Buglioni, Alessia

Background. Mesangial expansion (ME) is an understudied histologic lesion in renal allografts. The current Banff mm score is not reproducible and may miss important ME features. The study aimed to improve the quantification of ME using morphometry, assess changes over time, and determine its association with allograft loss. Methods. We studied ME in 1-y and 5-y surveillance biopsies in 835 kidney transplants performed between January 2000 and December 2013. ME was assessed using the Banff mm score by a central pathologist and by morphometry. We derived 3 different morphometric measures: (1) %ME mm (%glomeruli with ME in ≥2 lobules, like Banff mm); (2) %MEany (%glomeruli with any ME lesion); and (3) %ME area (sum of all ME areas/all glomerular tuft areas). Unadjusted and adjusted Cox models assessed the risk of death-censored allograft loss. Results. From 1- to 5-y biopsies, the mean Banff mm score increased from 0.18 to 0.34, whereas %ME mm increased from 2.5% to 13.3%. Banff mm score had modest correlations with morphometric ME measures. Moderate-severe %ME mm was present in 20.1% of 5-y biopsies, whereas only 6.6% of Banff mm scores were. In general, higher ME on both 1- and 5-y biopsies was associated with a deceased donor, older recipient age, recipient diabetes/obesity (present in >50% of severely affected biopsies), higher hemoglobin A1c at 5 y posttransplant, and recurrent kidney disease. Higher ME on 5-y biopsies was associated with delayed graft function. A higher Banff mm score at 1-y biopsy and morphometry ME measures at 5-y biopsy were associated with rejection during the first year posttransplant. Morphometric ME measures were associated with allograft loss independent of Banff scores and all clinical characteristics, including kidney function and recurrent disease. The model with %MEany had the highest c-statistic (0.872). Conclusions. Banff mm score underestimates the pervasiveness of ME in 5-y biopsies. ME is common and associated with alloimmune and nonalloimmune causes of graft loss.

Introduction: Aldosterone (Aldo) increases sodium and water reabsorption and mediates inflammatory processes. Its relationship to co-morbidities has not been well defined in the general population. Hypothesis: We addressed the hypothesis that plasma aldosterone concentration (PAC) is associated with metabolic and cardiorenal diseases in the general community. Methods: We determined PAC in a general community-based cohort from Olmsted County, MN (n=1,674), and defined correlations with cardiorenal, metabolic diseases and myocardial function and structure. Results: PAC was detected in all subjects and ranged from 2.5 to 91 ng/dl (mean value 6.7 ng/dl, +/- SD 6.6). Multivariate analysis adjusted for age, gender, BMI, ANP and BNP showed highly significant correlations between PAC and hypertension (HTN) (p<.0001, OR=2.28, CI:1.91-2.71), atrial fibrillation (p<.01), chronic kidney disease (CKD) (p<.0001, OR=1.64, CI:1.33-2.02), central obesity (p<.0001, OR=1.61, CI:1.27-2.05), hypertriglyceridemia (p<.003), prevalence of metabolic syndrome (MetS) (p<.0007, OR=1.40, CI=1.15-1.71), obesity (p<.0001, OR=1.48, CI:1.27-1.73), and concentric left ventricular hypertrophy (cLVH) (p=.01). Recognizing that ANP and BNP are known inhibitors of Aldo, we defined the relationship between these cardiac hormones (NTpro-ANP and NT-proBNP) and PAC and observed highly significant inverse relationships with NTpro-ANP (p=0.0009), NTpro-BNP (p=0.0003) in the general population. Conclusions: In conclusion, in the general community, PAC is strongly correlated with renal, metabolic and cardiovascular diseases as well as cardiac remodeling. Specifically, increasing PAC is strongly associated with HTN, CKD, obesity, MetS, cLVH, and lower levels of natriuretic peptides. Our data suggest a key role of Aldo as a possible biomarker and/or mediator of human cardiorenal and metabolic diseases even in the general adult population, prior to overt disease. This study also supports a possible counter regulatory relationship between cardiac natriuretic peptides and PAC. Further studies are warranted to evaluate possible therapeutic interventions targeting Aldo as a strategy to delay disease progression.

