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Device-related thrombosis (DRT) is one of the greatest challenges of transcatheter left atrial appendage device occlusion. Due to the invasive nature of transesophageal echocardiography (TEE), cardiac computed tomography angiography (CCTA) is being increasingly utilized in several centers for assessing adequate left atrial appendage closure and monitoring for DRT. There is a paucity of data regarding the standardized definition of DRT on CCTA for the WATCHMAN FLX™ device.A retrospective review was conducted on 43 patients receiving WATCHMAN FLX™ device implantation with CCTA performed at the first follow-up at our institution. A comparative review of DRT predictors was performed on 10 patients who had both CCTA and TEE at the time of follow-up.Hypoattenuated thickening (HAT) was a common finding on CCTA and was noted to be present in 95.35% of the patients. The combination of a large device size, peridevice gap4 mm, and HAT located on the device gutter and 1 shoulder were characteristics present on CCTA observed in 2 patients with confirmed DRT on TEE.CCTA is a noninvasive imaging modality for DRT monitoring, with guidelines still in development. We report potential predictors of DRT on CCTA. Additional studies are needed to further determine standardized parameters for DRT detection on CCTA and the significance of HAT with multimodality correlation.

Technetium-99 pyrophosphate scintigraphy (99mTc-PYP) provides qualitative and semiquantitative diagnosis of ATTR cardiac amyloidosis (ATTR-CA) using the Perugini scoring system and heart/contralateral heart ratio (H/CL) on planar imaging. Standardized uptake values (SUV) with quantitative single photon emission computed tomography (xSPECT/CT) can offer superior diagnostic accuracy and quantification through precise myocardial contouring that enhances assessment of ATTR-CA burden. We examined the correlation of xSPECT/CT SUVs with Perugini score and H/CL ratio. We also assessed SUV correlation with cardiac magnetic resonance (CMR), echocardiographic, and baseline clinical characteristics. Retrospective review of 78 patients with suspected ATTR-CA that underwent 99mTc-PYP scintigraphy with xSPECT/CT. Patients were grouped off Perugini score (Grade 0-1 and Grade 2-3), H/CL ratio (≥ 1.5 and1.5). Two cohorts were also created: myocardium SUV

Background Ibrutinib is a Bruton’s tyrosine kinase inhibitor used in the treatment of hematological malignancies. The most common cardiotoxicity associated with ibrutinib is atrial arrhythmia (atrial fibrillation and flutter). It is known that patients with cardiovascular disease (CVD) are at an increased risk for developing atrial arrhythmia. However, the rate of atrial arrhythmia in patients with pre-existing CVD treated with ibrutinib is unknown. Objective This study examined whether patients with pre-existing CVD are at a higher risk for developing atrial arrhythmias compared to those without prior CVD. Methods A single-institution retrospective chart review of patients with no prior history of atrial arrhythmia treated with ibrutinib from 2012 to 2020 was performed. Patients were grouped into two cohorts: those with CVD (known history of coronary artery disease, heart failure, pulmonary hypertension, at least moderate valvular heart disease, or device implantation) and those without CVD. The primary outcome was incidence of atrial arrhythmia, and the secondary outcomes were all-cause mortality, risk of bleeding, and discontinuation of ibrutinib. The predictors of atrial arrhythmia (namely atrial fibrillation) were assessed using logistic regression. A Cox-Proportional Hazard model was created for mortality. Results Patients were followed for a median of 1.1 years. Among 217 patients treated with ibrutinib, the rate of new-onset atrial arrhythmia was nearly threefold higher in the cohort with CVD compared to the cohort without CVD (17% vs 7%, p = 0.02). Patients with CVD also demonstrated increased adjusted all-cause mortality (OR 1.9, 95% CI 1.06-3.41, p = 0.01) and decreased survival probability (43% vs 54%, p = 0.04) compared to those without CVD over the follow-up period. There were no differences in risk of bleeding or discontinuation between the two cohorts. Conclusions Pre-existing cardiovascular disease was associated with significantly higher rates of atrial arrhythmia and mortality in patients with hematological malignancies managed with ibrutinib.

