Three Dimensional Glomerular Reconstruction: A Novel Approach to Evaluate Renal Microanatomy in Diabetic Kidney Disease.

Mesangial metrics reflect glomerular filtration surface area in diabetes. The point-sampled intercept (PSI) method is the conventional method to calculate these parameters. However, this is time consuming and subject to underestimation. We introduce a novel three-dimensional (3D) reconstruction method applicable to light microscopy to measure mesangial metrics. Transmission electron microscopy (TEM), PSI and our new 3D imaging methods were used to quantify mesangial metrics from 22 patients with type 2 diabetes, normo-, micro- and macroalbuminuria and an estimated glomerular filtration rate of <60 mL/min/1.73 m2. Repeated-measures ANOVA test was used to test the equality of the measurement means from the three methods and the degree of inter method variability. Repeated-measures and post-estimation ANOVA tests together with correlation coefficient measurements were used to compare the methods with TEM as reference. There was a statistically significant difference in mesangial volume measurements (F(2, 16) = 15.53, p = 0.0002). The PSI method underestimated measurements compared to TEM and 3D methods by 30% (p = 0.001) and 15%, respectively (p < 0.001). 3D and TEM measurements did not differ significantly. 3D reconstruction is a reliable and time efficient method for calculating mesangial metrics. It may prove to be a useful tool in clinical and experimental diabetic kidney disease.

Involvement of Rho-kinase in P2Y-receptor-mediated contraction of renal glomeruli.

The involvement of Rho-kinase in P2Y-receptor induced contraction of isolated rat renal glomeruli was investigated. The contraction effects have been investigated based on changes in the intracapillary volume of isolated glomeruli. ATP was found to induce time- and concentration-dependent contraction of isolated glomeruli. Other tested nucleotides (ADP, UTP) and ATP analogues (beta,gamma-methylene-ATP, 2-methylothio-ATP) contracted glomeruli in similar magnitude whereas AMP had no effect. Furthermore, the contractive effect of ATP was prevented in the presence of an antagonist of P2Y-receptors, reactive blue 2. However, a selective antagonist of A1-receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), had no effect. Contraction induced by ATP, ADP, and UTP, in contrast to 2-methylothio-ATP and beta,gamma-methylene-ATP, was prevented in the presence of Rho-kinase's inhibitor, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632). These findings suggest the involvement of Rho-kinase pathways in P2Y-induced contraction of isolated glomeruli.

[Concise information on the structure and function of intraglomerular mesangia]. / Strucná informace o strukture a funkcích intraglomerulárního mesangia.

The area between glomerular capillaries is filled with special cellular structure-mesangium. Intraglomerular mesangium is composed from mononuclear stellar cells embedded into an amorphous extracellular matrix. It has several functions: firm and compliant structural sustenance of capillaries, regulation of the ultrafiltration coefficient by means of the capillary surface area control, phagocytosis and removal of macromolecules and immune complexes, hormone secretion and production of bioactive substances (endothelin, prostaglandins, renin, cytokines, immunomodulatory peptides, components of the extracellular matrix etc.).

Three-dimensional analysis of the whole mesangium in the rat.

The three-dimensional structure of the mesangium was analyzed by means of reconstruction from serial semithin and ultrathin sections of the rat glomerulus. The mesangial domains traced on light micrographs of semithin sections were transferred to styrene models, which were stacked up to reconstruct the whole mesangium. The reconstructed mesangium was tree-like in shape and was divided into three lobes that were connected to the vascular pole by a slender neck. The glomerulus contained no islets of mesangium which were not connected to the vascular pole. The mesangium contained 64 mesangial loops that were penetrated by capillaries. Reliability of the findings on the mesangial loops was ascertained by various methods including reconstruction of part of the mesangium from ultrathin sections. Electron microscopic observations revealed that the arms of the mesangial loops were frequently very slender and consisted of mesangial cell processes containing prominent bundles of actin filaments. The mesangial loops were distributed evenly within the mesangium. Considering previous reports showing about 400 capillary branches in the rat glomerulus as well as the present findings, we concluded that the mesangial loops may change the distribution of intraglomerular blood flow by dynamic contraction of the mesangial cells, or serve as an additional safety device to prevent the expansion of glomerular capillaries.