Introduction: As importantly underscored by the SPRINT Trial, innovative blood pressure (BP) lowering and multiorgan protective drugs are needed for treating hypertension (HTN). We engineered a Mayo Clinic designer atrial natriuretic peptide (ANP)-based drug, ZD100, which possesses a unique 12 amino acid (AA) carboxy-terminus (CT) extension while maintaining the N-terminus (NT) of ANP. ZD100 is more potent than ANP in reducing BP, inducing natriuresis and inhibiting aldosterone via pGC-A and its second messenger cGMP. To date, the in vivo actions of ZD100 in a model of essential HTN are undefined. Further, in attempt to enhance and/or minimize pGC-A activation, the importance of specific AAs in the CT and NT of ZD100 is unknown and is critical to define. Hypothesis: We hypothesize that ZD100 is a potent anti-HTN drug activating pGC-A/cGMP system and that specific AA variations in the CT and/or NT of ZD100 will modify the ability of activating pGC-A. Methods: Spontaneously hypertensive rats (SHRs) received intravenous (IV) ZD100 or vehicle and BP, plasma and urinary cGMP and the natriuretic effects were determined. We then engineered 15 strategic ZD100 variants (Vs) consisting of mutated peptides in the CT or NT or both. We stimulated HEK293 cells overexpressing pGC-A with ZD100 and the 15 Vs (10-8 M) and measured intracellular cGMP generation. Results: In SHRs, acute IV ZD100 reduced BP, increased plasma and urinary cGMP generation and induced natriuresis demonstrating efficacy in HTN. In vitro, we found two more potent pGC-A activators (+100% cGMP activation) only among the CT-only mutated ZD100 Vs. Importantly, key mutations of either the CT or NT resulted in 6 loss-of-function Vs identifying importance of peptide-receptor dependency. Conclusions: We demonstrate for the first time in a genetic model of HTN that ZD100 reduces BP, activates systemic and renal pGC-A/cGMP and induces natriuresis. Enhanced or reduced pGC-A activation with targeted AA mutations in the CT and NT of ZD100 provides key structure function insights into ZD100 which may lead to next generation ZD100 therapeutic peptides. These findings support ZD100 as a novel peptide therapeutic strategy for HTN and will help guide the selection of best-in-class ZD100 analogues.

A 74-year-old male presented with rectal pain; workup uncovered an anal mass, and a diagnosis of melanoma was rendered via histologic examination and immunohistochemical (IHC) studies. Droplet digital PCR (ddPCR)–based BRAF testing was performed and revealed the presence of BRAF V600E, which is a common targetable genetic alteration in melanoma. Interestingly, the ratio of mutant to wild-type copy number was low (0.3%), whereas tumor cell percentage on tissue slides was 90%. With additional workup, BRAF V600E IHC confirmed a very small subset of BRAF V600E–positive cells, and a next-generation sequencing (NGS) panel revealed a pathogenic KIT variant, p.L576P, with an allele frequency of 63%. It was initially hypothesized that the main driver of the melanoma was the KIT alteration, whereas a small subclone (not detected by NGS, which has a 5% limit of detection) was driven by the BRAF V600E detected by ddPCR. To determine whether there were morphologic differences between the 2 clones, a careful review of the histology was performed and revealed distinct morphology of the BRAF V600E–positive cells, including pale cytoplasm, nuclear grooves, and infiltrating eosinophils. Additional IHC workup of the BRAF V600E–positive cells showed coexpression of CD1a, Langerin, and S100, diagnostic of Langerhans cell histiocytosis (LCH). This diagnosis was unexpected and would have been missed without highly sensitive molecular testing; yet it is of clinical importance for the patient. This case raises interesting biology questions regarding the relationship between melanoma and LCH; moreover, it highlights the importance of integrating quantitative information in molecular data interpretation.

