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Juvenile xanthogranuloma (JXG) is a histiocytic neoplasm that usually presents in the skin. Rarely, extracutaneous localizations occur; the genetic drivers of this clinical variant of JXG remain incompletely characterized. We present detailed clinicopathologic and molecular data of 16 children with extracutaneous JXG and 5 adults with xanthogranulomas confined to the central nervous system (CNS) or soft tissue. Tissue samples were obtained through the Dutch Nationwide Pathology Databank and analyzed with an innovative sequencing technique capable of detecting both small genomic variants and gene rearrangements. Targetable kinase alterations were detected in 16/16 children and 1/5 adults. Alterations included CLTC::SYK fusions in 6 children and CSF1R mutations in 7 others - all below 2 years old with soft tissue tumors. One child had a CSF1R mutation and MRC1::PDGFRB fusion. Most were treated surgically, although spontaneous regression occurred in 1/6 with CLTC::SYK and 2/7 with CSF1R mutations - underscoring that treatment is not always necessary. Tumors with CLTC::SYK fusions generally lacked Touton giant cells, but exhibited many other histologic features of JXG and concordant methylation profiles. Using multispectral immunofluorescence, phosphorylated-SYK expression was localized to CD163+ histiocytes; tumors with CLTC::SYK fusions also demonstrated mTOR activation, Cyclin D1 expression, and variable phosphorylated-ERK expression. BRAFV600E was detected in 1 child and 1 adult with CNS xanthogranulomas; both responded to BRAF inhibition. Finally, a TPM3::NTRK1 fusion or MAP2K1 deletion were detected in 2 children with systemic JXG who experienced spontaneous disease regression. This study advances the molecular understanding of histiocytic neoplasms and may guide diagnostics and clinical management., (Copyright © 2024 American Society of Hematology.)

TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-β1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-β1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1)., Methods: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees., Results: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198&#95;199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries., Conclusion: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Initial cysts that are formed upon Pkd1 loss in mice impose persistent stress on surrounding tissue and trigger a cystic snowball effect, in which local aberrant PKD-related signaling increases the likelihood of new cyst formation, ultimately leading to accelerated disease progression. Although many pathways have been associated with PKD progression, the knowledge of early changes near initial cysts is limited. To perform an unbiased analysis of transcriptomic alterations in the cyst microenvironment, microdomains were collected from kidney sections of iKsp-Pkd1 del mice with scattered Pkd1-deletion using Laser Capture Microdissection. These microdomains were defined as F4/80-low cystic, representing early alterations in the cyst microenvironment, F4/80-high cystic, with more advanced alterations, or non-cystic. RNA sequencing and differential gene expression analysis revealed 953 and 8088 dysregulated genes in the F4/80-low and F4/80-high cyst microenvironment, respectively, when compared to non-cystic microdomains. In the early cyst microenvironment, several injury-repair, growth, and tissue remodeling-related pathways were activated, accompanied by mild metabolic changes. In the more advanced F4/80-high microdomains, these pathways were potentiated and the metabolism was highly dysregulated. Upstream regulator analysis revealed a series of paracrine factors with increased activity in the early cyst microenvironment, including TNFSF12 and OSM. In line with the upstream regulator analysis, TWEAK and Oncostatin-M promoted cell proliferation and inflammatory gene expression in renal epithelial cells and fibroblasts in vitro. Collectively, our data provide an overview of molecular alterations that specifically occur in the cyst microenvironment and identify paracrine factors that may mediate early and advanced alterations in the cyst microenvironment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: D.J.M. Peters reports financial support was provided by Dutch Kidney Foundation. D.J.M. Peters reports a relationship with Mironid Ltd. that includes: consulting or advisory. D.J.M. Peters reports a relationship with Crown Bioscience Inc. that includes: consulting or advisory. D.J.M. Peters reports a relationship with Innoser Laboratories BV that includes: consulting or advisory., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Transmembrane protein 14A (TMEM14A) is a relatively unknown protein that is now identified to be required for maintaining the integrity of the glomerular filtration barrier. It is an integral transmembrane protein of 99 amino acids with three transmembrane domains. TMEM14A has been implied to suppress Bax-mediated apoptosis in other studies. Other than that, little is currently known of its function. Here, we show that its expression is diminished before onset of proteinuria in a spontaneously proteinuric rat model. Knocking down tmem14a mRNA translation results in proteinuria in zebrafish embryos without affecting tubular reabsorption. Also, it is primarily expressed by podocytes. Lastly, an increase in glomerular TMEM14A expression is exhibited in various proteinuric renal diseases. Overall, these results suggest that TMEM14A is a novel factor in the protective mechanisms of the nephron to maintain glomerular filtration barrier integrity., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns -/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H + symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS -/- kidney cells and injection into ctns -/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns -/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns -/- larvae, and restoration of the zebrafish pronephros function., (© 2023. The Author(s).)

Metabolic reprogramming is a driver of autosomal dominant polycystic kidney disease (ADPKD) progression and a potential therapeutic intervention route. We showed before that the AMP-associated protein kinase (AMPK) activator salsalate attenuates cystic disease progression. Here, we aim to study the early, direct effects of short salsalate treatment in adult-onset conditional Pkd1 deletion mice. Cystic mice were treated with salsalate for two weeks, after which NMR metabolomics and RNA sequencing analyses were performed. Pkd1 deletion resulted in clear metabolomic dysregulation. Short salsalate treatment has small, but significant, effects, reverting acetylcarnitine and phosphocholine concentrations back to wildtype levels, and showing associations with altered purine metabolism. RNA sequencing revealed that short salsalate treatment, next to restoring energy metabolism toward wildtype levels, also affects cell proliferation and inflammation, in PKD. We show that salsalate positively affects major dysregulated processes in ADPKD: energy metabolism, cell proliferation, and inflammation, providing more insights into its working mechanisms., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Aims: Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in association with cemento-osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement., Methods and Results: Out of the 48 cases, 36 could be used for fluorescence in-situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex., Conclusion: In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD-associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non-neoplastic, reactive origin., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Insufficient immune tolerance during pregnancy is associated with pathological conditions such as preeclampsia (PE). Soluble fms-like tyrosine kinase-1 (sFLT1), which exerts a role in the late stage of PE, has shown its beneficial anti-inflammatory effects in inflammation-associated diseases. Macrophage migration inhibitory factor (MIF) was reported to upregulate sFLT1 production in experimental congenital diaphragmatic hernia. However, the placental sFLT1 expression in early uncomplicated pregnancy and whether MIF can regulate sFLT1 expression in uncomplicated and preeclamptic pregnancy are unclear. We collected first-trimester placentas and term placentas from uncomplicated and preeclamptic pregnancies to investigate sFLT1 and MIF expression in vivo. Primary cytotrophoblasts (CTBs) and a human trophoblast cell line (Bewo) were used to study the regulation of MIF on sFLT1 expression in vitro. In placentas from first-trimester pregnancy, we observed a high expression of sFLT1, specifically in extravillous trophoblasts (EVTs) and syncytiotrophoblast (STB) cells. MIF mRNA levels strongly correlated with sFLT1 expression in term placentas from preeclamptic pregnancies. In in vitro experiments, sFLT1 and MIF levels increased significantly in CTBs during their differentiation to EVTs and STBs, and MIF inhibitor (ISO-1) significantly reduced sFLT1 expression in a dose-dependent manner during this process. sFLT1 showed significant upregulation with increasing doses of MIF in Bewo cells. Our results show that sFLT1 is highly expressed at the maternal-fetal interface during early pregnancy and that MIF can increase sFLT1 expression in early uncomplicated pregnancy and PE, which suggests that sFLT1 plays an essential role in the modulation of inflammation in pregnancy.

