Homoarginine test for evaluation of metabolic renal dysfunction

Low plasma L-homoarginine (hArg) concentration is an independent predictor of adverse cardiovascular outcomes and overall mortality, as well as the progression of chronic kidney disease (CKD). The enzyme L-arginine:glycinamidinotransferase (AGAT, EC 2.1.4.1) acts in the mitochondrial membrane of the renal tubular epithelium, forming the precursor of creatine, guanidinoacetic acid, and additionnaly by-product hArg. As it was shown recently, there is a decreased level of hArg in the late stages of CKD, however, the the level of hArg in the early stages of CKD remained unexplored. The aim of this study was to determine the diagnostic threshold levels of hArg in the blood of patients with stages 1 and 2 of CKD. In patients with the initial stages of CKD (n = 44) at the age of 58 (45-67) years, compared with the group of donors of 55 (42-58) years (n = 30), a significant decrease of hArg level was found. In the subgroup with stage CKD 2, the cut-off point of 1.59 μM threshold was characterized by greater sensitivity and specificity than in the subgroup with stage CKD 1 with 1.66 μM threshold level of hArg. For the full group, the hArg cut-off threshold was 1.60 μM, which is about to 0.2 μM lower than the lower limit of the reference interval for healthy individuals. It can be assumed that even before the formation of symptoms of proteinuria and albuminuria, a significant part of individuals from population cohort develops a state of decreased AGAT activity, since the expression of this enzyme is associated with a certain regulatory feedback inhibition at the body level. As a result of the study, it can be noted that in patients with early stages of CKD in the age group 45-67 years, there is a disturbance of the kidneys metabolic function. These metabolic changes can be detected by testing the level of hArg.

Toxicological studies of aqueous and ethanol leaf extract of Spondias purpurea (red plum) in rats

Background Spondias purpurea is a flowering plant of the cashew family commonly found in South Western Nigeria. The plant is used in folk medicine for treatment of gastric disorders and diarrhoea. The purpose of the study was to investigate the haematological and histopathological effects of aqueous and ethanol leaf extract of S. purpurea (red plum) in rats. Methods Female wistar rats weighing (121.5 ± 30.41 g) were administered 500, 1000 and 1500 mg/kg body weight of aqueous and ethanol extracts of S. purpurea leaf (orally) daily for 14 days, while the control group was administered 0.5 ml of normal saline (vehicle). At the end of the study, the rats were euthanized; blood samples were collected for haematological parameters. The liver, kidney and spleen were harvested from the rats for photomicrographic examination. Result The result of the acute toxicity test revealed no death with dose up to 5000 mg/kg body weight. The administration of the extracts showed no significant difference (p > 0.05) in the hematologic parameters of the animals. The liver sections showed congestion, mononuclear infiltration, widened sinusoidal space and congestions with hemosiderin. Similar changes were observed in the kidney showing slight necrosis of renal tubular epithelium, widened Bowman’s space, and collapsed renal tubules and adhesion of the parietal layer of glomerulus to the Bowman’s space. The spleen showed congestion, lymphocyte proliferation at the germinal centre. Conclusions The result of this study showed that the alterations observed in the organs intensified with increase in the doses of the extracts administered. It can be inferred that the prolonged consumption of S. purpurea leaf maybe associated with significant tissue damage of some vital organs.

Expression of ACE2 in the Intact and Acutely Injured Kidney

Background. The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. Evidence supports ACE2 as a cytoprotectant in some tissues. This study examined ACE2 expression in models of acute kidney injury (AKI). Methods. ACE2 mRNA and protein expression, ACE2 activity, and ACE2 expression by immunofluorescence were assessed following ischemic AKI in mice. Renal ACE2 mRNA expression was evaluated in lipopolysaccharide-induced AKI in wildtype (C57BL/6J) mice, in heme oxygenase-1+/+ and heme oxygenase-1-/- mice, and following unilateral urinary tract obstruction (UUO) in wildtype mice. The effect of sex and age on renal ACE2 protein expression was also assessed. Results. In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the renal tubular epithelium, especially in proximal tubules, exhibited decreased expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR per se as ACE2 mRNA expression was unaltered after UUO. Lipopolysaccharide induced renal ACE2 mRNA expression in wildtype mice, but this effect of lipopolysaccharide did not occur in heme oxygenase-1 deficient mice. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. Conclusion. We conclude that renal ACE2 expression is decreased in ischemic AKI, one characterized by markedly reduced GFR and abundant cell death, but is upregulated in lipopolysaccharide-induced AKI; this latter effect requires heme oxygenase-1. Determining the significance of ACE2 expression in models of AKI merits further study. We also suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation promotes inflammation in COVID-19; and AKI commonly occurs and determines outcomes in COVID-19.