Abstract Background and Aims Immune-checkpoint inhibitors (ICI) are antineoplastic therapies that unleash the immune-system and have revolutionized cancer outcomes. Though they are a drug class with great potential, their effects are not always limited to the malignant cells and can cause a spectrum of immune-related adverse events (irAEs). Roughly 5% of patients will experience acute kidney injury (AKI) with acute interstitial nephritis (AIN) being the predominant pathologic lesion. Glomerular and vascular lesions are thought to be rare and, as a result, not well-defined. We aim to document glomerular and vascular pathology in this context at our institution. Method We performed a retrospective review of internal native renal biopsies from 2010-2022 that had known ICI treatment. Standard processing of renal biopsies included light microscopy (LM), immunofluorescence (IF), and electron microscopy (EM). We reviewed the biopsies and pathology reports, as well as clinical data from patient electronic medical records. Results Our search yielded 32 kidney biopsies; of those, most had AIN (27/32), 8 cases had glomerular pathology, and 1 case had arterial endotheliitis. Of the cases with glomerular pathology, 4 had subacute glomerular thrombotic microangiopathy (TMA). Among the cases with TMA, 2 were also treated with vascular endothelial growth factor inhibitor (VEGFi),1 with gemcitabine, and 1 with BRAF-inhibitor therapy. Further, only one of these patients had complement testing, which did not reveal definitive deficiency. One patient had proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID), IgG-κ. One patient had IgA nephropathy (IgAN) and another had membranous nephropathy (MN). Most patients were biopsied for rising serum creatinine (SCr) (median: 1.6 mg/dL; range: 0.55-5.5); 1 had nephrotic-range proteinuria (median urine protein/creatinine ratio (UPCR): 0.3; range: 0.12-5.64). One, with thin basement membrane disease, had significant hematuria (&gt;100 RBC/hpf). Five patients had irAEs in other organs. 7/9 patients died or entered hospice care (median: 14 months post-biopsy; range: 2-48 months) due to malignancy progression. Of the deceased patients, 2 were dialysis-dependent. Of the living patients, 1 has dialysis-independent chronic kidney disease (CKD). Conclusion Overall, glomerular and vascular pathology due to ICI-use appears rare though difficult to isolate since other drugs and neoplasia can cause similar lesions. In our cohort, TMA was the most common glomerular lesion, though direct causation due to ICI is difficult to prove. In all patients with TMA, we identified concurrent use of other known causative drugs. IgAN and MN have been reported in association with neoplasia. PGNMID has been reported in a variety of malignant and nonmalignant conditions, and ICI-use could be a triggering factor. In conclusion, the patterns of glomerular injury seen in our cohort may be secondary to other concurrent risk factors other than ICI use. Arterial endotheliitis appears to be a rare but significant secondary effect to ICI-use.

