Alteration of cytosolic calcium induced by angiotensin II and norepinephrine in mesangial cells from diabetic rats.

To evaluate functional alterations of mesangial cells induced by diabetes (DMC), we observed the changes of cytosolic calcium ([Ca]i) in response to the vasoconstrictor agonists angiotensin II (Ang II) and norepinephrine (NOR). DMC were obtained from rats with streptozotocin-induced diabetes, cultured in normal medium and identified as mesangial cells (MC) in the third subculture. [Ca]i was measured using fura-2 as a fluorophore. Basal calcium levels (60 to 80 nM) in DMC were not different from control mesangial cells (CMC). The high glucose (30 mM) medium concentration reduced the response of CMC and DMC to Ang II and NOR. This was not an osmotic effect since mannitol did not alter these responses. When DMC were stimulated with Ang II, a desensitized response was always observed, with a transient variation of [Ca]i (N = 6, P < 0.05). In contrast, a non-desensitized response with a sustained pattern of [Ca]i increases was obtained in NOR-stimulated DMC. Therefore, the present results suggest that DMC show a modified response to stimulation of the Ang II receptor, which is expressed phenotypically in culture by desensitization. Furthermore, these alterations induced by diabetes environment in MC in vivo were maintained in vitro despite a long period (approximately 5 months) in which the cells were grown in normal culture medium.

Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats.

In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. The dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. In contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. The dermatan sulfate was secreted to the medium as proteoglycan. Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. The proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.

FK 506: effects on glomerular hemodynamics and on mesangial cells in culture.

FK 506 is a new immunosuppressive drug that, like cyclosporine A (CsA), presents nephrotoxicity. Glomerular hemodynamic studies showed that acute FK 506 infusion (N = 9, 3 mg/kg body wt, i.v. in bolus) caused a 57% reduction in glomerular filtration rate (GFR) (0.74 +/- 0.03 to 0.32 +/- 0.02 ml/min, P < 0.05) and a 40% reduction in single nephron glomerular filtration rate (SNGFR; 43.0 +/- 5.2 to 26.0 +/- 2.5 nl/min, P < 0.05) due to a 25% reduction in glomerular plasma flow rate (QA) (133.4 +/- 19.8 to 99.8 +/- 12.0 nl/min) and a 22% reduction in glomerular ultrafiltration coefficient (Kf; 0.1009 +/- 0.0203 to 0.0790 +/- 0.0130 nl/sec. mm Hg). After 10 days of FK treatment (N = 8, 0.6 mg/kg body wt, i.p.), we observed a reduction of 23% in GFR (0.97 +/- 0.02 to 0.75 +/- 0.04 ml/min, P < 0.05) and of 23% in SNGFR (37.9 +/- 3.0 to 29.1 +/- 1.9 nl/min, P < 0.05) due to a 42% reduction in Kf (0.1486 +/- 0.0101 to 0.0870 +/- 0.0110 nl/sec.mm Hg, P < 0.05) and a 38% reduction in QA (117.6 +/- 10.2 to 73.5 +/- 6.1 nl/min, P < 0.05). The latter was consequent to the increment of 72% in total arteriolar resistance (RT) (3.1 +/- 0.2 to 5.2 +/- 0.5 +/- 0.5 10(10).dyn.sec.cm-5, P < 0.05). Thus, the pattern of FK 506 effect on glomerular hemodynamics was similar in both acute and chronic treatments.(ABSTRACT TRUNCATED AT 250 WORDS)