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Glomerular endothelial cell (GEnC) dysfunction is important in the pathogenesis of glomerular sclerotic diseases, including Focal Segmental Glomerulosclerosis (FSGS) and overt diabetic nephropathy (DN). GEnCs form the first cellular barrier in direct contact with cells and factors circulating in the blood. Disturbances in these circulating factors can induce GEnC dysfunction. GEnC dysfunction occurs in early stages of FSGS and DN, and is characterized by a compromised endothelial glycocalyx, an inflammatory phenotype, mitochondrial damage and oxidative stress, aberrant cell signaling, and endothelial-to-mesenchymal transition (EndMT). GEnCs are in an interdependent relationship with podocytes and mesangial cells, which involves bidirectional cross-talk via intercellular signaling. Given that GEnC behavior directly influences podocyte function, it is conceivable that GEnC dysfunction may culminate in podocyte damage, proteinuria, subsequent mesangial activation, and ultimately glomerulosclerosis. Indeed, GEnC dysfunction is sufficient to cause podocyte injury, proteinuria and activation of mesangial cells. Aberrant gene expression patterns largely contribute to GEnC dysfunction and epigenetic changes seem to be involved in causing aberrant transcription. This review summarizes literature that uncovers the importance of cross-talk between GEnCs and podocytes, and GEnCs and mesangial cells in the context of the development of FSGS and DN, and the potential use of GEnCs as efficacious cellular target to pharmacologically halt development and progression of DN and FSGS.

Abstract Context Systemic inflammation plays a pivotal role in the development of type 2 diabetes (T2D). Objective We hypothesized that circulating levels of calprotectin, a myeloid cell-derived biomarker of inflammation, is associated with the development of new-onset T2D in the general population. Methods A total of 4815 initially nondiabetic participants of the Prevention of Renal and Vascular End-stage Disease (PREVEND), a prospective population-based cohort study, were assessed for plasma levels of calprotectin at baseline. Circulating levels of calprotectin were investigated for potential associations with the risk of new-onset T2D, defined as a fasting plasma glucose level of 7.0 mmol/L or greater, a random plasma glucose level of 11.1 mmol/L or greater, a self-reported physician-based diagnosis of T2D, the use of glucose-lowering drugs, or any combinations thereof. Results Median plasma calprotectin levels were 0.49 (0.35-0.69) mg/L. Plasma calprotectin levels were significantly associated with the risk of new-onset T2D (hazard ratio [HR] per doubling 1.42 [95% CI, 1.22-1.66]; P < .001). The association remained independent of adjustment for age and sex (HR 1.34 [95% CI, 1.14-1.57]; P < .001), but not after further adjustment for potentially confounding factors (HR 1.11 [95% CI, 0.90-1.37]; P = .326), with adjustment for hyperlipidemia and high-sensitivity C-reactive protein explaining the loss of significance. Stratified analyses showed significant effect modification by hypertension, history of cardiovascular disease (CVD), the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) (Pinteraction ≤ .001 for each), and the use of lipid-lowering drugs (Pinteraction ≤ .05), with higher HRs in individuals without hypertension, without history of CVD, with below-median HOMA-IR, and in those not using lipid-lowering drugs. Conclusion Elevated plasma levels of calprotectin are associated with a higher risk of developing T2D in the general population and may represent a moveable inflammatory biomarker. This association, however, does not represent a direct effect, and seems dependent on hyperlipidemia and systemic inflammation.

During brain-death, increased numbers of neutrophils are recruited to organs as part of the inflammatory response. In the organ microenvironment, the recruited neutrophils may release neutrophil extracellular traps (NETs) through interaction with various pro-inflammatory stimuli, contributing to brain-death-induced endothelial activation, microthrombus formation and ultimately a decline in organ quality. To investigate whether NETs form in organs from brain-dead donors; kidneys, hearts, livers, and plasma samples were collected from brain-dead or sham-operated rats. The presence of NET-specific components, neutrophils and macrophages were analyzed through immunofluorescent microscopy. Endothelial activation and platelet infiltration were analyzed through immunohistochemistry and qRT-PCR analysis. Plasma free thiol levels were used to evaluate systemic oxidative stress. Increased neutrophils, NETs and NET/neutrophil ratios were observed in kidneys, hearts and livers of brain-dead rats compared to sham-operated rats. Numbers of NETs positively correlated with the extent of endothelial cell activation. Brain-dead animals also had increased kidney and liver macrophages, increased infiltrated platelets in the liver, and elevated systemic oxidative stress, compared to sham-operated animals. Our findings established the presence of NETs in organs from a brain-dead donor model and suggest that NETs, alongside increased inflammation and a redox imbalance, might prime organs for microvascular endothelial dysfunction and increased injury during brain-death.

Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques ( n  = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques ( n  = 11) than in the non-culprit plaques ( n  = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques ( p  < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis ( p  < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo.

Background: Optimizing organ preservation techniques is imperative in the face of donor kidney shortage and high waiting list mortality. Hypothermic machine perfusion (HMP) has emerged as an effective method to improve graft function post-transplantation, particularly for deceased donor kidneys, prone to ischemia reperfusion injury (IRI). The perfusion solution includes glucose to support kidney metabolism; however, its effect on mitochondrial function remains unclear. The present study investigated the effect of glucose supplementation during 24 h of oxygenated HMP on mitochondrial function in porcine kidneys. Methods: After 30 min of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 h using HMP with one of the following three solutions: the standard HMP preservation solution, University of Wisconsin machine perfusion (UW-MP) solution, which contains glucose; the solution used for static cold storage, University of Wisconsin cold storage (UW-CS) solution, which lacks glucose; or the UW-CS supplemented with 10 mmol/L glucose. Tissue and perfusate samples were collected before, during, and after perfusion for further analysis. Results: ATP production, mitochondrial respiration, and oxidative stress markers were not significantly different between groups. Glucose was released into the perfusion solution even from kidneys without exogenous glucose supplementation in the perfusate. Conclusions: These results suggest that kidney mitochondrial respiration does not depend on the presence of glucose in the HMP perfusion solution at the start of perfusion, underscoring the need for further exploration of nutrient supplementation and mitochondrial function in organ preservation strategies.

Nephrocalcinosis is present in up to 43% of kidney allograft biopsies at one-year after transplantation and is associated with inferior graft function and poor graft survival. We studied [ 18 F]-sodium fluoride ([ 18 F]-NaF) imaging of microcalcifications in donor kidneys (n = 7) and explanted kidney allografts (n = 13). Three µm paraffin-embedded serial sections were used for histological evaluation of calcification (Alizarin Red; Von Kossa staining) and ex-vivo [ 18 F]-NaF autoradiography. The images were fused to evaluate if microcalcification areas corresponded with [ 18 F]-NaF uptake areas. Based on histological analyses, tubulointerstitial and glomerular microcalcifications were present in 19/20 and 7/20 samples, respectively. Using autoradiography, [ 18 F]-NaF uptake was found in 19/20 samples, with significantly more tracer activity in kidney allograft compared to deceased donor kidney samples ( p  = 0.019). Alizarin Red staining of active microcalcifications demonstrated good correlation (Spearman’s rho of 0.81, p  < 0.001) and Von Kossa staining of consolidated calcifications demonstrated significant but weak correlation (0.62, p  = 0.003) with [ 18 F]-NaF activity. This correlation between ex-vivo [ 18 F]-NaF uptake and histology-proven microcalcifications, is the first step towards an imaging method to identify microcalcifications in active nephrocalcinosis. This may lead to better understanding of the etiology of microcalcifications and its impact on kidney transplant function.