There is emerging recent data that has shown women to be more prone to in-hospital major adverse events after transcatheter left atrial appendage occlusion.Institutional LAAO registry at West Virginia University (WVU) was reviewed from January 2016 to October 2021 to identify 271 women and 293 men who underwent successful LAAO device implantation. Patients were evaluated for gender-based differences in baseline characteristics, CHA₂DS₂-VASc Score, HAS-BLED score, procedural data, in-hospital, and follow-up outcomes.Compared to men, women had lower baseline comorbidities including coronary artery disease (135 (49.6%) vs. 172 (58.7%), p=0.03), myocardial infarction (MI) (56 (20.5%) vs. 85 (29%), p=0.02) and coronary artery bypass surgery (10 (3.6%) vs. 27 (9.2%), p=0.008). Women were noted to have a higher CHA₂DS₂-VASc Score (5.3 ± 1.4 vs. 4.4 ± 1.4, p0.001), and left ventricular ejection fraction (57.9 ± 7.7 vs. 52.7 ± 12.4, p0.001). Women were noted to have a significantly higher rate of in-hospital composite adverse events (74 (27.2%) vs. 58 (19.8%), p=0.03); bleeding events (38 (10.2%) vs. 19 (6.4%), p=0.003) and associated blood transfusion (6 vs. 0, p=0.001) compared with men. No statistically significant differences were noted between both genders regarding the follow-up outcome.Our single center study shows women to have higher in-hospital composite adverse events as well as higher bleeding events during the index hospital admission.

Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-Gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsy-confirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

The Kidney Precision Medicine Project (KPMP) is a multisite study designed to improve understanding of CKD attributed to diabetes or hypertension and AKI by performing protocol-driven kidney biopsies. Study participants and their kidney tissue samples undergo state-of-the-art deep phenotyping using advanced molecular, imaging, and data analytical methods. Few patients participate in research design or concepts for discovery science. A major goal of the KPMP is to include patients as equal partners to inform the research for clinically relevant benefit. The purpose of this report is to describe patient and community engagement and the value they bring to the KPMP. Patients with CKD and AKI and clinicians from the study sites are members of the Community Engagement Committee, with representation on other KPMP committees. They participate in KPMP deliberations to address scientific, clinical, logistic, analytic, ethical, and community engagement issues. The Community Engagement Committee guides KPMP research priorities from perspectives of patients and clinicians. Patients led development of essential study components, including the informed consent process, no-fault harm insurance coverage, the ethics statement, return of results plan, a "Patient Primer" for scientists and the public, and Community Advisory Boards. As members across other KPMP committees, the Community Engagement Committee assures that the science is developed and conducted in a manner relevant to study participants and the clinical community. Patients have guided the KPMP to produce research aligned with their priorities. The Community Engagement Committee partnership has set new benchmarks for patient leadership in precision medicine research.

Due to multiple compensating mechanisms, the serum bicarbonate concentration is a relatively insensitive marker of acid-base status; especially in chronic kidney disease (CKD). This is a major drawback that impairs the ability to diagnose acid excess or monitor alkali therapy. We postulated that it is more logical to measure the compensatory defense mechanism(s) rather than the defended parameter, which remains normal if the compensation is successful. Therefore, a retrospective cross-sectional study was performed in 1733 stone formers along with a prospective cross-sectional study of 22 individuals with normal kidney function and 50 patients in different stages of CKD. While serum bicarbonate was flat and did not fall below the reference range until near CKD stage 5, citrate excretion (24-hour urinary citrate excretion rate; urinary citrate-to-creatinine ratio, in the retrospective analysis, and spot urinary citrate-to-creatinine ratio in the prospective study) progressively and significantly declined starting from CKD stage 2. Following an acute acid load in 25 participants with a wide range of estimated glomerular filtration rates, the urinary citrate-to-creatinine ratio inversely and significantly associated with acid accumulation, whereas serum bicarbonate did not. We compared changes in serum bicarbonate and urinary citrate-to-creatinine ratio in response to alkali therapy in patients with CKD stage 3 or 4 started on potassium citrate in our kidney stone database. With alkali therapy, there was no change in serum bicarbonate, but the urinary citrate-to-creatinine ratio rose consistently in all patients adherent to potassium citrate therapy. Thus, the urinary citrate-to-creatinine ratio (the defense mechanism) is a potential easily implementable, pragmatic, and a superior parameter to serum bicarbonate (the defended entity) to assess acid-base status, and monitor alkali therapy. Additional studies are needed before a clinical test can be devised.