O25: TARGETING THE RENAL TUBULAR EPITHELIUM WITH ANTI-MIRNA THERAPY: A POTENTIAL MECHANISM FOR MINIMISING ISCHAEMIA REPERFUSION INJURY

Introduction Ischaemia reperfusion injury (IRI) is an unavoidable, significant consequence of renal transplantation. MicroRNAs are small, non-coding RNA molecules that regulate multiple downstream mRNA targets. MiRNA-21-5p and miRNA-24-3p have been previously implicated in IRI. Antisense oligonucleotides (ASOs) block specific microRNAs, with previous work by our group demonstrating their delivery to kidneys using normothermic machine perfusion. Imaging these kidneys revealed ASO localisation around proximal tubule epithelial cells (PTECs). This project aimed to characterise ASO blockade against miRNA-21-5p and miRNA-24-3p in PTECs. Method HKC8 cells, a human PTEC cell line, were used throughout these experiments. Cells were placed in a hypoxic incubator for 24hrs, followed by 6hrs of reoxygenation to mimic IRI. HKC8s were transfected with ASOs using lipofectamine. RT-qPCR and Western Blots were used to evaluate expression of antioxidant targets, SOD2 and HMOX1. Result MiRNA-21-5p and miRNA-24-3p levels were high throughout hypoxia and reoxygenation. Single blockade with anti-miRNA-21-5p resulted in a significant increase in its downstream target SOD2 (P<0.05). Anti-miRNA-24-3p treatment resulted in no change in either of its downstream targets, HMOX1 or SOD2. This was reflected in the failure of dual blockade to produce a synergistic effect on the shared target, SOD2. Conclusion Anti-miRNA-21-5p results in a significant increase of SOD2, which is well characterised as protective during IRI. Anti-miRNA-24-3p appears to have no effect on PTECs, contrary to previous work in endothelial cells, perhaps suggesting a cell specific response of microRNAs. Normothermic machine perfusion could be used to deliver dual ASOs; allowing simultaneous targeting of different kidney cell types. Take-home message The delivery of anti-miRNA-21-5p therapy pre-transplant, using normothermic machine perfusion, has the potential to reduce ischaemia reperfusion injury and improve kidney transplant outcomes.

Histomorphology and Immunohistochemistry of a Congenital Nephromegaly Demonstrate Concurrent Features of Heritable and Acquired Cystic Nephropathies in a Girgentana Goat (Capra falconeri)

Polycystic kidney diseases (PKD) represent frequent congenital and adult nephropathies in humans and domestic animals. This report illustrates an uncommon state of congenital PKD in a girgentana goat (Capra falconeri). A stillborn female goat kid was submitted for postmortem examination and underwent macroscopic and microscopic examination. The kidneys showed a bilateral nephromegaly and a perpendicular polycystic altered texture of the renal parenchyma. Renal tissue sections were comprehensively investigated by histopathology (overview and special stains), immunohistochemistry (CD10, CD117, pan-cytokeratin, cytokeratin 7, E-cadherin, Pax2, Pax8, and vimentin), and electron microscopy (SEM, TEM). Histopathology of renal tissue sections revealed polycystic alterations of the renal parenchyma as well as conspicuous polypoid proliferates/projections of the renal tubular epithelium, which showed clear cell characteristics. Furthermore, epithelial projections were indicative for epithelio-mesenchymal-transition, cellular depolarization, and strong expression of differentiation markers Pax2, Pax8, and CD10. Ultrastructural morphology of the projections was characterized by numerous diffusely distributed, demarked round cytoplasmic structures and several apico-lateral differentiations. Additionally, hepatic malformations comprising biliary duct proliferation with saccular dilation and bridging fibrosis were observed. Notably, this report describes the first case of a congenital cystic nephropathy with overlapping features of heritable and acquired nephropathies in any species. Epithelio-mesenchymal-transition and altered cadherin expression seem to be crucial components of a suspected pathomechanism during cystogenesis.

Mesenchymal stem cell-derived exosome miR-21-3p prevents renal fibrosis

Background: Renal fibrosis is the pathological result of excessive deposition of extracellular matrix (ECM) in the process of chronic kidney disease, but its mechanism is not clear. Mesenchymal stem cells (MSCs) exerts its therapeutic effect mainly through paracrine effects, such as exosome, to change the cellular microenvironment. Here, we explore the function of exosome derived MSCs in renal fibrosis.Methods: UUO model was constructed to simulate renal fibrosis in mice. Heat map and RT-PCR were used to explore the differential expression of miRNAs. RT-PCR and western blot were performed to detect the expression levels of fibrosis-associated genes and proteins in vivo and vitro. Transmission electron microscope and particle size detection were used to confirm the exosome construct. Then we forced expression of miR-21-3p in MSCs and isolated the exosomes. Then the fibrosis-associated genes and proteins were explored after exosomes injection.Results: In this study, we observed that exogenous miR-21-3p, interacted with smad2, the downstream target of miR-21-3p, which prevented renal fibrosis in UUO mice, and alleviate fibrosis in TGF-β1-induced renal tubular epithelium cells (HK-2). The extractive exosome-miR-21-3p treatment blocked renal fibrosis in UUO mice and alleviated fibrosis in TGF-β1-induced HK-2 cells and renal fibrosis mice. Conclusion: Taken together, Overexpression of miR-21-3p prevented CKD-induced renal fibrosis via exosome-mediated miR-21-3p transfer. These results suggest possible therapeutic strategies for using exosome delivery of miR-21-3p to treat complications of CKD.

