showed significant overlap with KTRs in these functions. Serum inflammatory cytokines followed an opposite trend (i.e., increased with disease severity), indicating that blood lymphocytes are not the primary source. CONCLUSIONS: The blood transcriptome of KTRs affected by COVID-19 shows decreases in T cell and adaptive immune activation pathways during acute disease that, despite reduced immunosuppressant use, associate with severity. These pathways show recovery after acute illness.

Blood transcriptomes of SARS-CoV-2 infected kidney transplant recipients demonstrate immune insufficiency.

BACKGROUND: Kidney transplant recipients (KTRs) with COVID-19 have poor outcomes compared to non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort.□. METHODS: Clinical data were collected by chart review. PAXgene blood RNA was poly-A selected and RNA sequencing was performed to evaluate transcriptome changes. RESULTS: A total of 64 cases of COVID-19 in KTRs were enrolled, including 31 acute cases (< 4 weeks from diagnosis) and 33 post-acute cases (>4 weeks). In the blood transcriptome of acute cases, we identified differentially expressed genes (DEGs) in positive or negative association COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways, but downregulation of T-cell and adaptive immune-activation pathways proportional to severity score. This finding was independent of lymphocyte count and despite reduction in immunosuppression (IS) in most KTRs. Comparison with post-acute cases showed "normalization" of these enriched pathways after >4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of IS. The latter analysis was adjusted for COVID-19 severity score and lymphocyte count. DEGs associated with worsening disease severity in a non-KTR cohort with COVID-19 (GSE152418) showed significant overlap with KTRs in these identified enriched pathways. CONCLUSION: Blood transcriptome of KTRs affected by COVID-19 shows decrease in T-cell and adaptive immune activation pathways during acute disease that associate with severity despite IS reduction and show recovery after acute illness. SIGNIFICANCE STATEMENT: Kidney transplant recipients (KTRs) are reported to have worse outcomes with COVID-19, and empiric reduction of maintenance immunosuppression is pursued. Surprisingly, reported rates of acute rejection have been low despite reduced immunosuppression. We evaluated the peripheral blood transcriptome of 64 KTRs either during or after acute COVID-19. We identified transcriptomic signatures consistent with suppression of adaptive T-cell responses which significantly associated with disease severity and showed evidence of recovery after acute disease, even after adjustment for lymphocyte number. Our transcriptomic findings of immune-insufficiency during acute COVID-19 provide an explanation for the low rates of acute rejection in KTRs despite reduced immunosuppression. Our data support the approach of temporarily reducing T -cell-directed immunosuppression in KTRs with acute COVID-19.

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.

Honokiol regulates mitochondrial substrate utilization and cellular fatty acid metabolism in diabetic mice heart.

Type 2 diabetes mellitus is strongly associated with cardiac mitochondrial dysfunction, which is one of the main reasons for cardiovascular diseases. Among the mitochondrial metabolic changes, fatty acid metabolism is of great importance as cardiac tissues depend primarily on fatty acids. Honokiol, a constituent of Magnolia tree bark extract, is reported to strongly influence cardiac mitochondrial functions, via various mechanisms. The current study showed that honokiol decreased fatty acid-mediated complex I respiration and increased carbohydrate-mediated complex I and II respiration in diabetic C57BL/6 mice cardiac mitochondria. It was also found that honokiol treatment decreased expression of Cluster of Differentiation 36, AMP-activated kinases and nuclear transcription factors like, Peroxisome proliferator-activated receptor γ co-activator 1α/ß and Peroxisome proliferator-activated receptor α, surrogating the evidence of decreased fatty acid-mediated complex I respiration. Honokiol treatment also reduced the levels of mitochondrial acetylated proteins, suggesting the possible action of honokiol via acetylation/deacetylation mechanism of regulation of protein functions in diabetic mitochondria. The antioxidant effect of honokiol is evidenced by the augmented expression of Manganese super oxide dismutase. In conclusion, honokiol imparts beneficial effect on diabetic cardiac mitochondria by decreasing the oxidant burden via regulating mitochondrial fatty acid respiration and expression of oxidant response factors.

Are nitric oxide-mediated protein modifications of functional significance in diabetic heart? ye'S, -NO', wh'Y-NO't?

Protein modifications effected by nitric oxide (NO) primarily in conjunction with reactive oxygen species (ROS) include tyrosine nitration, cysteine S-nitrosylation, and glutathionylation. The physiological and pathological relevance of these three modifications is determined by the amino acids on which these modifications occur -cysteine and tyrosine, for instance, ranging from altering structural integrity/catalytic activity of proteins or by altering propensity towards protein degradation. Even though tyrosine nitration is a well-established nitroxidative stress marker, instilled as a footprint of oxygen- and nitrogen-derived oxidants, newer data suggest its wider role in embryonic heart development and substantiate the need to focus on elucidating the underlying mechanisms of reversibility and specificity of tyrosine nitration. S-nitrosylation is a covalent modification in specific cysteine residues of proteins and is suggested as one of the ways in which NO contributes to its ubiquitous signalling. Several sensitive and specific techniques including biotin switch assay and mass spectrometry based analysis make it possible to identify a large number of these modified proteins, and provide a great deal of potential S-nitrosylation sites. The number of studies that have documented nitrated proteins in diabetic heart is relatively much less compared to what has been published in the normal physiology and other cardiac pathologies. Nevertheless, elucidation of nitrated proteome of diabetic heart has revealed the presence of many mitochondrial and cytosolic proteins of functional importance. But, the existence of different models of diabetes and analyses at diverse stages of this disease have impeded scientists from gaining insights that would be essential to understand the cardiac complications during diabetes. This review summarizes NO mediated protein modifications documented in normal and abnormal heart physiology including diabetes.