Chronic kidney disease (CKD) and end-stage renal disease are traditionally associated with worse outcomes after endovascular and open repair of abdominal aortic aneurysm (AAA). This study stratifies outcomes of AAA repair by approach, CKD severity, and dialysis dependence.All patients undergoing elective infrarenal open aneurysm repair (OAR) and endovascular aortic repair (EVAR) with preoperative renal function data captured by the Vascular Quality Initiative between January 2003 and September 2020 were analyzed. Patients were stratified by CKD class as follows: CKD stages 1 and 2, CKD stage 3a, CKD stage 3b, CKD stages 4 and 5, and dialysis. Primary outcomes were perioperative and 1-year mortality. Predictors of survival were identified by Cox multivariate regression models.In total, 53,867 elective AAA repairs were identified: 5396 (10%) OARs and 48,471 (90%) EVARs. Most patients were White (90%) and male (81%), with a mean age of 73 ± 9 years. Patients who underwent EVAR were older and had more comorbidities. The use of elective EVAR for AAA increased from 52% in 2003 to 91% in 2020 (P .001). The OAR cohort had more perioperative complications and short-term mortality. The CKD 1 and 2 group had the highest 1-year survival compared with the other groups after both OAR and EVAR. On Cox regression analysis, after EVAR, compared with CKD 1 and 2, worsening CKD stage (CKD 3a: hazard ratio [HR], 1.25; 95% confidence interval [CI], 0.93-1.68; P = .13; CKD 3b: HR, 1.74; 95% CI, 1.23-2.45; P .050; CKD 4-5: HR, 3.23; 95% CI, 2.13-4.88; P .001), and dialysis (HR, 4.48; 95% CI, 1.90-10.6; P .001) were independently associated with worse 1-year survival rates. After OAR, compared with CKD 1 and 2, worsening CKD stage (CKD 3a: HR, 1.08; 95% CI, 0.96-1.20; P = .20; CKD 3b: HR, 1.60; 95% CI, 1.41-1.81; P .001; CKD 4-5: HR, 2.85; 95% CI, 2.39-3.41; P .001), and dialysis (HR, 3.79; 95% CI, 3.01-4.76; P .001) were independently associated with worse 1-year survival rates.Regardless of the treatment approach, CKD severity is an important predictor of perioperative and 1-year mortality rates after infrarenal AAA repair and may reflect the natural history of CKD. Open repair is associated with high perioperative mortality risk in patients with CKD stages 4 and 5, as well as end-stage renal disease. Individualization of patient decision-making is especially important in patients with a glomerular filtration rate of less than 45 and perhaps consideration should be given to raising the threshold for elective AAA repair in these patients. Further studies focusing on appropriate size threshold for repair in these patients may be warranted.

An understanding of the ethical underpinnings of human subjects research that involves some risk to participants without anticipated direct clinical benefit-such as the kidney biopsy procedure as part of the Kidney Precision Medicine Project (KPMP)-requires a critical examination of the risks as well as the diverse set of countervailing potential benefits to participants. This kind of deliberation has been foundational to the development and conduct of the KPMP. Herein, we use illustrative features of this research paradigm to develop a more comprehensive conceptualization of the types of benefits that may be important to research participants, including respecting pluralistic values, supporting the opportunity to act altruistically, and enhancing benefits to a participant's community. This approach may serve as a model to help researchers, ethicists, and regulators to identify opportunities to better respect and support participants in future research that entails some risk to these participants as well as to improve the quality of research for people with kidney disease.