Leptin is a hormone that plays a key role in controlling food intake and energy homeostasis. Skeletal muscle is an important target for leptin and recent studies have shown that leptin deficiency may lead to muscular atrophy. However, leptin deficiency-induced structural changes in muscles are poorly understood. The zebrafish has emerged as an excellent model organism for studies of vertebrate diseases and hormone response mechanisms. In this study, we explored ex-vivo magnetic resonance microimaging (μMRI) methods to non-invasively assess muscle wasting in leptin-deficient (lepb-/-) zebrafish model. The fat mapping performed by using chemical shift selective imaging shows significant fat infiltration in muscles of lepb-/- zebrafish compared to control zebrafish. T2 relaxation measurements show considerably longer T2 values in the muscle of lepb-/- zebrafish. Multiexponential T2 analysis detected a significantly higher value and magnitude of long T2 component in the muscles of lepb-/- as compared to control zebrafish. For further zooming into the microstructural changes, we applied diffusion-weighted MRI. The results show a significant decrease in the apparent diffusion coefficient indicating increased constraints of molecular movements within the muscle regions of lepb-/- zebrafish. The use of the phasor transformation for the separation of diffusion-weighted decay signals showed a bi-component diffusion system which allows us to estimate each fraction on a voxel-wise basis. A substantial difference was found between the ratio of two components in lepb-/- and control zebrafish muscles, indicating alterations in diffusion behavior associated with the tissue microstructural changes in muscles of lepb-/- zebrafish as compared to control zebrafish. Taken together, our results demonstrate that the muscles of lepb-/- zebrafish undergo significant fat infiltration and microstructural changes leading to muscle wasting. This study also demonstrates that μMRI provides excellent means to non-invasively study the microstructural changes in the muscles of the zebrafish model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Eeza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Chronic histiocytic intervillositis (CHI) is a rare histopathological lesion in the placenta that is associated with poor reproductive outcomes. The intervillous infiltrate consists mostly of maternal mononuclear cells and fibrin depositions, which are both indicators for the severity of the intervillous infiltrate. The severity of the intervillous infiltrate as well as the clinical outcomes of pregnancy differ between cases. Our objective is to determine the relation between the severity of the intervillous infiltrate and the clinical outcomes of CHI., Methods: Cases of CHI were semi-quantitatively graded based on histopathological severity scores. Hereto, CD68 positive mononuclear cells were quantified, fibrin depositions visualized by both a PTAH stain and an immuohistochemical staining, and placental dysfunction was assessed via thrombomodulin staining., Results: This study included 36 women with CHI. A higher CD68 score was significantly associated with a lower birthweight. Loss of placental thrombomodulin was associated with lower gestational age, lower birthweight, and a lower placenta weight. The combined severity score based on CD68 and PTAH was significantly associated with fetal growth restriction, and the joint score of CD68 and fibrin was associated with birthweight and placental weight., Discussion: More severe intervillous infiltrates in CHI placentas is associated with a lower birth weight and placental weight. Furthermore, this study proposes thrombomodulin as a possible new severity marker of placental damage. More research is needed to better understand the pathophysiology of CHI., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chronic kidney disease (CKD) is a slow-developing, progressive deterioration of renal function. The final common pathway in the pathophysiology of CKD involves glomerular sclerosis, tubular atrophy and interstitial fibrosis. Transforming growth factor-beta (TGF-β) stimulates the differentiation of fibroblasts towards myofibroblasts and the production of extracellular matrix (ECM) molecules, and thereby interstitial fibrosis. It has been shown that endoglin (ENG, CD105), primarily expressed in endothelial cells and fibroblasts, can function as a co-receptor of TGF signaling. In several human organs, endoglin tends to be upregulated when chronic damage and fibrosis is present. We hypothesize that endoglin is upregulated in renal interstitial fibrosis and plays a role in the progression of CKD. We first measured renal endoglin expression in biopsy samples obtained from patients with different types of CKD, i.e., IgA nephropathy, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN) and patients with chronic allograft dysfunction (CAD). We showed that endoglin is upregulated in CAD patients (p < 0.001) and patients with DN (p < 0.05), compared to control kidneys. Furthermore, the amount of interstitial endoglin expression correlated with eGFR (p < 0.001) and the amount of interstitial fibrosis (p < 0.001), independent of the diagnosis of the biopsies. Finally, we investigated in vitro the effect of endoglin overexpression in TGF-β stimulated human kidney fibroblasts. Overexpression of endoglin resulted in an enhanced ACTA2, CCN2 and SERPINE1 mRNA response (p < 0.05). It also increased the mRNA and protein upregulation of the ECM components collagen type I (COL1A1) and fibronectin (FN1) (p < 0.05). Our results suggest that endoglin is an important mediator in the final common pathway of CKD and could be used as a possible new therapeutic target to counteract the progression towards end-stage renal disease (ESRD).

