Deep-Learning based segmentation and quantification in experimental kidney histopathology

BACKGROUND: Nephropathologic analyses provide important outcomes-related data in experiments with the animal models that are essential for understanding kidney disease pathophysiology. Precision medicine increases the demand for quantitative, unbiased, reproducible, and efficient histopathologic analyses, which will require novel high-throughput tools. A deep learning technique, the convolutional neural network, is increasingly applied in pathology because of its high performance in tasks like histology segmentation. METHODS: We investigated use of a convolutional neural network architecture for accurate segmentation of periodic acid-Schiff-stained kidney tissue from healthy mice and five murine disease models and from other species used in preclinical research. We trained the convolutional neural network to segment six major renal structures: glomerular tuft, glomerulus including Bowman\u27s capsule, tubules, arteries, arterial lumina, and veins. To achieve high accuracy, we performed a large number of expert-based annotations, 72,722 in total. RESULTS: Multiclass segmentation performance was very high in all disease models. The convolutional neural network allowed high-throughput and large-scale, quantitative and comparative analyses of various models. In disease models, computational feature extraction revealed interstitial expansion, tubular dilation and atrophy, and glomerular size variability. Validation showed a high correlation of findings with current standard morphometric analysis. The convolutional neural network also showed high performance in other species used in research-including rats, pigs, bears, and marmosets-as well as in humans, providing a translational bridge between preclinical and clinical studies. CONCLUSIONS: We developed a deep learning algorithm for accurate multiclass segmentation of digital whole-slide images of periodic acid-Schiff-stained kidneys from various species and renal disease models. This enables reproducible quantitative histopathologic analyses in preclinical models that also might be applicable to clinical studies

A systematic review and meta-analysis of murine models of uremic cardiomyopathy

Chronic kidney disease (CKD) triggers the risk of developing uremic cardiomyopathy as characterized by cardiac hypertrophy, fibrosis and functional impairment. Traditionally, animal studies are used to reveal the underlying pathological mechanism, although variable CKD models, mouse strains and readouts may reveal diverse results. Here, we systematically reviewed 88 studies and performed meta-analyses of 52 to support finding suitable animal models for future experimental studies on pathological kidney-heart crosstalk during uremic cardiomyopathy. We compared different mouse strains and the direct effect of CKD on cardiac hypertrophy, fibrosis and cardiac function in "single hit" strategies as well as cardiac effects of kidney injury combined with additional cardiovascular risk factors in "multifactorial hit" strategies. In C57BL/6 mice, CKD was associated with a mild increase in cardiac hypertrophy and fibrosis and marginal systolic dysfunction. Studies revealed high variability in results, especially regarding hypertrophy and systolic function. Cardiac hypertrophy in CKD was more consistently observed in 129/Sv mice, which express two instead of one renin gene and more consistently develop increased blood pressure upon CKD induction. Overall, "multifactorial hit" models more consistently induced cardiac hypertrophy and fibrosis compared to "single hit" kidney injury models. Thus, genetic factors and additional cardiovascular risk factors can "prime" for susceptibility to organ damage, with increased blood pressure, cardiac hypertrophy and early cardiac fibrosis more consistently observed in 129/Sv compared to C57BL/6 strains

Peptide YY (PYY) Is Associated with Cardiovascular Risk in Patients with Acute Myocardial Infarction

Aims: Recent studies have found circulating concentrations of the gastrointestinal hormone GLP-1 to be an excellent predictor of cardiovascular risk in patients with myocardial infarction. This illustrates a yet not appreciated crosstalk between the gastrointestinal and cardiovascular systems, which requires further investigation. The gut-derived hormone Peptide YY (PYY) is secreted from the same intestinal L-cells as GLP-1. Relevance of PYY in the context of cardiovascular disease has not been explored. In this study, we aimed to investigate PYY serum concentrations in patients with acute myocardial infarction and to evaluate their association with cardiovascular events. Material and Methods: PYY levels were assessed in 834 patients presenting with acute myocardial infarction (553 Non-ST-Elevation Myocardial Infarction (NSTEMI) and 281 ST-Elevation Myocardial Infarction (STEMI)) at the time of hospital admission. The composite outcomes of first occurrence of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke (3-P-MACE), and all-cause mortality were assessed with a median follow-up of 338 days. Results: PYY levels were significantly associated with age and cardiovascular risk factors, including hypertension, diabetes, and kidney function in addition to biomarkers of heart failure (NT-pro BNP) and inflammation (hs-CRP). Further, PYY was significantly associated with 3-P-MACE (HR: 1.7; 95% CI: 1–2.97; p = 0.0495) and all-cause mortality (HR: 2.69; 95% CI: 1.61–4.47; p = 0.0001) by univariable Cox regression analyses, which was however lost after adjusting for multiple confounders. Conclusions: PYY levels are associated with parameters of cardiovascular risk as well as cardiovascular events and mortality in patients presenting with acute myocardial infarction. However, this significant association is lost after adjustment for further confounders