Introduction: Plasma aldosterone (Aldo) increases Na and water reabsorption and mediates inflammation. Our recent findings in a general population cohort showed Aldo strongly associated with hypertension (HTN), obesity, chronic kidney disease (CKD) and lower natriuretic peptide levels (NPs) during a first visit (V1), even when Aldo was within the normal range. It is undefined whether Aldo itself (V1) and/or its increase after 4-year (second visit, V2) can predict new onset cardiovascular and metabolic diseases. Hypotheses: We hypothesized that: 1) Aldo-V1 predicts new onset disease at V2; 2) The increase of Aldo between V1 and V2 predicts new onset disease-V2; and 3) Aldo-V2 remains associated with cardiovascular and metabolic disease-V2. Methods: We defined the correlations between Aldo-V1 and Aldo-V2 with cardiometabolic diseases and medications at V2, in a general community cohort (N=1368) from Olmsted County, MN. Associations were adjusted for age, sex and BMI; Aldo was analyzed as continuous variable and according to tertiles. Results: After 4 years, Aldo-V1 continuous variable predicted new onset of central obesity (p=0.0113, OR=1.36, CI=1.07-1.73), HTN (p=0.0012, OR=1.36, CI=1.13-1.63) and use of antilipemic drugs (p=0.0119, OR=1.25, CI=1.05-1.48); while Aldo-V1 in the 3 rd tertile predicted new onset of type 2 diabetes (T2DM) (p=0.0392, OR=1.96, CI=1.03-3.70), use of antilipemic therapy (p=0.0162, OR=1.59, CI=1.09-2.31) and HTN (p=0.049, OR1.44, CI=1.00-2.08) at V2. Secondly, we observed that the increase in Aldo between V1 and V2 predicted new HTN-V2. Finally, we found higher Aldo-V2, but still within the normal range and Aldo-V2 remained associated with HTN, obesity and CKD, when analyzed as continuous variable and when in the 3 rd tertile. Conclusions: In the general community, Aldo-V1 predicts new onset HTN, central obesity, T2DM and use of antilipemic therapy. Importantly, the increase of aldosterone between V1 and V2 also predicts new onset HTN. Finally, Aldo-V2 correlates with HTN, obesity and CKD as we found at V1. Further studies are warranted to evaluate aldosterone as therapeutic target to delay disease progression, and to support its role as prognostic biomarker to identify high-risk subjects.

Abstract Background: Discovery of methylated DNA markers (MDM) of esophageal squamous cell carcinoma (ESCC) has sparked interest in assessing these markers in tissue. We evaluated MDMs in ESCC from three geographically and ethnically distinct populations, and explored the feasibility of assaying MDMs from DNA obtained by swallowed balloon devices. Methods: MDMs were assayed in ESCC and normal tissues obtained from the populations of United States, Iran, and China, and from exfoliative cytology specimens obtained by balloons in a Chinese population. Areas under the receiver operating curve (AUC) of MDMs discriminating ESCC from normal tissues were calculated. Random forest prediction models were built, trained on U.S. cases and controls, and calibrated to U.S.-only controls (model 1) and three-country controls (model 2). Statistical tests were used to assess the relationship between dysplasia and MDM levels in balloons. Results: Extracted DNA from 333 ESCC and 322 normal tissues was analyzed, in addition to archival DNA from 98 balloons. For ESCC, model 1 validated in Iranian and Chinese tissues with AUCs of 0.90 and 0.87, and model 2 yielded AUCs of 0.99, 0.96, and 0.94 in tissues from the United States, Iran, and China, respectively. In Chinese balloons, MDMs showed a statistically significant trend of increasing levels with increasing grades of dysplasia (P &lt; 0.004). Conclusions: MDMs accurately discriminate ESCC from normal esophagus in tissues obtained from high- and low-incidence countries. Preliminary data suggest that levels of MDMs assayed in DNA from swallowed balloon devices increase with dysplasia grade. Larger studies are needed to validate these results. Impact: MDMs coupled with minimally invasive collection methods have the potential for worldwide application in ESCC screening.

Abstract Objectives To examine the immunohistochemical and ultrastructural features of hepatocellular cytoplasmic globules in venous outflow impairment (VOI). Methods Sixty-four liver core biopsies were screened. Patients with α-1 antitrypsin (AAT) deficiency were excluded. All biopsies were stained with H&E, Masson trichrome, periodic acid-Schiff with diastase digestion (PAS-D), phosphotungstic acid hematoxylin (PTAH), complement protein 4d (C4d) immunostain, and AAT immunostain. Electron microscopy was also performed. Results Hepatocellular globules were identified in 8% of in-house cases. Causes of VOI included heart failure and Budd-Chiari syndrome. The hepatocellular cytoplasmic globules showed size variability, random distribution, and positivity for PAS-D, PTAH, and AAT. C4d was inconsistently positive. Electron microscopy showed that the globules were lysosome-bound inclusions containing microfibrillar material and fibrinogen. Conclusions PAS-D–positive hepatocellular globules occur in VOI. They cross-react with AAT but have different appearance, localization, and ultrastructural composition from globules in AAT deficiency.