Objectives: We aimed to determine the presence, amount and origin of microchimerism in peripheral blood of pregnant and non-pregnant parous women with systemic lupus erythematosus (SLE) as compared to control subjects., Methods: We performed a comparative study in which peripheral blood was drawn from eleven female non-pregnant SLE-patients and 22 control subjects, and from six pregnant SLE-patients and eleven control subjects during gestation and up to six months postpartum. Quantitative PCR for insertion-deletion polymorphisms and null alleles was used to detect microchimerism in peripheral blood mononuclear cells and granulocytes., Results: Microchimerism was detected more often in non-pregnant SLE-patients than control subjects (54.4% vs. 13.6%, respectively; p=0.03). When present, the median total number of foetal chimeric cells was 5 gEq/106 in patients and 2.5gEq/106 in control subjects (p=0.048). Microchimerism was mostly foetal in origin; maternal microchimerism was detected in one patient and one control subject. In control subjects, microchimerism was always derived from only one source whereas in 50% of patients it originated from multiple sources. The pregnant patients had a significantly higher median number of foetal chimeric cells in the granulocyte fraction just after delivery than control subjects (7.5 gEq/106 vs. 0 gEq/106, respectively; p=0.02)., Conclusions: Just after delivery, SLE-patients had more microchimerism than control subjects. Three months post-partum, microchimerism was no longer detectable, only to reappear many years after the last pregnancy, more often and at higher levels in SLE-patients than in control subjects. This suggests that these chimeric cells may originate from non-circulating foetal chimeric stem cells.

There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta-Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top two genes-RPL36AL and TMBIM6, and the bottom two genes-UBA1 and B2M, were further studied in iPSC-EC with and without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC-derived endothelial cells as well as genetically modified iPSC for drug treatment., (© 2022. The Author(s).)

(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we studied whether transfection with sFlt1 aggravates experimental AKI-to-CKD transition and whether sFLT1 is increased in human kidney fibrosis. (2) Methods: Mice were transfected via electroporation with sFlt1 . After confirming transfection efficacy, mice underwent unilateral ischemia/reperfusion injury (IRI) and were sacrificed 28 days later. Kidney histology and RNA were analyzed to study renal fibrosis, PTC damage and inflammation. Renal sFLT1 mRNA expression was measured in CKD biopsies and control kidney tissue. (3) Results: sFlt1 transfection did not aggravate renal fibrosis, PTC loss or macrophage recruitment in IRI mice. In contrast, higher transfection efficiency was correlated with reduced expression of pro-fibrotic and pro-inflammatory markers. In the human samples, sFLT1 mRNA levels were similar in CKD and control kidneys and were not correlated with interstitial fibrosis or PTC loss. (4) Conclusion: As we previously found that sFLT1 has therapeutic potential in diabetic nephropathy, our findings indicate that sFLT1 can be administered at a dose that is therapeutically effective in reducing inflammation, without promoting maladaptive kidney damage.

Background: Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function., Methods: We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment., Results: Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r2 = 0.48, P < 0.0005) but also to plasma sFlt-1 (r2 = 0.56, P < 0.0001 and r2 = 0.23, P = 0.03, respectively)., Conclusions: In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia., (© The Author(s) 2021. Published by Oxford University Press on behalf of ERA-EDTA.)

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors., (© 2022 The Author(s).)

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology., (© 2022. The Author(s).)

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.

Leptin is a hormone which functions in the regulation of energy homeostasis via suppression of appetite. In zebrafish, there are two paralogous genes encoding leptin, called lepa and lepb. In a gene expression study, we found that the lepb gene, not the lepa gene, was significantly downregulated under the state of insulin-resistance in zebrafish larvae, suggesting that the lepb plays a role in glucose homeostasis. In the current study, we characterised lepb-deficient (lepb-/-) adult zebrafish generated via a CRISPR-CAS9 gene editing approach by investigating whether the disruption of the lepb gene would result in the development of type 2 diabetes mellitus (T2DM) and diabetic complications. We observed that lepb-/- adult zebrafish had an increase in body weight, length and visceral fat accumulation, compared to age-matched control zebrafish. In addition, lepb-/- zebrafish had significantly higher blood glucose levels compared to control zebrafish. These data collectively indicate that lepb-/- adult zebrafish display the features of T2DM. Furthermore, we showed that lepb-/- adult zebrafish had glomerular hypertrophy and thickening of the glomerular basement membrane, compared to control zebrafish, suggesting that lepb-/- adult zebrafish develop early signs of diabetic nephropathy. In conclusion, our results demonstrate that lepb regulates glucose homeostasis and adiposity in zebrafish, and suggest that lepb-/- mutant zebrafish are a promising model to investigate the role of leptin in the development of T2DM and are an attractive model to perform mechanistic and therapeutic research in T2DM and its complications.

The endothelial glycoprotein thrombomodulin regulates coagulation, inflammation, and apoptosis. In diabetic mice, reduced thrombomodulin function results in diabetic nephropathy (DN). Furthermore, thrombomodulin treatment reduces renal inflammation and fibrosis. Herein, thrombomodulin expression was examined in human kidney samples to investigate the possibility of targeting thrombomodulin in patients with DN. Glomerular thrombomodulin was analyzed together with the number of glomerular macrophages in 90 autopsied diabetic cases with DN, 55 autopsied diabetic cases without DN, and 37 autopsied cases without diabetes or kidney disease. Thrombomodulin mRNA was measured in glomeruli microdissected from renal biopsies from patients with DN and nondiabetic controls. Finally, glomerular thrombomodulin was measured in diabetic mice following treatment with the selective endothelin A receptor (ET A R) blocker, atrasentan. In diabetic patients, glomerular thrombomodulin expression was increased at the mRNA level, but decreased at the protein level, compared with nondiabetic controls. Reduced glomerular thrombomodulin was associated with an increased glomerular influx of macrophages. Blocking the ET A R with atrasentan restored glomerular thrombomodulin protein levels in diabetic mice to normal levels. The reduction in glomerular thrombomodulin in diabetes likely serves as an early proinflammatory step in the pathogenesis of DN. Thrombomodulin protein may be cleaved under diabetic conditions, leading to a compensatory increase in transcription. The nephroprotective effects of ET A R antagonists in diabetic patients may be attributed to the restoration of glomerular thrombomodulin., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The endothelial glycoprotein thrombomodulin regulates coagulation, vascular inflammation and apoptosis. In the kidney, thrombomodulin protects the glomerular filtration barrier by eliciting crosstalk between the glomerular endothelium and podocytes. Several glomerular pathologies are characterized by a loss of glomerular thrombomodulin. In women with pre-eclampsia, serum levels of soluble thrombomodulin are increased, possibly reflecting a loss from the glomerular endothelium. We set out to investigate whether thrombomodulin expression is decreased in the kidneys of women with pre-eclampsia and rats exposed to an angiogenesis inhibitor. Thrombomodulin expression was examined using immunohistochemistry and qPCR in renal autopsy tissues collected from 11 pre-eclamptic women, 22 pregnant controls and 11 hypertensive non-pregnant women. Further, kidneys from rats treated with increasing doses of sunitinib or sunitinib in combination with endothelin receptor antagonists were studied. Glomerular thrombomodulin protein levels were increased in the kidneys of women with pre-eclampsia. In parallel, in rats exposed to sunitinib, glomerular thrombomodulin was upregulated in a dose-dependent manner, and the upregulation of glomerular thrombomodulin preceded the onset of histopathological changes. Selective ET A R blockade, but not dual ET A/B R blockade, normalised the sunitinib-induced increase in thrombomodulin expression and albuminuria. We propose that glomerular thrombomodulin expression increases at an early stage of renal damage induced by antiangiogenic conditions. The upregulation of this nephroprotective protein in glomerular endothelial cells might serve as a mechanism to protect the glomerular filtration barrier in pre-eclampsia.