Introduction: Device related thrombosis (DRT) is an indication for continuation of anticoagulation for patients undergoing left atrial appendage (LAA) occlusion. There is a paucity of data in regards to the definition of DRT on coronary CT angiography (CTA) in patients with WATCHMAN FLX TM (Boston scientific) device implantation. Methods: This is a single center experience of using CTA for follow up in patients post WATCHMAN FLX TM implantation. 37 consecutive patients who underwent WATCHMAN FLX TM implantation at our center and had CTA performed at first follow up were included. CTA images were analyzed for the presence of hypo-attenuated thickening (HAT) on the device, device positioning, peridevice gap, angulation, compression percentage, depth of implant from coumadin ridge and enhancement in the LAA compared to left atrium (LA). Results: Among 37 patients with CTA follow up, HAT was noted in 28 patients (75%). 38% of these patients had HAT >3 mm. LAA patency (LAA/LA ratio of >25%) was seen in 7 patients (10%), 4 with gaps around the device and the rest secondary to trans-fabric leak. Gap around the device was noted in 13 patients (35%). 6 of these 13 patients had >4 mm gap; 5/6 patients had HAT and 3/6 patients had >25% enhancement in the LAA compared to LA. Increased compression % on both CTA and intraprocedural TEE was associated with absence of HAT (p=0.0003) as well as better sealing on CTA (LAA/LA ratio of >25%) (p=0.04). 6 months follow up is available on only 18 patients; only one of these patients had stoke and that patient did not have HAT on the CT scan. Conclusions: Definition of thrombus on WATCHMAN FLX TM device is not clearly defined on CTA. HAT on CTA could be artifact from the device knob and gutter or visible endothelialization around the device. Further studies with multimodality imaging and clinical correlation are needed to help determine normal cut offs for HAT on these CTAs.

Pre-procedural chronic kidney disease (CKD) and in-hospital acute kidney injury (AKI) are associated with worse outcomes following transcatheter aortic valve replacement (TAVR). We tested the feasibility of reducing overall AKI by avoiding pre-procedural cardiac CT angiography (CCTA) by using direct 3D-TEE guidance in TAVR patients with known CKD.An institutional TAVR database was examined from January 2016 to June 2020 to identify 396 patients in whom CCTA sizing was performed and 54 patients with creatinine (Cr) of1.6 mg/dL in whom direct 3D-TEE, without prior CCTA, was used for TAVR guidance. Baseline demographics, procedural, echocardiographic, and clinical endpoints were compared as defined by the Valve Academic Research Consortium-2 criteria.Baseline demographics and risk factors were similar in both groups other than the creatinine level in CCTA vs. TEE groups (1.33 ± 1.1 vs 1.76 ± 0.7 mg/dL, p = 0.005). Procedural contrast volume was significantly lower in the TEE group compared to the CCTA group. No differences were noted in echocardiographic and clinical endpoints for both groups. Despite higher baseline Cr, patents in the TEE group experienced a similar pattern of changes in Cr compared to the CCTA group, with an overall renal improvement noted at the time of discharge for both groups.In patients with baseline CKD, careful avoidance of large contrast loads associated with CCTA and intra-procedural aortography by using TEE guidance may help reduce AKI following TAVR.

A gust simulation and modeling capability will support the aircraft certification process and therefore reduces the development cost. Two different gust modeling techniques are considered in this article: 1) a user-defined boundary condition available in the commercial flow solver of Cobalt and 2) a field-velocity method implemented in the open-source flow solver of FlowPsi . Specifically, this article describes implementation of gust models in the FlowPsi code including a new time-stepping scheme and a new grid-motion capability that could be used in the simulation of responding vehicles to wind gusts. In more detail, the three stage time-stepping scheme, as here developed, provides much better convergence properties than the Crank–Nicolson scheme available in the code. The grid motion using an external code, as here implemented, is a part of larger development plans to make FlowPsi communicate with external processes such as a finite element code. In addition, the FlowPsi code uses a field-velocity approach to simulate the wind gust which offers a reduced computational expense for simulating wind gust responses when compared to Cobalt . Instead of propagating the gust perturbation from the far inflow boundary as used in Cobalt , an artificial velocity profile is imposed on the grid cells to induce the gust effects on the vehicle. This article aims to determine the accuracy of FlowPsi in predicting gust responses when compared to Cobalt which has already been verified to produce accurate results. The two methods in different codes are compared for a number of gust profiles such as one-minus-cosine, step (sharp-edged), and a random gust profile. FlowPsi shows close convergence and agreement to Cobalt predictions for smooth gust profiles such as the one-minus-cosine, however, the code produces a numerical oscillation pattern at low free-stream Mach numbers when modeling gusts with a rapid change of velocity such as the step gust.