Hypertension contributes to cardiac damage and remodeling. Despite the availability of renin-angiotensin system inhibitors and other antihypertensive therapies, some patients still develop heart failure. Novel therapeutic approaches are required that are effective and without major adverse effects. Sodium Thiosulfate (STS), a reversible oxidation product of hydrogen sulfide (H 2 S), is a promising pharmacological entity with vasodilator and anti-oxidant potential that is clinically approved for the treatment of calciphylaxis and cyanide poisoning. We hypothesized that Sodium Thiosulfate improves cardiac disease in an experimental hypertension model and sought to investigate its cardioprotective effects by direct comparison to the ACE-inhibitor lisinopril, alone and in combination, using a rat model of chronic nitric oxide (NO) deficiency. Systemic nitric oxide production was inhibited in Sprague Dawley rats by administering N-ω-nitro- l -arginine (L-NNA) with the food for three weeks, leading to progressive hypertension, cardiac dysfunction and remodeling. We observed that STS, orally administered via the drinking water, ameliorated L-NNA-induced heart disease. Treatment with STS for two weeks ameliorated hypertension and improved systolic function, left ventricular hypertrophy, cardiac fibrosis and oxidative stress, without causing metabolic acidosis as is sometimes observed following parenteral administration of this drug. STS and lisinopril had similar protective effects that were not additive when combined. Our findings indicate that oral intervention with a H 2 S donor such as STS has cardioprotective properties without noticeable side effects.

Engulfment and cell motility 1 (ELMO1) functions as a guanine exchange factor for Rac1 and was recently found to protect endothelial cells from apoptosis. Genome wide association studies suggest that polymorphisms within human elmo1 act as a potential contributing factor for the development of diabetic nephropathy. Yet, the function of ELMO1 with respect to the glomerulus and how this protein contributes to renal pathology was unknown. Thus, this study aimed to identify the role played by ELMO1 in renal development in zebrafish, under hyperglycaemic conditions, and in diabetic nephropathy patients. In zebrafish, hyperglycaemia did not alter renal ELMO1 expression. However, hyperglycaemia leads to pathophysiological and functional alterations within the pronephros, which could be rescued via ELMO1 overexpression. Zebrafish ELMO1 crispants exhibited a renal pathophysiology due to increased apoptosis which could be rescued by the inhibition of apoptosis. In human samples, immunohistochemical staining of ELMO1 in nondiabetic, diabetic and polycystic kidneys localized ELMO1 in glomerular podocytes and in the tubules. However, ELMO1 was not specifically or distinctly regulated under either one of the disease conditions. Collectively, these results highlight ELMO1 as an important factor for glomerular protection and renal cell survival via decreasing apoptosis, especially under diabetic conditions.

Renal fibrosis is a serious clinical problem forming the utmost cause of need for renal replacement therapy. No adequate preventive or curative therapy is available that can be clinically used to specifically target renal fibrosis. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, here we evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study development of fibrosis and the prevention thereof using anti-fibrotic compounds. Precision-cut slices (200-300 µm thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml) for 48 hours in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate which is targeted to the PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-SMA, fibronectin, collagen I, and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 hours of incubation and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin, and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. Major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue.

Summary Several studies show evidence for the genetic basis of renal disease, which renders some individuals more prone than others to accelerated renal aging. Studying the genetics of renal aging can help us to identify genes involved in this process and to unravel the underlying pathways. First, this opinion article will give an overview of the phenotypes that can be observed in age-related kidney disease. Accurate phenotyping is essential in performing genetic analysis. For kidney aging, this could include both functional and structural changes. Subsequently, this article reviews the studies that report on candidate genes associated with renal aging in humans and mice. Several loci or candidate genes have been found associated with kidney disease, but identification of the specific genetic variants involved has proven to be difficult. CUBN,UMOD, and SHROOM3 were identified by human GWAS as being associated with albuminuria, kidney function, and chronic kidney disease (CKD). These are promising examples of genes that could be involved in renal aging, and were further mechanistically evaluated in animal models. Eventually, we will provide approaches for performing genetic analysis. We should leverage the power of mouse models, as testing in humans is limited. Mouse and other animal models can be used to explain the underlying biological mechanisms of genes and loci identified by human GWAS. Furthermore, mouse models can be used to identify genetic variants associated with age-associated histological changes, of which Far2, Wisp2, and Esrrg are examples. A new outbred mouse population with high genetic diversity will facilitate the identification of genes associated with renal aging by enabling high-resolution genetic mapping while also allowing the control of environmental factors, and by enabling access to renal tissues at specific time points for histology, proteomics, and gene expression.