An evaluation of the renal protective effect of manidipine in the uninephrectomized spontaneously hypertensive rat.

The effects of calcium antagonism on the development and progression of renal disease are controversial. To address this problem, studies were performed on young, uninephrectomized spontaneously hypertensive rats (SHRs) with the dihydropyridine calcium antagonist, manidipine, to assess its effect on the early pathogenesis of focal glomerulosclerosis. Male SHRs underwent uninephrectomy at age 10 to 11 weeks and were subsequently assigned to no treatment (control), a predetermined subvasodepressor (low) dose of manidipine (2.5 mg/kg body weight), or a predetermined antihypertensive (high) dose of manidipine (20 mg/kg body weight). All animals received a diet containing 0.4% sodium and 23% protein. Serial determinations of body weight, systolic tail cuff pressure, and 24-hour urinary excretion of creatinine, sodium, and protein (UprotV) were made at 1- to 6-week intervals, for a total treatment period of 12 weeks. In final experiments plasma was obtained for creatinine, angiotensin I, and angiotensin II determinations, and renal tissue was harvested for histologic and morphometric analysis. Compared with the untreated control, low-dose manidipine therapy had no effect on body weight, systolic blood pressure, creatinine clearance, UprotV, renal histologic findings, glomerular volume, or plasma angiotensin I or II concentrations. In contrast, high-dose manidipine therapy decreased systolic blood pressure from 194 +/- 3 to 160 +/- 4 mm Hg (p < 0.01). Creatinine clearance and UprotV were unchanged. Although body weight was not different, kidney weight was higher. However, mean glomerular volume was lower. More importantly, the prevalence of mesangial expansion with proliferation was lower: 6.7% (control) versus 2.8% (high-dose manidipine) (p < 0.01). Finally, plasma angiotensin I and angiotensin II concentrations did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

Species-specific properties of the glomerular mesangium.

Mesangial cells play a central role in the physiology and pathophysiology of the glomerulus. To date, most of the in vitro studies have been performed in cultured rat mesangial cells, with only 10% of them performed in human mesangial cells. In this article, the major differences between results obtained with these two types of cultured cells will be reviewed. In rats and in humans, most of the mesangial cells appear to be of smooth muscle origin. In the rat, some of the cultured cells also express a phenotype suggesting a monocyte/macrophage origin. Phagocytosis and synthesis of cytokines or proinflammatory proteins that have been described in cultured rat cells seem mostly linked to this monocyte/macrophage subtype of resident mesangial cells. In humans, macrophages are only detected in pathologic conditions, suggesting that they are not resident but rather infiltrating cells. Mesangial receptors, most notably angiotensin II receptors, are clearly present on mesangial cell membranes and are linked to prostaglandin E2 synthesis and to cell contraction. In humans, spontaneous prostanoid synthesis is low and is increased by the induction of cyclooxygenase by sodium butyrate in the medium. Even so, the amount of prostaglandin E2 synthesized by human mesangial cells is quantitatively low comparatively with that in rats. In rats, accordingly, mesangial cells play a role in the regulation of single-nephron GFR. In humans, angiotensin II also exerts a control on GFR but it is more difficult to demonstrate its contractile effect on human than on rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathology of the human mesangium in situ.