Mapping the cardiac vascular niche in heart failure

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling

Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in CKD

BACKGROUND: In patients with chronic kidney disease (CKD), atrial fibrillation (AF) is highly prevalent and represents a major risk factor for stroke and death. CKD is associated with atrial proarrhythmic remodeling and activation of the sympathetic nervous system. Whether reduction of the sympathetic nerve activity by renal denervation (RDN) inhibits AF vulnerability in CKD is unknown. METHODS: Left atrial (LA) fibrosis was analyzed in samples from patients with AF and concomitant CKD (estimated glomerular filtration rate [eGFR], <60 mL/min per 1.73 m2) using picrosirius red and compared with AF patients without CKD and patients with sinus rhythm with and without CKD. In a translational approach, male Sprague Dawley rats were fed with 0.25% adenine (AD)-containing chow for 16 weeks to induce CKD. At week 5, AD-fed rats underwent RDN or sham operation (AD). Rats on normal chow served as control. After 16 weeks, cardiac function and AF susceptibility were assessed by echocardiography, radiotelemetry, electrophysiological mapping, and burst stimulation, respectively. LA tissue was histologically analyzed for sympathetic innervation using tyrosine hydroxylase staining, and LA fibrosis was determined using picrosirius red. RESULTS: Sirius red staining demonstrated significantly increased LA fibrosis in patients with AF+CKD compared with AF without CKD or sinus rhythm. In rats, AD demonstrated LA structural changes with enhanced sympathetic innervation compared with control. In AD, LA enlargement was associated with prolonged duration of induced AF episodes, impaired LA conduction latency, and increased absolute conduction inhomogeneity. RDN treatment improved LA remodeling and reduced LA diameter compared with sham-operated AD. Furthermore, RDN decreased AF susceptibility and ameliorated LA conduction latency and absolute conduction inhomogeneity, independent of blood pressure reduction and renal function. CONCLUSIONS: In an experimental rat model of CKD, RDN inhibited progression of atrial structural and electrophysiological remodeling. Therefore, RDN represents a potential therapeutic tool to reduce the risk of AF in CKD, independent of changes in renal function and blood pressure

Pro-oxidative priming but maintained cardiac function in a broad spectrum of murine models of chronic kidney disease

AIMS: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular events and exhibit myocardial changes including left ventricular (LV) hypertrophy and fibrosis, overall referred to as 'uremic cardiomyopathy'. Although different CKD animal models have been studied for cardiac effects, lack of consistent reporting on cardiac function and pathology complicates clear comparison of these models. Therefore, this study aimed at a systematic and comprehensive comparison of cardiac function and cardiac pathophysiological characteristics in eight different CKD models and mouse strains, with a main focus on adenine-induced CKD. METHODS AND RESULTS: CKD of different severity and duration was induced by subtotal nephrectomy or adenine-rich diet in various strains (C57BL/6J, C57BL/6 N, hyperlipidemic C57BL/6J ApoE-/-, 129/Sv), followed by the analysis of kidney function and morphology, blood pressure, cardiac function, cardiac hypertrophy, fibrosis, myocardial calcification and inflammation using functional, histological and molecular techniques, including cardiac gene expression profiling supplemented by oxidative stress analysis. Intriguingly, despite uremia of variable degree, neither cardiac dysfunction, hypertrophy nor interstitial fibrosis were observed. However, already moderate CKD altered cardiac oxidative stress responses and enhanced oxidative stress markers in each mouse strain, with cardiac RNA sequencing revealing activation of oxidative stress signaling as well as anti-inflammatory feedback responses. CONCLUSION: This study considerably expands the knowledge on strain- and protocol-specific differences in the field of cardiorenal research and reveals that several weeks of at least moderate experimental CKD increase oxidative stress responses in the heart in a broad spectrum of mouse models. However, this was insufficient to induce relevant systolic or diastolic dysfunction, suggesting that additional "hits" are required to induce uremic cardiomyopathy. TRANSLATIONAL PERSPECTIVE: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular adverse events and exhibit myocardial changes, overall referred to as 'uremic cardiomyopathy'. We revealed that CKD increases cardiac oxidative stress responses in the heart. Nonetheless, several weeks of at least moderate experimental CKD do not necessarily trigger cardiac dysfunction and remodeling, suggesting that additional "hits" are required to induce uremic cardiomyopathy in the clinical setting. Whether the altered cardiac oxidative stress balance in CKD may increase the risk and extent of cardiovascular damage upon additional cardiovascular risk factors and/or events will be addressed in future studies

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR–Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling