Computational Identification and 3D Morphological Characterization of Renal Glomeruli in Optically Cleared Murine Kidneys

The aim of this study was to establish an accessible methodology for the objective identification and 3D morphological characterization of renal glomeruli in mice. 3D imaging of the renal cortex was performed by light sheet microscopy on iDISCO+ optical cleared kidneys of six C57BL/6J mice after labelling of the capillary endothelium by lectin injection. 3D images were processed with the open source software ImageJ, and statistical analysis done with GraphPad Prism. Non-visual delimitation of the external surface of the glomeruli was ensured by greyscale-based thresholding, the value of which was determined from the statistical analysis of the voxel frequency distribution. Exclusion of false-positive identification was done by successive volume- and shape-based segmentation. Renal glomeruli were characterized by their number, surface area, volume, and compactness. Average data were expressed as mean ± SD. The number of glomeruli was equal to 283 ± 35 per mm3 of renal tissue, representing 1.78 ± 0.49% of the tissue volume. The surface area, volume and compactness were equal to 20,830 ± 6200 µm², 62,280 ± 14,000 µm3 and 0.068 ± 0.026, respectively. The proposed standardized methodology allows the identification of the renal glomeruli and their 3D morphological characterization, and is easily accessible for biologists.

Curcumin Reinforces MiR-29a Expression, Reducing Mesangial Fibrosis in a Model of Diabetic Fibrotic Kidney via Modulation of CB1R Signaling

Renal fibrosis is a hallmark event in the pathogenesis of diabetic nephropathy. Considerable evidence now supports that multiple intracellular signaling pathways are critically involved in renal fibrosis. Previously, our studies have shown that dysregulation of the MicroRNA 29a (miR-29a)- or cannabinoid type 1 receptor (CB1R)-mediated signaling cascade in renal glomeruli substantially contributes to diabetic renal fibrosis. The purpose of the current study was to explore whether curcumin, a natural polyphenolic compound with potential renoprotective activity, could modulate the miR-29a/CB1R signaling axis to attenuate renal fibrosis. In this study, rat renal mesangial cells cultured in high glucose (HG) and the diabetic db/db mice were used as an in vitro and in vivo model of diabetes, respectively. Our results showed that in rat renal mesangial cells, curcumin treatment substantially counteracted HG-induced changes in the expressions of miR-29a, CB1R, peroxisome proliferator-activated receptor gamma (PPAR-γ), and a profibrotic marker type IV collagen (collagen IV), as assessed by quantitative Real-Time Polymerase chain reaction (RT-PCR). Furthermore, in the db/db mouse model, administration of curcumin markedly lowered urinary albumin excretion, and reduced deposition of extracellular matrices including collagen IV in renal tissues. Importantly, quantitative RT-PCR, in situ hybridization, and immunohistochemical analysis revealed that curcumin treatment consistently blocked diabetes-induced downregulation of miR-29a and upregulation of CB1R in renal glomeruli. Collectively, our study provides novel evidence showing that curcumin can rescue the dysregulated miR-29a/CB1R signaling pathway in glomerular mesangium to ameliorate diabetic renal fibrosis.

Diagnostic Value of Type IV Collagen Expression in Renal Glomeruli at Alport’s Syndrome

The Alport’s syndrome is the hereditary multisystem disease characterized by the development of the progressive nephropathy. The early diagnosis and subsequent prescription of nephroprotective therapy improves significantly the nephrological prognosis. Purpose of the Study. Determine the value of the immunohistochemical method for the Alport’s syndrome diagnosis. Material and methods. The clinical, laboratory and morphological data of 35 patients with suspected Alport’s syndrome (13 years of age [11; 16]; 18 boys and 17 girls) examined in the Nephrology Department in 2013–2019 were summarized. The study of the renal tissue included the light, immunofluorescence, electron microscopy of the kidney biopsy sample, determination of the expression of α1, α3 and α5 chains of type IV collagen in the renal glomeruli using the immunohistochemical method; the genetic testing was carried out for 26 patients. The children were divided into groups depending on the glomerular expression of α5 chain of type IV collagen: normal (group 1, n=18), decreased (group 2, n=4), negative (group 3, n=13). Results are as the following: The disorder of the expression of α5 chain was detected in ¾ (q = 0.78) patients with genetically confirmed Alport’s syndrome and in almost all children with the X-linked variant of the disease (q = 0.94). Results. Based on the genetic testing, the Alport’s syndrome was confirmed in ¼ of the children of the 1st group (the children with the heterozygous variants of COL4A3, COL4A5 genes) and in all children of the 2nd and 3rd groups (COL4A5 variants). The sensitivity/ specificity of the immunohistochemical study for the Alport’s syndrome diagnosis was 78% /100%, that of the electron microscopy – 93% /87%. The predictive value of the positive/negative result of the immunohistochemical study was 100% /66%, that of the electron microscopy – 95% / 88% compared with 100% / 88% with the combine use of two methods. Conclusion. The determination of the expression of α5 chain of type IV collagen in the renal glomeruli has the independent diagnostic value, but it is inferior to the electron microscopy in the heterozygous variants of the Alport’s syndrome. The high specificity of the immunohistochemical method makes it possible to confirm the Alport’s syndrome in the case of the change in the expression of α5 chain of type IV collagen in the renal glomeruli.

