Thiazides stimulate calcium absorption in urinary bladder of winter flounder

Thiazides inhibit voltage-independent NaCl absorption in the urinary bladder of the winter flounder presumably by blocking an electroneutral mucosal Na/Cl co-transporter. As thiazides stimulate calcium absorption in mammalian distal convoluted tubule while inhibiting NaCl absorption, we studied the effects of hydrochlorothiazide (HCTZ) on unidirectional 45Ca fluxes and intracellular electrical potential in short-circuited bladders to examine possible mechanisms of HCTZ effects on calcium transport. Basal secretory calcium flux was, on average, slightly larger than absorptive flux, reflecting small net calcium secretion. Mucosal addition of HCTZ (10(-4) M) stimulated absorptive calcium flux by 46% while the secretory flux was unaltered. Thus, HCTZ tended to induce net calcium absorption. Pre-treatment with serosal ouabain (10(-4) M) attenuated the HCTZ-induced increase in absorptive calcium flux. Moreover, HCTZ hyperpolarized the mucosal membrane potential by 18% as measured by conventional open-tip microelectrodes. These effects of HCTZ are consistent with the hypothesis that HCTZ indirectly stimulates Na/Ca exchange located at the serosal membrane. In conclusion, HCTZ in flounder urinary bladder, as in mammalian distal convoluted tubule, simultaneously inhibits NaCl absorption and stimulates calcium absorption. This study expands on the functional similarities between the flounder urinary bladder and the mammalian distal convoluted tubule.     

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.     

RAGE mRNA expression in the diabetic mouse kidney

Receptors for advanced glycation end products (RAGE), which bind and internalize AGE-modified proteins formed from oxidation and other products of the nonenzymatic glycation reaction, have been mechanistically implicated in the development of the chronic complications of diabetes. In the present experiments, we sought evidence for the participation of RAGE in diabetic nephropathy by analysis of steady state levels of mRNA encoding RAGE in the renal cortex of a well-defined animal model (the db/db mouse) that develops renal pathology similar to that found in human diabetes. In these animals, increased AGE-product formation was confirmed by measurement of fluorescence in serum and renal cortex proteins. Renal involvement was confirmed by demonstration of increased urine albumin excretion and elevated serum creatinine concentrations relative to nondiabetic (db/m) littermate controls. Despite elevated concentrations of circulating and tissue AGE-modified proteins, the level of RAGE mRNA expression in renal cortex of diabetic mice did not significantly differ from that in nondiabetic littermate controls. The findings militate against changes in RAGE expression in the pathogenesis of renal abnormalities in this animal model.     

Global Renal Gene Expression Profiling Analysis in B2-Kinin Receptor Null Mice: Impact of Diabetes

Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B₂-receptors (B₂R) in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B₂R knockout (B₂R^−/−) mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER) were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B₂R^−/−D null mice had a significantly decreased AER levels compared to wild type B₂R^+/+D mice (P = 0.0005). Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B₂R^+/+C, B₂R^+/+D, B₂R^−/−C and B₂R^−/−D) highlighted the role of several altered pathological pathways in response to disruption of B₂R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.     

Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

pCMBS-induced swelling of dogfish (Squalus acanthias) rectal gland cells: role of the Na+,K(+)-ATPase and the cytoskeleton

(1) 0.1-1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark (Squalus acanthias) rectal glands, with an uptake of cell Na+ and a loss of K+. In contrast, 1 mM N-ethylmaleimide (NEM) does not swell rectal gland cells (RGC), while affecting cell cations. (2) The slow entry of [203Hg]pCMBS is linearly related to its external concentration (10 microM-1 mM) and a small accumulation of pCMBS (apparent gradient about 3) in the cells occurs in 2 h. Cell 203Hg rapidly washes out of the cells (fast rate constant 0.153.min-1; slow rate constant 0.0067.min-1), and this efflux is accelerated by 1mM dithiothreitol. Thus, a major portion of pCMBS inter-acts rather loosely with cell components. (3) pCMBS and NEM share: (a) a negligible effect on the efflux of 86Rb+ and of [14C]urea; (b) a gradual inhibition of the cell Na+,K(+)-ATPase activity. (4) NEM as well as agents lowering cell glutathione accelerate and increase the pCMBS-induced cell swelling. Conditions inhibiting the Na+,K(+)-ATPase (ouabain, absence of Na+) have the same effect. (5) pCMBS, but not NEM produce a disappearance of the F-actin-phalloidin fluorescence independent of cell volume changes, particularly at the basolateral RGC membrane. (6) The data are consistent with the following set of events: (a) pCMBS (but not NEM) affects the cell membrane by increasing the efflux of the cell osmolyte taurine (Ziyadeh et al. (1988) Biochim. Biophys. Acta 943, 43-52 and unpublished data); (b) on entry into the cells, pCMBS and NEM interact with cell -SH, including those of the Na+,K(+)-ATPase; this action produces the observed changes in cell cations. Also, pCMBS, but not NEM, decrease F-actin at the membrane; (c) the inhibition of the Na+,K(+)-ATPase activity together with the decreased resistance of the cell membrane to stretch (absence of F-actin) produces the observed pCMBS-induced cell swelling by osmotic forces (intracellular non-diffusible anions).     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

20-HETE and EETs in Diabetic Nephropathy: A Novel Mechanistic Pathway

Diabetic nephropathy (DN), a major complication of diabetes, is characterized by hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. Hypertrophy is a major factor inducing proximal tubular epithelial cells injury. However, the mechanisms leading to tubular injury is not well defined. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. In conclusion, we show that hyperglycemia in diabetes has a significant effect on the expression of Arachidonic Acid (AA)-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites.     

Prevalence of endoparasites in dog faecal deposits in Jordan.

Of 756 dog faecal deposits collected from the road side and public places in five Jordanian Governorates 466 (61.6%) harboured parasite ova, oocysts and/or larvae. Of these, 67.7%, 26.8%, 4.1% and 1.4% revealed one, two, three and four different types of parasite ova, oocysts or larvae in the same sample, respectively. Examination of the infected samples revealed that 44.1% contained taeniid ova, 19.8% Dipylidium caninum, 19% Toxocara canis, 8% Sarcocystis spp. oocysts, 5% hookworm larvae, 1.5% Diphyllobothrium latum, 0.1% Capillaria spp, and 0.1% Trichuri vulpis eggs.