Oocyte donation (OD) pregnancies are characterized by a complete immunogenetic dissimilarity between mother and fetus, which requires enhanced immunoregulation compared to naturally conceived (NC) pregnancies. The trophoblast expresses co-inhibitory ligands crucial for regulation of the maternal T cell response. Therefore, we studied the role of placental immune checkpoint inhibitors for the establishment of fetal tolerance and their relation to the development of preeclampsia in OD compared to NC pregnancies. Placental tissue from uncomplicated OD (n = 21) and NC (n = 21) pregnancies, and OD (n = 9) and NC (n = 15) pregnancies complicated with preeclampsia were studied. Protein expression of co-inhibitory ligands PD-L1 and CD200 was double blind semi-quantitatively determined by immunohistochemistry. Messenger RNA expression of PD-L1, CD200 and indoleamine 2,3-dioxygenase (IDO) was determined using qPCR. Decreased PD-L1 and CD200 protein expression and increased IDO mRNA expression was observed in uncomplicated OD versus NC pregnancies (all p < 0.05). CD200 protein expression was positively correlated with PD-L1 expression in all groups, with the number of HLA total mismatches and with HLA class I mismatches in uncomplicated OD cases (all p < 0.05). Preeclamptic cases showed lower PD-L1 protein and CD200 protein and mRNA expression in OD compared to NC pregnancies (all p < 0.05). This study shows that signaling by co-inhibitory PD-L1 and CD200 and by immunosuppressive IDO is altered in the placenta of OD pregnancies, suggesting a contribution to the higher risk for preeclampsia. These insights provide future prospects in unraveling the immune paradox of oocyte pregnancy, which are applicable for better risk management and treatment of uncomplicated and preeclamptic pregnancies., Competing Interests: Declaration of Competing Interest None of the authors has any conflict of interest related to this manuscript., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Diabetic nephropathy (DN) is a complication of diabetes mellitus that can lead to proteinuria and a progressive decline in renal function. Endoglin, a co-receptor of TGF-β, is known primarily for regulating endothelial cell function; however, endoglin is also associated with hepatic, cardiac, and intestinal fibrosis. This study investigates whether endoglin contributes to the development of interstitial fibrosis in DN. Kidney autopsy material from 80 diabetic patients was stained for endoglin and Sirius Red and scored semi-quantitatively. Interstitial endoglin expression was increased in samples with DN and was correlated with Sirius Red staining ( p < 0.001). Endoglin expression was also correlated with reduced eGFR ( p = 0.001), increased creatinine ( p < 0.01), increased systolic blood pressure ( p < 0.05), hypertension ( p < 0.05), and higher IFTA scores ( p < 0.001). Biopsy samples from DN patients were also co-immunostained for endoglin together with CD31, CD68, vimentin, or α-SMA Endoglin co-localized with both the endothelial marker CD31 and the myofibroblast marker α-SMA. Finally, we used shRNA to knockdown endoglin expression in a human kidney fibroblast cell line. We found that TGF-β1 stimulation upregulated SERPINE1 , CTGF , and ACTA2 mRNA and α-SMA protein, and that these effects were significantly reduced in fibroblasts after endoglin knockdown. Taken together, these data suggest that endoglin plays a role in the pathogenesis of interstitial fibrosis in DN., Competing Interests: The authors declare no conflict of interest.

Clusterin, a glycoprotein encoded by the CLU gene, is expressed in many tissues, including the kidney, and clusterin expression is upregulated in the glomeruli of patients with various forms of kidney disease. Here, we investigated the role of clusterin in diabetic nephropathy (DN). In this study, we found that glomerular clusterin expression was increased in both patients with DN and streptozotocin-induced diabetic mice and that it co-localised with the podocyte marker WT1, indicating clusterin is expressed in podocytes. In our in vitro analysis, we found no significant change in CLU mRNA expression in podocytes following stimulation with high glucose and angiotensin II; in contrast, CLU mRNA expression was significantly upregulated following methylglyoxal stimulation. Methylglyoxal treatment also significantly decreased the mRNA expression of the slit diaphragm markers ZO-1 and NEPH1 and significantly increased the mRNA expression of the oxidative stress marker HO-1. Lastly, we showed that pre-incubating podocytes with recombinant human clusterin protein increased podocyte survival, prevented slit diaphragm damage, and reduced oxidative stress‒induced apoptosis following methylglyoxal stimulation. Taken together, our results indicate that glomerular clusterin is upregulated in DN, and this increase in clusterin expression may protect against oxidative stress-induced apoptosis in podocytes, providing a possible new therapeutic target for DN and other kidney diseases.

Aims: Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria., Methods and Results: Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan., Conclusions: Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Manipulation of circulating histidine-containing dipeptides (HCD) has been shown to affect the development of diabetes and early-stage diabetic nephropathy (DN). The aim of the present study was to investigate whether such interventions, which potentially alter levels of circulating HCD, also affect the development of advanced-stage DN. Two interventions, aerobic exercise training and overexpression of the human carnosinase-1 (hCN1) enzyme, were tested. BTBR ob/ob mice were either subjected to aerobic exercise training (20 wk) or genetically manipulated to overexpress hCN1, and different diabetes- and DN-related markers were compared with control ob/ob and healthy (wild-type) mice. An acute exercise study was performed to elucidate the effect of obesity, acute running, and hCN1 overexpression on plasma HCD levels. Chronic aerobic exercise training did not affect the development of diabetes or DN, but hCN1 overexpression accelerated hyperlipidemia and aggravated the development of albuminuria, mesangial matrix expansion, and glomerular hypertrophy of ob/ob mice. In line, plasma, kidney, and muscle HCD were markedly lower in ob/ob versus wild-type mice, and plasma and kidney HCD in particular were lower in ob/ob hCN1 versus ob/ob mice but were unaffected by aerobic exercise. In conclusion, advanced glomerular damage is accelerated in mice overexpressing the hCN1 enzyme but not protected by chronic exercise training. Interestingly, we showed, for the first time, that the development of DN is closely linked to renal HCD availability. Further research will have to elucidate whether the stimulation of renal HCD levels can be a therapeutic strategy to reduce the risk for developing DN.

Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.

Complement factor C4d was recently observed in renal biopsies from patients who had IgA nephropathy and a poor prognosis. We previously reported that C4d is a common denominator in microangiopathies. In this retrospective cohort study, we investigated whether C4d is a marker of microangiopathy in both IgA nephropathy and IgA vasculitis with nephritis, and whether patients with C4d and microangiopathy have poor renal outcome. We examined 128 renal biopsies from adult and pediatric patients, including normotensive and hypertensive patients, who presented with IgA nephropathy or IgA vasculitis with nephritis. Biopsies were re-evaluated in accordance with the Oxford classification, scored for additional lesions, and stained for complement proteins using immunohistochemistry, including C4d and C5b-9. Clinical data were collected with a mean (±SD) follow-up period of 51 ± 39 months. Changes in estimated glomerular filtration rate over time were compared using linear mixed-effects models. Renal survival was analyzed using multivariable Cox regression. Microangiopathic lesions were present in 20% of all biopsies (23% and 9% of patients with IgA nephropathy and IgA vasculitis with nephritis, respectively). Microangiopathy was associated with C4d and C5b-9 deposits, a higher number of chronic lesions, and hypertension (all p < 0.05). Patients with C4d and microangiopathic lesions had significantly poorer renal survival than patients without these findings, corrected for hypertension (p < 0.01). In conclusion, patients with IgA nephropathy or IgA vasculitis with nephritis with a combination of C4d positivity and microangiopathy comprise a clinical subgroup with an increased number of chronic lesions, lower estimated glomerular filtration rate, and poorer renal survival, even when corrected for hypertension. These data suggest that complement activation is involved in the development of microangiopathy in patients with IgA nephropathy and IgA vasculitis with nephritis, and that complement-mediated microangiopathy contributes to disease progression.

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

In diabetic nephropathy, differential expression of growth factors leads to vascular changes, including endothelial cell activation, monocyte infiltration, and inflammation. Endoglin plays an important role in endothelial function and is also associated with inflammation. In the kidney, vascular endoglin expression is increased in animal models of renal injury, where it contributes to disease severity, possibly by promoting endothelial cell activation and inflammation. Herein, we investigated whether endoglin expression is associated with diabetic nephropathy. In addition, we examined whether reducing endothelial endoglin expression in vitro affects endothelial cell activation and monocyte adhesion and, if so, which intracellular pathways are involved. Finally, we analyzed whether glomerular endoglin expression is correlated with endothelial cell activation in patients with diabetic nephropathy. Endoglin levels were significantly increased in mice with type 1 diabetes compared with control mice. Reducing endoglin expression in cultured endothelial cells significantly impaired the vascular endothelial growth factor-A-induced up-regulation of activation markers and monocyte adhesion. This was mediated by increased phosphorylation of Akt, thereby inhibiting activating transcription factor 2 phosphorylation, which regulates vascular cell adhesion molecule-1 (VCAM1) gene transcription in these cells. Last, endoglin colocalized with VCAM-1 in the glomeruli of diabetic patients, glomerular VCAM-1 expression was significantly increased in these patients, and this increase in VCAM-1 expression was correlated with increased glomerular endoglin expression. Thus, targeting endoglin function may have therapeutic value in patients at risk for diabetic nephropathy., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Angiogenesis inhibition with bevacizumab, a monoclonal antibody against vascular endothelial growth factor A (VEGF-A), is an anticancer treatment associated with hypertension and renal glomerular toxicity referred to as a preeclampsia-like syndrome. In preeclampsia, podocyturia predates proteinuria and clinical features of preeclampsia, and is regarded as a biomarker of ongoing glomerular injury. Using a quantitative polymerase chain reaction of the podocyte-specific molecules nephrin, podocin, and VEGF-A in the urine, we examined whether podocyturia is present in bevacizumab-treated cancer patients, and whether it relates to proteinuria and the cumulative dose of bevacizumab. Urine samples were cross-sectionally collected from 43 bevacizumab-treated patients, 21 chemotherapy-treated patients, and 7 healthy controls. Urinary protein-to-creatinine ratio (mean and range) was 32.0 mg/mmol (5.2-284.4) in the bevacizumab group, compared with 11.4 mg/mmol (1.1-21.0) in the chemotherapy group and 7.4 mg/mmol (3.9-16.5) (P < .05) in healthy controls, whereas urinary albumin-to-creatinine ratio values in the three groups were, respectively, 18.9 mg/mmol (0.1-227.7), 1.5 mg/mmol (0.2-3.5), and 0.2 mg/mmol (0.1-0.4) (P < .05). The cumulative dose of bevacizumab ranged from 550 to 93,628 mg. Urinary podocin mRNA expression was undetectable in 59% of participants, urinary nephrin mRNA expression per mmol creatinine ranged from 0.0 to 5.3 and urinary VEGF-A mRNA expression from 0.0 to 2.7. Urinary nephrin mRNA expression did not correlate to the albumin-to-creatinine ratio or the cumulative dose of bevacizumab, whereas the latter correlated with the albumin-to-creatinine ratio (r = 0.77; P < .001). Our results demonstrate that the cumulative dose of bevacizumab is closely correlated with albuminuria but not with podocyturia as measured with the quantitative polymerase chain reaction technique, challenging the feasibility of this measurement to monitor ongoing glomerular injury in patients chronically treated with bevacizumab., (Copyright © 2018 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Background: Obesity and type 2 diabetes have not only been linked to fatty liver, but also to fatty kidney and chronic kidney disease. Since non-invasive tools are lacking to study fatty kidney in clinical studies, we explored agreement between proton magnetic resonance spectroscopy (1H-MRS) and enzymatic assessment of renal triglyceride content (without and with dietary intervention). We further studied the correlation between fatty kidney and fatty liver., Methods: Triglyceride content in the renal cortex was measured by 1H-MRS on a 7-Tesla scanner in 27 pigs, among which 15 minipigs had been randomized to a 7-month control diet, cafeteria diet (CAF) or CAF with low-dose streptozocin (CAF-S) to induce insulin-independent diabetes. Renal biopsies were taken from corresponding MRS-voxel locations. Additionally, liver biopsies were taken and triglyceride content in all biopsies was measured by enzymatic assay., Results: Renal triglyceride content measured by 1H-MRS and enzymatic assay correlated positively (r = 0.86, P < 0.0001). Compared with control diet-fed minipigs, renal triglyceride content was higher in CAF-S-fed minipigs (137 ± 51 nmol/mg protein, mean ± standard error of the mean, P < 0.05), but not in CAF-fed minipigs (60 ± 10 nmol/mg protein) compared with controls (40 ± 6 nmol/mg protein). Triglyceride contents in liver and kidney biopsies were strongly correlated (r = 0.97, P < 0.001)., Conclusions: Non-invasive measurement of renal triglyceride content by 1H-MRS closely predicts triglyceride content as measured enzymatically in biopsies, and fatty kidney appears to develop parallel to fatty liver. 1H-MRS may be a valuable tool to explore the role of fatty kidney in obesity and type 2 diabetic nephropathy in humans in vivo., (© The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Chronic intervillositis of unknown etiology (CIUE) is a poorly understood, relatively rare condition characterized histologically by the intervillous infiltration of mononuclear cells in the placenta. Clinically, CIUE is associated with poor pregnancy outcome (e.g., impaired fetal growth, preterm birth, fetal death) and high risk of recurrence in subsequent pregnancies. Because CIUE is not defined consistently, it is essential to clearly define this condition. We therefore review the published definitions of CIUE. In addition, we provide an overview of the reviewed histopathological and maternal characteristics, obstetric features, and pregnancy outcomes. Medical publication databases were searched for articles published through February 2017. Eighteen studies were included in our systematic review. The sole inclusion criterion used in all studies was the presence of intervillous infiltrates. Overall, CIUE was characterized by adverse pregnancy outcome. Miscarriage occurred in 24% of cases, with approximately half of these miscarriages defined as late. Impaired growth was commonly observed, 32.4% of pregnancies reached term, and the live birth rate was 54.9%. The high recurrence rate (25.1%) of the intervillous infiltrates in subsequent pregnancies underscores the clinical relevance of CIUE, the need for increased awareness among pathologists and clinicians, and the need for further research. Criteria for the diagnosis of CIUE are proposed and a Delphi study could be used to resolve any controversy regarding these criteria. Future studies should be designed to characterize the full clinical spectrum of CIUE., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Complement activation plays a role in various organs in patients with diabetes. However, in diabetic nephropathy (DN), the role of complement activation is poorly understood. We examined the prevalence and clinical significance of complement deposits in the renal tissue of cases with type 1 and type 2 diabetes with and without DN., Methods: We measured the prevalence of glomerular C4d, C1q, mannose-binding lectin (MBL), and C5b-9 deposits in 101 autopsied diabetic cases with DN, 59 autopsied diabetic cases without DN, and 41 autopsied cases without diabetes or kidney disease. The presence of complement deposits was scored by researchers who were blinded with respect to the clinical and histological data., Results: C4d deposits were more prevalent in cases with DN than in cases without DN in both the glomeruli (46% vs. 26%) and the arterioles (28% vs. 12%). C1q deposits were also increased in the glomerular hili (77% vs. 55%) and arterioles (33% vs.14%), and were correlated with DN ( P  < 0.01). MBL deposits were only rarely observed. C5b-9 deposits were more prevalent in the cases with diabetes mellitus (DM) than in the cases without DM (69% vs. 32%; P  < 0.001). Finally, glomerular C4d and C5b-9 deposits were correlated with the severity of DN (ρ = 0.341 and 0.259, respectively; P  < 0.001)., Conclusion: Complement activation is correlated with both the presence and severity of DN, suggesting that the complement system is involved in the development of renal pathology in patients with diabetes and is a promising target for inhibiting and/or preventing DN in these patients.

Aims/hypothesis: Animal models of diabetic nephropathy show increased levels of glomerular vascular endothelial growth factor (VEGF)-A, and several studies have shown that inhibiting VEGF-A in animal models of diabetes can prevent albuminuria and glomerular hypertrophy. However, in those studies, treatment was initiated before the onset of kidney damage. Therefore, the aim of this study was to investigate whether transfecting mice with the VEGF-A inhibitor sFlt-1 (encoding soluble fms-related tyrosine kinase 1) can reverse pre-existing kidney damage in a mouse model of type 1 diabetes. In addition, we investigated whether transfection with sFlt-1 can reduce endothelial activation and inflammation in these mice., Methods: Subgroups of untreated 8-week-old female C57BL/6J control (n = 5) and diabetic mice (n = 7) were euthanised 5 weeks after the start of the experiment in order to determine the degree of kidney damage prior to treatment with sFLT-1. Diabetes was induced with three i.p. injections of streptozotocin (75 mg/kg) administered at 2 day intervals. Diabetic nephropathy was then investigated in diabetic mice transfected with sFlt-1 (n = 6); non-diabetic, non-transfected control mice (n = 5); non-diabetic control mice transfected with sFlt-1(n = 10); and non-transfected diabetic mice (n = 6). These mice were euthanised at the end of week 15. Transfection with sFlt-1 was performed in week 6., Results: We found that transfection with sFlt-1 significantly reduced kidney damage by normalising albuminuria, glomerular hypertrophy and mesangial matrix content (i.e. glomerular collagen type IV protein levels) (p < 0.001). We also found that transfection with sFlt-1 reduced endothelial activation (p < 0.001), glomerular macrophage infiltration (p < 0.001) and glomerular TNF-α protein levels (p < 0.001). Finally, sFLT-1 decreased VEGF-A-induced endothelial activation in vitro (p < 0.001)., Conclusions/interpretation: These results suggest that sFLT-1 might be beneficial in treating diabetic nephropathy by inhibiting VEGF-A, thereby reducing endothelial activation and glomerular inflammation, and ultimately reversing kidney damage.

Diabetic nephropathy is the leading cause of end-stage renal disease. Diabetic patients have increased plasma concentrations of apolipoprotein C-I (apoCI), and meta-analyses found that a polymorphism in APOC1 is associated with an increased risk of developing nephropathy. To investigate whether overexpressing apoCI contributes to the development of kidney damage, we studied renal tissue and peritoneal macrophages from APOC1 transgenic (APOC1-tg) mice and wild-type littermates. In addition, we examined renal material from autopsied diabetic patients with and without diabetic nephropathy and from autopsied control subjects. We found that APOC1-tg mice, but not wild-type mice, develop albuminuria, renal dysfunction, and glomerulosclerosis with increased numbers of glomerular M1 macrophages. Moreover, compared to wild-type macrophages, stimulated macrophages isolated from APOC1-tg mice have increased cytokine expression, including TNF-alpha and TGF-beta, both of which are known to increase the production of extracellular matrix proteins in mesangial cells. These results suggest that APOC1 expression induces glomerulosclerosis, potentially by increasing the cytokine response in macrophages. Furthermore, we detected apoCI in the kidneys of diabetic patients, but not in control kidneys. Moreover, patients with diabetic nephropathy have significantly more apoCI present in glomeruli compared to diabetic patients without nephropathy, suggesting that apoCI could be involved in the development of diabetic nephropathy. ApoCI co-localized with macrophages. Therefore, apoCI is a promising new therapeutic target for patients at risk of developing nephropathy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Objective: To investigate whether thrombomodulin dysregulation is involved in the development of preeclampsia after oocyte donation (OD). Women who become pregnant after OD are prone to develop preeclampsia, a syndrome characterized by an aberrant immunologic response, hypercoagulability, and endothelial dysfunction. A mediator of inflammation and coagulation is thrombomodulin, which has a possible role to play in this syndrome., Design: Case-control study., Setting: Not applicable., Patient(s): Placentas from 82 women with an uncomplicated pregnancy (48 naturally conceived, 21 IVF, and 33 OD pregnancies) and 9 women with an OD pregnancy complicated by preeclampsia have been studied., Intervention(s): None., Main Outcome Measure(s): Abundances of thrombomodulin protein and vitamin D receptor (VDR) were determined using immunohistochemistry; mRNA expression was determined using quantitative polymerase chain reaction., Result(s): Placental thrombomodulin protein abundance was lower in OD pregnancies (diffuse pattern in 45%) than in controls (diffuse pattern in 96%). Placental thrombomodulin mRNA expression was lower in OD pregnancies complicated by preeclampsia (0.72 ± 0.47) compared with in uncomplicated OD pregnancies (0.43 ± 0.18). Thrombomodulin expression correlated with inflammation and coagulation. VDR expression was decreased in OD pregnancies complicated by preeclampsia and was correlated with thrombomodulin mRNA., Conclusion(s): Pregnancies conceived through OD lose placental thrombomodulin expression. This loss is associated with an increased coagulation and inflammation and indicates that endothelial protection is diminished in OD pregnancies, which might be an explanation for the increased risk for preeclampsia. Vitamin D metabolism is dysregulated in OD pregnancies and might be a target for therapy., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A 121 was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples., Competing Interests: The authors have declared that no competing interests exist.

Background: Antiangiogenic treatment with the multitargeted vascular endothelial growth factor (VEGF) receptor inhibitor sunitinib associates with a blood pressure (BP) rise and glomerular renal injury. Recent evidence indicates that VEGF derived from tubular cells is required for maintenance of the peritubular vasculature. In the present study, we focussed on tubular and glomerular pathology induced by sunitinib and explored whether a high salt (HS) diet augments the BP rise and renal abnormalities., Methods: Normotensive Wistar Kyoto (WKY) rats were exposed to a normal salt (NS) or HS diet for 2 weeks and subsequently for 8 days to sunitinib or vehicle administration after which the rats were euthanized and kidneys excised. Mean arterial pressure (MAP) was telemetrically measured. Urine was sampled for proteinuria and endothelinuria, and blood for measurement of endothelin-1, creatinine and cystatin C., Results: Compared with the NS diet, MAP rapidly rose by 27 ± 3 mmHg with the HS diet. On sunitinib, MAP rose further by 15 ± 1 with the NS and by 23 ± 4 mmHg with the HS diet (P < 0.05). The HS diet itself had no effect on proteinuria, endothelinuria or the plasma levels of endothelin-1, creatinine and cystatin C. Only with the HS diet, sunitinib administration massively increased proteinuria and endothelinuria and these two parameters were related (r = 0.50, P < 0.01). Likewise, renal glomerular pathology was enhanced during sunitinib with the HS diet, whereas tubulointerstitial injury or reduced peritubular capillary density did not occur., Conclusions: An HS diet induces a marked BP rise in WKY rats and exacerbates both the magnitude of the BP rise and glomerular injury induced by sunitinib., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective: Preeclampsia is a pregnancy-specific syndrome characterized by placental dysfunction and an angiogenic imbalance. Systemically, levels of thrombomodulin, an endothelium- and syncytiotrophoblast-bound protein that regulates coagulation, inflammation, apoptosis, and tissue remodeling, are increased. We aimed to investigate placental thrombomodulin dysregulation and consequent downstream effects in the pathogenesis of preeclampsia., Approach and Results: Placentas from 28 preeclampsia pregnancies, 30 uncomplicated pregnancies, and 21 pregnancies complicated by growth restriction as extra controls were included. Immunohistochemical staining of thrombomodulin, caspase-3, and fibrin was performed. Placental mRNA expression of thrombomodulin, inflammatory markers, matrix metalloproteinases 2 and 9, and soluble Flt-1 were measured with quantitative polymerase chain reaction. Thrombomodulin mRNA expression was determined in vascular endothelial growth factor-transfected trophoblast cell lines. Thrombomodulin protein and mRNA expression were decreased in preeclampsia as compared with both control groups (P=0.001). Thrombomodulin mRNA expression correlated with maternal body mass index (P<0.01) and diastolic blood pressure (P<0.05) in preeclampsia. An increase in placental apoptotic cells was associated with preeclampsia (P<0.001). Thrombomodulin expression correlated positively with matrix metalloproteinase expression (P<0.01) in preeclampsia, but not with fibrin deposits or inflammatory markers. Placental soluble Flt-1 expression correlated with decreased thrombomodulin expression. Vascular endothelial growth factor induced upregulation of thrombomodulin expression in trophoblast cells., Conclusions: Decreased thrombomodulin expression in preeclampsia may play a role in placental dysfunction in preeclampsia and is possibly caused by an angiogenic imbalance. Hypertension and obesity are associated with thrombomodulin downregulation. These results set the stage for further basic and clinical research on thrombomodulin in the pathogenesis of preeclampsia and other syndromes characterized by endothelial dysfunction., (© 2016 American Heart Association, Inc.)

Histidine-containing dipeptides like carnosine and anserine have protective functions in both health and disease. Animal studies suggest that carnosine can be metabolized within the kidney. The goal of this study was to obtain evidence of carnosine metabolism in the human kidney and to provide insight with regards to diabetic nephropathy. Expression, distribution, and localization of carnosinase-1 (CNDP1), carnosine synthase (CARNS), and taurine transporters (TauT) were measured in human kidneys. CNDP1 and CARNS activities were measured in vitro. CNDP1 and CARNS were located primarily in distal and proximal tubules, respectively. Specifically, CNDP1 levels were high in tubular cells and podocytes (20.3 ± 3.4 and 15 ± 3.2 ng/mg, respectively) and considerably lower in endothelial cells (0.5 ± 0.1 ng/mg). CNDP1 expression was correlated with the degradation of carnosine and anserine (r = 0.88 and 0.81, respectively). Anserine and carnosine were also detectable by HPLC in the renal cortex. Finally, TauT mRNA and protein were found in all renal epithelial cells. In diabetic patients, CNDP1 seemed to be reallocated to proximal tubules. We report compelling evidence that the kidney has an intrinsic capacity to metabolize carnosine. Both CNDP1 and CARNS are expressed in glomeruli and tubular cells. Carnosine-synthesizing and carnosine-hydrolyzing enzymes are localized in distinct compartments in the nephron and increased CNDP1 levels suggest a higher CNDP1 activity in diabetic kidneys.

Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency of these side effects. Normotensive Wistar Kyoto rats were exposed to 3 different doses of sunitinib or vehicle. After 8 days, rats were euthanized. Telemetrically measured blood pressure rose dose dependently, from 13 to 30 mm Hg. Proteinuria was present at all doses, but a rise in cystatin C occurred only at the intermediate and high doses. Compared with vehicle circulating endothelin-1 increased dose dependently, whereas 24-hour urinary endothelin excretion decreased. Light and electron microscopy revealed glomerular endotheliosis and ischemia with the intermediate and high doses of sunitinib but completely absent histological abnormalities with the low dose. Podocyte number per glomerular circumference did not change. Glomerular nephrin, Neph1, podocin, and endothelin-converting enzyme gene expression were downregulated in a dose-dependent manner. We conclude that the sunitinib-induced rise in blood pressure requires lower doses than its induction of renal function impairment and that functional changes in glomerular filtration barrier contribute to the occurrence of proteinuria, given the lack of histopathologic changes with the low dose of sunitinib., (© 2015 American Heart Association, Inc.)

Complement activation has a major role in thrombotic microangiopathy (TMA), a disorder that can occur in a variety of clinical conditions. Promising results of recent trials with terminal complement-inhibiting drugs call for biomarkers identifying patients who might benefit from this treatment. The primary aim of this study was to determine the prevalence and localization of complement factor C4d in kidneys of patients with TMA. The secondary aims were to determine which complement pathways lead to C4d deposition and to determine whether complement activation results in deposition of the terminal complement complex. We examined 42 renal sections with histologically confirmed TMA obtained from a heterogeneous patient group. Deposits of C4d, mannose-binding lectin, C1q, IgM, and C5b-9 were scored in the glomeruli, peritubular capillaries, and arterioles. Notably, C4d deposits were present in 88.1% of TMA cases, and the various clinical conditions had distinct staining patterns within the various compartments of the renal vasculature. Classical pathway activation was observed in 90.5% of TMA cases. C5b-9 deposits were present in 78.6% of TMA cases and in 39.6% of controls (n=53), but the staining pattern differed between cases and controls. In conclusion, C4d is a common finding in TMA, regardless of the underlying clinical condition. Moreover, C5b-9 was present in >75% of the TMA samples, suggesting that terminal complement inhibitors may have a beneficial effect in these patients. C4d and C5b-9 should be investigated as possible diagnostic biomarkers in the clinical work-up of patients suspected of having complement-mediated TMA., (Copyright © 2015 by the American Society of Nephrology.)

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy., (Copyright © 2015 by the American Society of Nephrology.)

Anserine (β-alanyl-N(Pi)-methyl-L-histidine), a methylated derivative of carnosine (β-alanyl-L-histidine), is an abundant constituent of vertebrate skeletal muscles. Although it has been suggested to serve as a proton buffer and radical scavenger, its physiological function remains mysterious. The formation of anserine is catalyzed by carnosine N-methyltransferase, recently identified in chicken as histamine N-methyltransferase-like (HNMT-like) protein. Although the HNMT-like gene is absent in mammalian genomes, the activity of carnosine N-methyltransferase was reported in most mammalian species. In the present investigation, we purified carnosine N-methyltransferase from rat muscles about 2600-fold. Three polypeptides of ∼ 45, 50, and 70 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of UPF0586 protein C9orf41 homolog as the only meaningful candidate. Rat UPF0586 and its yeast, chicken, and human orthologs were expressed in COS-7 cells and purified to homogeneity. Although all recombinant proteins catalyzed the formation of anserine, as confirmed by chromatographic and mass spectrometry analysis, rat UPF0586 was more active on carnosine than other orthologs. Confocal microscopy of HeLa cells expressing recombinant UPF5086 proteins revealed their presence in both cytosol and nucleus. Carnosine and Gly-His were the best substrates for all UPF0586 orthologs studied, although the enzymes also methylated other l-histidine-containing di- and tripeptides. Finally, cotransfection of COS-7 cells with rat or human UPF0586 and carnosine synthase transformed the cells into efficient anserine producers. We conclude that UPF0586 is mammalian carnosine N-methyltransferase and hypothesize that it may also serve as a peptide or protein methyltransferase in eukaryotes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)