Mesangial cells play an important role in the development and progression of human glomerular disease. This article summarizes some important aspects of mesangial properties and behaviour in situ. Intrinsic mesangial cells express alpha-smooth muscle actin and are best characterized as myofibroblasts or glomerular pericytes. The main integrin receptor in the mesangium is the alpha 1 beta 1 integrin. The beta 2 and beta 3 integrins have not been detected. Mesangial cells in situ fail to react with many monoclonal antibodies which stain human mesangial cells in culture, including leukocyte activation antigens. Prominent reactions in glomerular disease are mesangial expansion and progressive glomerular sclerosis, which are preceded by or associated with mesangial cell hypertrophy and/or proliferation. Mesangial enlargement is accompanied by an altered integrin expression and an abnormal composition of extracellular mesangial matrix. From the numerous autocrine and paracrine mediators identified in vitro which stimulate or inhibit mesangial cell growth and extracellular matrix synthesis, up to now only a few factors have been shown to be present in selected human glomerulopathies. These include platelet derived growth factors and platelet derived growth factor receptor beta, transforming growth factors beta, interleukin 1 beta, tumor necrosis factor alpha, and interleukin 6. Further identification of such mediators in situ will improve our understanding of pathological glomerular processes, particularly with respect to the multifunctional properties of the mesangial cell.

An approach to the structure and function of the glomerular mesangium.

The mesangium of the glomerulus is a connective tissue tree arising at the vascular pole of the glomerulus and supporting the glomerular capillaries. It is partly covered by a basement membrane that follows the epithelial cells from the peripheral glomerular capillary wall over the supporting tissue. The capillary endothelium does not normally have a separate basement membrane. The endothelium has fenestrations that open directly into the mesangium and allow blood plasma and tracers to flow into the mesangium. The fenestrations partially restrict (or sieve) particles over 405 A in mean length from entry. Tracers move in intercellular channels and are filtered and concentrated by the basement membrane at the sides of the mesangium or by mesangial matrix filaments in the channels between cells. The irregular distributions of flow, matrix, and concentrations of tracers may account for irregular lobular reactions in glomerular disease. Two main pathways of flow seem to be (1) through the basement membrane and between the epithelial foot processes to form part of the glomerular filtrate and (2) into the efferent capillaries through their mesangial fenestrations. Intrinsic mesangial cells can now be regarded as myofibroblasts associated with the production of the connective tissue matrix. These cells hold the basement membrane to maintain the shape of the glomerular capillaries, they swell readily, and they can constrict like smooth muscle cells with appropriate stimulation. These reactions may enable them to control the flow of blood through the capillary network in glomerular disease. Mesangial cells can take up large amounts of foreign material within 24 h. Intrinsic mesangial cells and monocytes can increase in numbers in disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in glomerular cross-sectional area induced by platelet activating factor.

Isolated glomeruli from normal rats were incubated with platelet-activating factor 10(-6) M at variable incubation times (8, 15, 30 and 45 min) and with different concentrations (0 to 10(-6) M) for 20 min. In addition, the platelet-activating factor effect (10(-6) or 10(-8) M, 20 min) was tested in the presence of BN-52021 (10 or 50 micrograms/ml), verapamil (10(-5) M), acetylsalicylate of lysine (10(-3) M), and in a free-calcium media with EGTA 2 mM. Glomerular microphotographs were taken before and 20 min after adding the substances, and glomerular cross-sectional area was measured using a computerised technique. Platelet-activating factor induced a significant time-dependent reduction in glomerular cross-sectional area, from a concentration of 10(-8) M. BN-52021, verapamil, and the free-calcium media inhibited platelet-activating factor-induced reduction of glomerular cross-sectional area, but acetylsalicylate of lysine did not. Platelet-activating factor-induced reduction in glomerular cross-sectional area seems to be dependent on the interaction of platelet-activating factor with a specific glomerular receptor, with a subsequent modification of the intracellular concentration of calcium. Arachidonic acid metabolites from the cyclo-oxygenase pathway do not seem to be involved in these phenomena. Results suggest that platelet-activating factor could modulate glomerular filtration rate not only by inducing changes in systemic or intrarenal haemodynamics, but also by modifying the filtration surface, thus reducing the Kf.