The thermal efficiency of an Otto cycle engine is directly related to the compression ratio (CR). However, in a spark-ignited engine, the CR is often restricted by full load knock, thus limiting part load efficiency. A proof of concept design and experimental study has been conducted on a 4-cylinder naturally aspirated spark-ignited (SI) engine whereby a four-bar linkage mechanism has been implemented to vary the CR. The base engine selected was a production 2.0L GM-LNF SI 4-cylinder engine with a stock CR of 9.2:1 and with a bore and stroke of 86mm and 86mm, respectively. The engine was modified to allow the centerline axis of rotation of the crankshaft to translate in an arc about a fixed point. With the use of the four-bar mechanism, and larger dome volume pistons, a range of 8:1 to 11.5:1 CR was achieved. The prototype VCR engine was tested and analyzed at three different CR’s at a fixed load of 600 kPa net indicated mean effective pressure gross (IMEPGROSS) at an engine speed of 1000 revolutions per minute (RPM). At this condition, a sweep of combustion phasing was conducted. with a stoichiometric air to fuel mixture for each case. The CR’s selected for engine testing were 8.7:1, 10.2:1, and 11.1:1. The processed data includes averaged cycle analysis of each of the test conditions including combustion phasing, combustion duration, and cycle variation. The combustion data was also analyzed to determine overall heat release, indicated gross, net, pumping mean effective pressures, and indicated fuel conversion efficiency for each of the CR’s. The studies show an indicated fuel conversion efficiency of 31.2% for the 8.7:1 CR. As the CR was increased to 10.2:1 and 11.1:1 the relative increase in efficiency was 7.1% and 9.7% respectively at MBT combustion phasing.

Background: Ibrutinib is a Bruton’s tyrosine kinase inhibitor used in the treatment of hematological malignancies. The most common cardiotoxicity associated with ibrutinib is atrial arrhythmia (atrial fibrillation and flutter). It is known that patients with cardiovascular disease (CVD) are at an increased risk for developing atrial arrhythmia. However, the rate of atrial arrhythmia in patients with pre-existing CVD treated with ibrutinib is unknown. Objective: This study examined whether patients with pre-existing CVD are at a higher risk for developing atrial arrhythmias compared to those without prior CVD. Methods: A single-institution retrospective chart review of patients with no prior history of atrial arrhythmia treated with ibrutinib from 2012 to 2020 was performed. Patients were grouped into two cohorts: those with CVD (known history of coronary artery disease, heart failure, pulmonary hypertension, at least moderate valvular heart disease, or device implantation) and those without CVD. The primary outcome was incidence of atrial arrhythmia, and the secondary outcomes were all-cause mortality, risk of bleeding, and discontinuation of ibrutinib. Results: Patients were followed for a median of 1.1 years. Among 217 patients treated with ibrutinib, the rate of new-onset atrial arrhythmia was nearly threefold higher in the cohort with CVD compared to the cohort without CVD (17% vs 7%, p = 0.02). Patients with CVD also demonstrated increased adjusted all-cause mortality (OR 1.9, 95% CI 1.06–3.41, p = 0.01) and decreased survival probability (43% vs 54%, p = 0.04) compared to those without CVD over the follow-up period. Conclusions: Pre-existing cardiovascular disease was associated with significantly higher rates of atrial arrhythmia and mortality in patients with hematological malignancies managed with ibrutinib.