Nobiletin represses change in the levels of blood coagulation markers in the LPS-induced rat DIC model

Background: Nobiletin is contained in Shiikuwasa fruit, a popular citrus fruit from Okinawa Prefecture in Japan. Nobiletin reportedly acts as a strong antioxidant, an anti-inflammatory agent, and an anti-cancer agent, and it suppresses the expression of TF which triggers blood coagulation. However, in vivo verification of in vitro reports is necessary. This study used a rat model of LPS-induced microthrombosis based on the in vivo studies as previously reported. Sustained intravenous injection of LPS changed all blood coagulation indicators in the direction of thrombus formation. The aim of this study was to determine if intake of nobiletin could suppress DIC-like symptoms.Methods: Experimental SD rats were fully anesthetized and fixed to an operating table. Either LPS alone or nobiletin (50 mg/kg) plus LPS was given to rats to investigate the repressive effects of nobiletin on the expression of blood coagulation factors.Results: After 4 h of LPS infusion (12.5 mg/kg/h, i.v.), PLT counts and Fbg levels in rat plasma decreased by 80% and 74%, respectively. PT and APTT were extended by 180% and 256%, respectively. TF activity and PAI-1 antigen levels were remarkably increased (54- and 86-fold, respectively vs. control). Pretreatment on nobiletin (50 mg/kg, p.o.) reduced or suppressed fluctuations in blood coagulation indices caused by LPS. TF activity was repressed almost completely by nobiletin pretreatment. After 4 h, PAI-1 antigen levels in nobiletin-treated animals were repressed 82.6% compared to LPS-treated rats. Nobiletin repressed LPS-induced changes in TF and PAI-1 more effectively than other parameters. Further, nobiletin repressed fibrin thrombi formation in the renal glomeruli induced by LPS treatment.Conclusions: Nobiletin was found to reduce LPS-induced DIC-like symptoms in rats. In the fluctuations of blood indices related to the coagulation cascade, nobiletin suppressed the LPS-induced expression of PAI-1 and TF more effectively than other indices. The binding sites of transcription factors that are activated by LPS-induced signals reside in the promoter areas of TF and PAI-1 gene sequences. Thus, the suppression of TF and PAI-1 expression by nobiletin appears similar to mechanisms previously evaluated during in vitro experiments. Importantly, nobiletin repressed fibrin deposition in the renal glomeruli induced by LPS treatment and improved overall health. Nobiletin may function as an anti-thrombogenic agent when ingested daily. Keywords: nobiletin; LPS; DIC model; blood coagulation; anti-thrombogenic

Systemic AAV10.COMP-Ang1 rescues renal glomeruli and pancreatic islets in type 2 diabetic mice

IntroductionDiabetic hyperglycemia causes progressive and generalized damage to the microvasculature. In renal glomeruli, this results in the loss of podocytes with consequent loss of constitutive angiopoietin-1 (Ang1) signaling, which is required for stability of the glomerular endothelium. Repeated tail vein injection of adenovirus expressing COMP-Ang1 (a stable bioengineered form of Ang1) was previously reported to improve diabetic glomerular damage despite the liver and lungs being primary targets of adenoviral infection. We thus hypothesized that localizing delivery of sustained COMP-Ang1 to the kidney could increase its therapeutic efficacy and safety for the treatment of diabetes.Research design and methodsUsing AAVrh10 adeno-associated viral capsid with enhanced kidney tropism, we treated 10-week-old uninephrectomized db/db mice (a model of type 2 diabetes) with a single dose of AAVrh10.COMP-Ang1 delivered via the intracarotid artery, compared with untreated diabetic db/db control and non-diabetic db/m mice.ResultsSurprisingly, both glomerular and pancreatic capillaries expressed COMP-Ang1, compensating for diabetes-induced loss of tissue Ang1. Importantly, treatment with AAVrh10.COMP-Ang1 yielded a significant reduction of glycemia (blood glucose, 241±193 mg/dL vs 576±31 mg/dL; glycosylated hemoglobin, 7.2±1.5% vs 11.3±1.3%) and slowed the progression of albuminuria and glomerulosclerosis in db/db mice by 70% and 61%, respectively, compared with untreated diabetic db/db mice. Furthermore, COMP-Ang1 ameliorated diabetes-induced increases of NF-kBp65, nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase-2 (Nox2), p47phox and productions of myeloperoxidase, the inflammatory markers in both renal and pancreatic tissues, and improved beta-cell density in pancreatic islets.ConclusionsThese results highlight the potential of localized Ang1 therapy for treatment of diabetic visceropathies and provide a mechanistic explanation for reported improvements in glucose control via Ang1/Tie2 signaling in the pancreas.

A sorafenib-induced model of glomerular kidney disease

Glomerular damage and proteinuria are important pathophysiological signs of chronic kidney disease. This study provides data obtained using a model developed based on the use of the anti-cancer drug sorafenib. Sorafenib is a tyrosine kinase inhibitor that acts through the signaling pathway associated with vascular endothelial growth factor and is widely used to treat various types of cancer. Sorafenib, on the other hand, causes serious side effects in patients, including the development of chronic kidney disease. This study was aimed at using the nephrotoxic properties of sorafenib to model chronic kidney disease in rats. We showed that rats treated with sorafenib for 8 weeks along with a diet high in salt (8% NaCl) develop hypertension with high systolic blood pressure of 80 mmHg, proteinuria with an increase in protein content of 75% higher , and a 4-fold increase in glomerular damage compared to the control group. In case of damage to the renal glomeruli caused by sorafenib, the level of transcripts that are involved in the synthesis of key glomerular proteins such as nephrine, podocin, synaptopodin and subplanin is significantly reduced. Also, when studying this model, activation of the endothelial-mesenchymal transition is observed. In the group of rats treated with sorafenib, the mRNA level for the WT-1 endothelial cell marker was reduced by 20%, while the concentration of the Col III, FSP-1, α-SMA and vimentin mesenchymal cell markers increased by 2–3 times. Thus, we developed a preclinical model of chronic kidney disease, expressed in damage to the renal glomeruli. We also demonstrated that glomerular damage in this model is associated with decreased expression of key structural glomerular proteins and activation of the endothelial-mesenchymal transition of the kidneys.

Pathomorphological picture of diabetic nephropathy in experimental diabetes mellitus

Aim. To conduct a morphological study of the renal glomerulus in experimental diabetes mellitus and to study the pathomorphological features of the development of diabetic nephropathy. Methods. The study was carried out on 25 male Wistar rats. Modeling of diabetes mellitus was carried out by intraperitoneal injection of streptozotocin at a dose of 65 mg/kg. For more selective modeling of type 2 diabetes, the rats of the control group were intraperitoneally administered a solution of cytoflavin, calculated by the dosage of nicotinamide 115 mg/kg. In the comparison group 1 ml of physiological solution was administered in a similar manner. On day 28 of the experiment, the animals were euthanized under ether anesthesia, the kidneys were extracted, purified and washed with physiological saline. The material was fixed in a 10% neutral formalin solution. Using the morphometric method, the area of the renal glomeruli and the area of the capillary lumens were measured, and after special computer processing of digital photographs, the total area of the vascular bed in the glomerulus and the area of mesangium in the renal glomerulus were estimated. Results. After four weeks of experiment on rats with diabetes mellitus, some characteristic changes in the morphofunctional state of renal tissues developed: an increase of the area of renal glomeruli, thickening of the basement membrane of glomerular capillaries, an increase in the size of podocytes with a decrease of their number, and foci of nephrosclerosis. Conclusion. Based on the results of the study, characteristic pathomorphological phenomena in the kidneys of rats with experimental diabetes mellitus were recorded, which indicate the development of diabetic nephropathy.

MicroRNA-29a Attenuates Diabetic Glomerular Injury through Modulating Cannabinoid Receptor 1 Signaling

Diabetic nephropathy often leads to end-stage renal disease and life-threatening morbidities. Simple control of risk factors is insufficient to prevent the progression of diabetic nephropathy, hence the need for discovering new treatments is of paramount importance. Recently, the dysregulation of microRNAs or the cannabinoid signaling pathway has been implicated in the pathogenesis of various renal tubulointerstitial fibrotic damages and thus novel therapeutic targets for chronic kidney diseases have emerged; however, the role of microRNAs or cannabinoid receptors on diabetes-induced glomerular injuries remains to be elucidated. In high-glucose-stressed renal mesangial cells, transfection of a miR-29a precursor sufficiently suppressed the mRNA and protein expressions of cannabinoid type 1 receptor (CB1R). Our data also revealed upregulated CB1R, interleukin-1β, interleukin-6, tumor necrosis factor-α, c-Jun, and type 4 collagen in the glomeruli of streptozotocin (STZ)-induced diabetic mice, whereas the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) was decreased. Importantly, using gain-of-function transgenic mice, we demonstrated that miR-29a acts as a negative regulator of CB1R, blocks the expressions of these proinflammatory and profibrogenic mediators, and attenuates renal hypertrophy. We also showed that overexpression of miR-29a restored PPAR-γ signaling in the renal glomeruli of diabetic animals. Collectively, our findings indicate that the interaction between miR-29a, CB1R, and PPAR-γ may play an important role in protecting diabetic renal glomeruli from fibrotic injuries.