Loss of filtration function in diabetic glomeruli is associated with ultrastructural changes in glomerular endothelial cell fenestrations

Background: The study of glomerular endothelial cell (GEnC) fenestrations including key regulatory factors is neglected despite their loss in diabetic nephropathy, a disease associated with decreased filtration function, being previously described. Methods: We comprehensively characterised GEnC fenestral and renal filtration functional changes including measurement of glomerular ultrafiltration coefficient and glomerular filtration rate (GFR) in diabetic mice and humans. We further evaluated Eps homology domain protein-3 (Ehd3) as a potential regulator of GEnC fenestrations. Results: This study identified loss of GEnC fenestration density which was associated with decreased renal filtration function in diabetic nephropathy. We also identified increased GEnC fenestration width, an ultrastructural change that may develop to maintain filtration surface area. GEnC fenestration width was negatively associated with renal filtration function considered a result of development of diaphragms in widening fenestrations providing resistance to filtration. The increased presence of diaphragmed fenestrations in diabetes was supported by increased PLVAP1 expression. We identified decreased glomerular Ehd3 expression in diabetes and demonstrated its association with GEnC fenestration measurements suggesting its role in regulating fenestrations. We further demonstrated reduced fenestration formation in vitro in an Ehd3 knockdown cell line. Ehd3 was positively associated with filtration function suggesting loss of glomerular Ehd3 expression in disease may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. Conclusions: This is the first study to demonstrate the critical role of GEnC fenestrations in renal filtration function and identify a key regulator, Ehd3, that may serve as a therapeutic target to retore filtration function in disease.

Diuretic activity of methylxanthine derivative furoxane with one- and long-time use on the excretory function system of rats on the background of spontaneous diuresis

The study of the effect of furoxane on renal activity under conditions of spontaneous diuresis was performed by the method of Berkhin. Hydrochlorothiazide was chosen as a reference drug. Furoxane and hydrochlorothiazide were administered intragastrically at a dose of 25 mg/kg for 7 days. The content of Na+ and K+in the urine was determined by flame photometry. The concentration of creatinine in the urine was studied by the method of Folin in the modification of Berkhin. The obtained results were calculated using the methods of variation statistics. The analysis of the results shows that under one-time action of furoxane in rats the daily urination increased by 210.4 % (p <0.001), the concentration of Na+ in the urine of rats increased by 51.9 % (p <0.05), and the concentration of K+ in the urine tended to increase by 5.6 %. Natriuresis increased 1.7 times (p <0.05), potassium - 1.23 times (p <0.05). Excretion of endogenous creatinine increased 1.49 times (p <0.05), indicating an improvement in glomerular ultrafiltration. After administration of hydrochlorothiazide there was an increase in diuresis by 92.2 % (p <0.05), urinary Na+ concentration increased by 49% (p <0.05) and increased Na+excretion from urine by 23.3 % (p <0.05). K+ concentration in urine increased by 12.6 %, and K+excretion - by 27.5 % (p <0.05). Thus, a single administration of furoxane at a dose of 25 mg/kg led to an increase in daily diuresis, Na+ and K+ diuresis, which indicates the potential diuretic and saluretic properties of the drug. Long-term (7 days) furoxane administration contributed to the increase of urination in animals. After 3 days diuresis increased by 226,1 % (p <0.001), on 7th day – by 236,3 % (p <0.001). Salures was increased: the excretion of Na+ after 3 days increased by 2.27 times (p <0,05), after 7 days - by 2,38 times (p <0,05), K+ - 1.83 times (p <0.05). The gradual increase in creatinine excretion in the dynamics of the use of furoxane, indicated a possible prolongation of the effect of furoxane. Thus, furoxane, which by its chemical structure belongs to methylxanthine derivatives is a promising pharmacologically active substance for further study of its specific activity and safety in order to create a highly effective diuretic and implementation in clinical practice.

Abstract 106: Signaling Mechanisms Affecting Nitric Oxide Production in Glomerular Podocytes