Acute kidney injury is a reversible medical condition commonly caused by nephrotoxic agents. The infrequency that a nebulized medication elicits a renal insult presents a rare diagnostic challenge. Within this case, we report a 57-year-old cystic fibrosis patient with chronic kidney disease (CKD) Stage G3b (baseline 1.5–1.6 mg/dL) who developed an acute kidney injury (AKI) with a serum creatinine elevation to 4.08 mg/dL and associated worsening vestibular dysfunction related to twice-daily nebulized tobramycin inhalation solution (TIS). The patient was found to have a tobramycin serum level of 4.2 μg/mL 2.5 h after TIS dosing, with elevation remaining present at 1.1 μg/mL 24 h after discontinuation of therapy. Laboratory values at one month continued to show elevated creatinine levels at 2.1 mg/dL, suggesting progression of his baseline CKD. This case supports the benefit of obtaining tobramycin serum levels and vestibular/audiology function testing when evaluating patients on chronic nebulized TIS who present with acute or chronic renal dysfunction. From these serum levels, adjustments to daily dosing, regular monitoring of tobramycin serum levels, or discontinuation of treatment should be made to prevent permanent renal damage in patients with CKD. Calculated Naranjo ADR Probability Scale: 9; Definite.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are common, heterogeneous, and morbid diseases. Mechanistic characterization of CKD and AKI in patients may facilitate a precision medicine approach to prevention, diagnosis, and treatment. The Kidney Precision Medicine Project aims to ethically and safely obtain kidney biopsies from participants with CKD or AKI, create a reference kidney atlas, and characterize disease subgroups to stratify patients based on molecular features of disease, clinical characteristics, and associated outcomes. An additional aim is to identify critical cells, pathways, and targets for novel therapies and preventive strategies. This project is a multicenter prospective cohort study of adults with CKD or AKI who undergo a protocol kidney biopsy for research purposes. This investigation focuses on kidney diseases that are most prevalent and therefore substantially burden the public health, including CKD attributed to diabetes or hypertension and AKI attributed to ischemic and toxic injuries. Reference kidney tissues (for example, living kidney donor biopsies) will also be evaluated. Traditional and digital pathology will be combined with transcriptomic, proteomic, and metabolomics analysis of the kidney tissue as well as deep clinical phenotyping for supervised and unsupervised subgroup analysis and systems biology analysis. Participants will be followed prospectively for ten years to ascertain clinical outcomes. Cell types, locations, and functions will be characterized in health and disease in an open, searchable, online kidney tissue atlas. All data from the Kidney Precision Medicine Project will be made readily available for broad use by scientists, clinicians, and patients.

Cytoskeletal structure and its regulation are essential for maintenance of the differentiated state of specific types of cells and their adaptation to physiologic and pathophysiologic conditions. Renal glomerular capillaries, composed of podocytes, endothelial cells, and the glomerular basement membrane, have distinct structural and biophysical properties and are the site of injury in many glomerular diseases. Calcineurin inhibitors, immunosuppressant drugs used for organ transplantation and auto-immune diseases, can protect podocytes and glomerular capillaries from injury by preserving podocyte cytoskeletal structure. These drugs cause complications including hypertension and hyperkalemia which are mediated by WNK (With No Lysine) kinases as well as vasculopathy with glomerulopathy. WNK kinases and their target kinases oxidative stress-responsive kinase 1 (OSR1) and SPS1-related proline/alanine-rich kinase (SPAK) have fundamental roles in angiogenesis and are activated by calcineurin inhibitors, but the actions of these agents on kidney vasculature, and glomerular capillaries are not fully understood. We investigated WNK1 expression in cultured podocytes and isolated mouse glomerular capillaries to determine if WNK1 contributes to calcineurin inhibitor-induced preservation of podocyte and glomerular structure. WNK1 and OSR1/SPAK are expressed in podocytes, and in a pattern similar to podocyte synaptopodin in glomerular capillaries. Calcineurin inhibitors increased active OSR1/SPAK in glomerular capillaries, the Young’s modulus (E) of glomeruli, and the F/G actin ratio, effects all blocked by WNK inhibition. In glomeruli, WNK inhibition caused reduced and irregular synaptopodin-staining, abnormal capillary and foot process structures, and increased deformability. In cultured podocytes, FK506 activated OSR1/SPAK, increased lamellipodia, accelerated cell migration, and promoted traction force. These actions of FK506 were reduced by depletion of WNK1. Collectively, these results demonstrate the importance of WNK1 in regulation of the podocyte actin cytoskeleton, biophysical properties of glomerular capillaries, and slit diaphragm structure, all of which are essential to normal kidney function.

The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell’s physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ. Tissue cytometry is a promising new microscopy technique that can be used to enumerate and characterize each cell in a tissue. Here the authors describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear. Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury. Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes. Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

Objective Autoimmune lymphocytic parathyroiditis and acquired hypocalciuric hypercalcemia associated with autoantibodies against the calcium-sensing receptor (anti-CaSR) are rare and poorly understood conditions. Here, we describe a patient with acquired parathyroid hormone (PTH)-dependent hypercalcemia with associated hypocalciuria, found to have true lymphocytic parathyroiditis on histopathology, and circulating anti-CaSR antibodies in serum. Design and methods A 64-year-old woman was referred to our clinic for persistent hypercalcemia after a subtotal parathyroidectomy. She was normocalcemic until the age of 63 years when she was diagnosed with primary hyperparathyroidism. She underwent subtotal parathyroidectomy with appropriate intraoperative PTH decline. Two weeks post-parathyroidectomy, she presented with persistent hypercalcemia and hyperparathyroidism. Urine studies revealed an inappropriately low 24-h urine calcium (Ca)/creatinine clearance ratio. Surgical pathology was consistent with true lymphocytic parathyroiditis with lymphoid follicles. The presence of circulating anti-CaSR antibodies was detected by immunoprecipitation of CaSR by the patient’s serum. After a 4-week course of prednisone, serum Ca and PTH normalized, and her anti-CaSR titers declined. She remains normocalcemic 10 months after the discontinuation of glucocorticoid therapy. We present this patient in the context of the relevant published literature on lymphocytic parathyroiditis and acquired hypocalciuric hypercalcemia related to anti-CaSR antibodies. Conclusions Autoimmune lymphocytic parathyroiditis and acquired hypocalciuric hypercalcemia associated with anti-CaSR antibodies is a very rare yet important condition to be considered in a patient with acquired PTH-dependent hypercalcemia with inappropriate hypocalciuria. Although subtotal parathyroidectomy is unlikely to correct the hypercalcemia, this entity may respond to a short course of prednisone therapy.

This paper presents the development of a novel shape memory alloy (SMA) actuated gripper for use in the biomedical applications. The use of SMA in surgical forceps can allow a surgical robot to accurately and repeatedly apply a force and grip small objects or perform minor surgeries that are less invasive and allow for quicker recovery times. Current designs of thermally actuated grippers use SMAs as the gripping parts, which limits their application due to the transfer of heat to the object being gripped. The design of the gripper illustrated in this paper isolates the SMA coil from the gripping jaws to maintain a constant surface temperature at the gripping end and prevent thermal contamination of soft tissues. Isolating the SMA from the grippers also simplifies automated surgical robots by centralizing all heating elements. A magnetic field exerted between a pair of permanent magnets is used to restore the SMA coil upon cooling. The gripper housing and jaws were fabricated using a 3-D printer to allow for modeling of small features with little down time. A Nitinol SMA wire with a transition temperature of 45°C was wrapped into a 2.5mm diameter coil and heat treated to set the predefined shape. The SMA coil and other parts were assembled to form the gripper. The gripper was successfully tested using an Interlink Electronics Force Sensor and data acquisition card (DAQ), and the forces between the gripper jaws as well as the response time to close and open the jaws were recorded. The gripper produced a force of 0.9N when reaching the transition temperature. The response time for the gripper to close and open the jaws was measured to be approximately 0.16 s and 0.12 s, respectively. It was found that the magnetic field had a faster actuation on the coil than the shape memory alloy force during opening and closing jaws.