THE ROLE OF DISORGANIZATION OF CONNECTIVE TISSUE AND BASEMENT MEMBRANE OF RENAL TUBULAR EPITHELIUM IN THE PATHOGENESIS OF UROLITHIASIS

According to a number of researchers, pathological changes in the structures of the renal papilla play a key role in the pathogenesis of urolithiasis. According to the results of existing studies, destructive processes in collagen are characterized by a change in the length, thickness of the fibers and their orientation in space. Collagen disorganization has enzymatic and non-enzymatic mechanisms. The aim of the study was to study the frequency and features of localization of Randall's plaques and morphological study of the state of connective tissue and the basement membrane of the epithelium of the renal tubules of different levels in urolithiasis. Microscopically studied preparations of 29 biopsies of renal tissue obtained from patients with fibro-uretero-pyeloscopy (11), percutaneous nephrolitholapaxy (18) and preparations obtained at 20 autopsies in cases without visible renal pathology. Histological preparations were stained with hematoxylin and eosin to determine the state of connective tissue fibers using Van Gieson, Mallory, Schiff-reagent, alcian blue, and Kos for calcifications. The results of the study show that the morphological signs of mucoid swelling of the connective tissue and basement membrane of the epithelium of the renal tubules of different levels in combination with the formation of microcalcifications require further study of the possible role of disorganization of the connective tissue of the renal stroma in the initiation of stone formation in the kidneys.

Mesenchymal Stem Cell-Derived Exosome miR-21-3p Prevents Renal Fibrosis

Background: Renal fibrosis is the pathological result of excessive deposition of extracellular matrix (ECM) in the process of chronic kidney disease, but its mechanism is not clear. Preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) can delay the progression of chronic kidney disease (CKD).MSCs exerts its therapeutic effect mainly through paracrine effects, such as exosome, to change the cellular microenvironment. Here, we would explore the function of MSCs derived exosome in renal fibrosis.Materials and Methods: MRNA, microRNAs and proteins carried by exosomes produced by MSCs could save damaged cells by regulating endogenous molecules to regulate apoptosis, inflammation, fibrosis and angiogenesis. Results: In this study, we observed that exogenous miR-21-3p, interacted with smad2, the downstream target of miR-21-3p, which prevented renal fibrosis in UUO mice, and alleviate fibrosis in TGF-β1-induced renal tubular epithelium cells (HK-2). Then we forced expression of miR-21-3p in MSCs and isolated the exosomes. The extractive exosome-miR-21-3p treatment blocked renal fibrosis in UUO mice and alleviated fibrosis in TGF-β1-induced HK-2 cells. Conclusion: Taken together, Overexpression of miR-21-3p prevented CKD-induced renal fibrosis via exosome-mediated miR-21-3p transfer. These results suggest possible therapeutic strategies for using exosome delivery of miR-21-3p to treat complications of CKD.

Ultrastructural Evidence for Direct Renal Infection with SARS-CoV-2

BackgroundA significant fraction of patients with coronavirus disease 2019 (COVID-19) display abnormalities in renal function. Retrospective studies of patients hospitalized with COVID-19 in Wuhan, China, report an incidence of 3%–7% progressing to ARF, a marker of poor prognosis. The cause of the renal failure in COVID-19 is unknown, but one hypothesized mechanism is direct renal infection by the causative virus, SARS-CoV-2.MethodsWe performed an autopsy on a single patient who died of COVID-19 after open repair of an aortic dissection, complicated by hypoxic respiratory failure and oliguric renal failure. We used light and electron microscopy to examine renal tissue for evidence of SARS-CoV-2 within renal cells.ResultsLight microscopy of proximal tubules showed geographic isometric vacuolization, corresponding to a focus of tubules with abundant intracellular viral arrays. Individual viruses averaged 76 µm in diameter and had an envelope studded with crown-like, electron-dense spikes. Vacuoles contained double-membrane vesicles suggestive of partially assembled virus.ConclusionsThe presence of viral particles in the renal tubular epithelium that were morphologically identical to SARS-CoV-2, and with viral arrays and other features of virus assembly, provide evidence of a productive direct infection of the kidney by SARS-CoV-2. This finding offers confirmatory evidence that direct renal infection occurs in the setting of AKI in COVID-19. However, the frequency and clinical significance of direct infection in COVID-19 is unclear. Tubular isometric vacuolization observed with light microscopy, which correlates with double-membrane vesicles containing vacuoles observed with electronic microscopy, may be a useful histologic marker for active SARS-CoV-2 infection in kidney biopsy or autopsy specimens.

BCL-XL is essential for the protection from secondary anemia caused by radiation-induced fatal kidney damage

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis, but less is known about the roles of these proteins in adults, including in the response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL-deficiency in erythroid cells. Unexpectedly, the combination of total-body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combinations with DNA damage-inducing drugs for cancer therapy.SummaryThe inducible loss of BCL-XL in all cells of adult mice causes primary anemia due to apoptosis of erythroid and megakaryocytic cell populations. In contrast γ-radiation plus loss of BCL-XL in all cells except hematopoietic cells causes secondary anemia resulting from kidney damage.