While Nitric Oxide (NO), a potent vasodilator and vital signaling molecule, has been shown to contribute to the regulation of glomerular ultrafiltration, its role in podocytes during the pathogenesis of salt-sensitive hypertension has not yet been thoroughly examined. Recent studies have demonstrated that the deficiency of eNOS (the enzyme responsible for synthesizing NO) exacerbates renal injury and accelerates development of proteinuria and glomerulosclerosis. Considering this, we hypothesized that the podocytes of hypertensive animals would exhibit reduced NO production in response to various paracrine factors and this directly contributes to proteinuria. To test this, we isolated glomeruli from the kidneys of Dahl salt-sensitive (SS) rats fed either a high salt (HS; 4% NaCl, 3 weeks) or low salt (LS; 0.4% NaCl) diet and loaded podocytes with a combination of NO and Ca 2+ ionophores (DAF-FM and Fura Red, respectively). Changes in fluorescence were observed with the use of confocal microscopy in response to adenosine triphosphate (ATP), angiotensin II (Ang II), and H 2 O 2 . Application of Ang II or H 2 O 2 resulted in activation of both NO and [Ca 2+ ] i ; fluorescent transients which were significantly elevated in the soma and foot processes of podocytes of LS fed rats. In contrast, ATP specifically activated only changes in [Ca 2+ ] i , but did not have any effects on NO production. Ang II treatment also caused hypertrophy of the podocytes, whereas ATP had no effect on cell volume (41.1±7.7 vs. 0.1±3.6% increase for Ang II and ATP, respectively; P <0.05). Collectively, our results show that in contrast to [Ca 2+ ] i , which is modulated by all studied paracrine molecules, NO is produced by podocytes only in response to Ang II and H 2 O 2 , but not ATP. SS rats fed a HS diet for 3 weeks demonstrated impaired NO production; the response to Ang II or H 2 O 2 on HS contained only 23.7±6.6 and 43.4±28.4% of total effects on LS, respectively ( P <0.05). Therefore, when fed a HS diet, SS podocytes had an impaired NO response to Ang II or oxidative stress, suggesting that NO signaling is dysfunctional and likely contributes to the development of kidney injury.

Functions of the podocyte proteins nephrin and Neph3 and the transcriptional regulation of their genes

Nephrin and Neph-family proteins [Neph1–3 (nephrin-like 1–3)] belong to the immunoglobulin superfamily of cell-adhesion receptors and are expressed in the glomerular podocytes. Both nephrin and Neph-family members function in cell adhesion and signalling, and thus regulate the structure and function of podocytes and maintain normal glomerular ultrafiltration. The expression of nephrin and Neph3 is altered in human proteinuric diseases emphasizing the importance of studying the transcriptional regulation of the nephrin and Neph3 genes NPHS1 (nephrosis 1, congenital, Finnish type) and KIRREL2 (kin of IRRE-like 2) respectively. The nephrin and Neph3 genes form a bidirectional gene pair, and they share transcriptional regulatory mechanisms. In the present review, we summarize the current knowledge of the functions of nephrin and Neph-family proteins and transcription factors and agents that control nephrin and Neph3 gene expression.

VEGF165b overexpression restores normal glomerular water permeability in VEGF164-overexpressing adult mice

Vascular endothelial growth factor (VEGF)-A, a family of differentially spliced proteins produced by glomerular podocytes, maintains glomerular filtration barrier function. The expression of VEGF molecules is altered in human nephropathy. We aimed to determine the roles of the angiogenic VEGF164 isoform, and the antiangiogenic VEGF165b isoform in mature, adult glomeruli in vivo using conditional, inducible transgenic overexpression systems in mice. Podocyte-specific VEGF164 overexpression (up to 100 days) was induced by oral administration of doxycycline to adult podocin-rtTA/TetO-VEGF164 double transgenic mice. The consequences of simultaneous overexpression of VEGF164 and VEGF165b were assessed in triple-transgenic podocin-rtTA/TetO-VEGF164/nephrin-VEGF165b mice. Persistent VEGF164 overexpression did not cause proteinuria but did increase glomerular ultrafiltration coefficient between days 3 and 7. Despite persistently increased VEGF164 levels, glomerular ultrafiltration coefficient normalized by day 14 and remained normal up to 100 days. Decreased subpodocyte space (SPS) coverage of the glomerular capillary wall accompanied increased glomerular hydraulic conductivity in VEGF164-overexpressing mice. The changes in glomerular ultrafiltration coefficient and SPS coverage induced by 7 days of overexpression of VEGF164 were not present in triple transgenic VEGF164 and VEGF165b overexpressing mice. These results indicate that 1) the adult mouse glomerulus is relatively resistant to induced VEGF164 overexpression. VEGF164 overexpression altered glomerular permeability but did not cause proteinuria in these mature, adult animals; 2) the SPS is a dynamic VEGF-responsive modulator of glomerular function; and 3) the balance of VEGF isoforms plays a critical role in the regulation of glomerular permeability. VEGF165b is capable of preventing VEGF164-induced changes in glomerular permeability and ultrastructure in vivo.

Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking

Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.