Receptor-Induced Dilatation in the Systemic and Intrarenal Adaptation to Pregnancy in Rats

Normal pregnancy is associated with systemic and intrarenal vasodilatation resulting in an increased glomerular filtration rate. This adaptive response occurs in spite of elevated circulating levels of angiotensin II (Ang II). In the present study, we evaluated the potential mechanisms responsible for this adaptation. The reactivity of the mesangial cells (MCs) cultured from 14-day-pregnant rats to Ang II was measured through changes in the intracellular calcium concentration ([Cai]). The expression levels of inducible nitric oxide synthase (iNOS), the Ang II-induced vasodilatation receptor AT2, and the relaxin (LGR7) receptor were evaluated in cultured MCs and in the aorta, renal artery and kidney cortex by real time-PCR. The intrarenal distribution of LGR7 was further analyzed by immunohistochemistry. The MCs displayed a relative insensitivity to Ang II, which was paralleled by an impressive increase in the expression level of iNOS, AT2 and LGR7. These results suggest that the MCs also adapt to the pregnancy, thereby contributing to the maintenance of the glomerular surface area even in the presence of high levels of Ang II. The mRNA expression levels of AT2 and LGR7 also increased in the aorta, renal artery and kidney of the pregnant animals, whereas the expression of the AT1 did not significantly change. This further suggests a role of these vasodilatation-induced receptors in the systemic and intrarenal adaptation during pregnancy. LGR7 was localized in the glomeruli and on the apical membrane of the tubular cells, with stronger labeling in the kidneys of pregnant rats. These results suggest a role of iNOS, AT2, and LGR7 in the systemic vasodilatation and intrarenal adaptation to pregnancy and also suggest a pivotal role for relaxin in the tubular function during gestation

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 (Q(A)) (133.4 +/- 19.8 to 99.8 +/- 12.0 nl/min) and a 22% reduction in glomerular ultrafiltration coefficient (K-f; 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 K-f (0.1486 +/- 0.0101 to 0.0870 +/- 0.0110 nl/sec . mm Hg, P < 0.05) and a 38% reduction in Q(A) (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 (R(T)) (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. Additionally, in order to evaluate the effect of FK 506 on mesangial cells (MC), we performed studies measuring intracellular calcium concentration ([Ca2+](i)) with Fura-2/AM as well as MC contraction by morphometric analysis. It was observed that FK 506 increases the [Ca2+](i) (R 340/380: 2.5 +/- 0.3 to 3.8 +/- 0.4, P < 0.05) due to mobilization of the extracellular calcium pool, via opening calcium type L voltage dependent channels, since verapamil blunted the increases of [Ca2+](i) caused by FK 506 (R 340/380: 3.5 +/- 0.9 to 2.8 +/- 0.8). the [Ca2+](i) was not changed after FK 506 incubation of MC with verapamil and thapsigargin, and thus no calcium release-activated channel (CRAC) was affected. the increase of [Ca2+](i) induced by FK 506 probably caused contraction of MC evaluated by reduction of the cross sectional area from 3772 +/- 106 to 1912 +/- 61 mu m(2) (P < 0.05). Thus, FX 506 caused a reduction in SNGFR by reducing Q(A) and K-f after both acute and chronic administration, and the mesangial cells potentially participate in this nephrotoxicity via reduction in K-f mainly during chronic treatment.ESCOLA PAULISTA MED,DEPT MED,DIV NEPHROL,BR-04023062 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT PATHOL,BR-04023062 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT BIOPHYS,BR-04023062 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT MED,DIV NEPHROL,BR-04023062 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT PATHOL,BR-04023062 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT BIOPHYS,BR-04023062 São Paulo,BRAZILWeb of Scienc

Intracellular calcium concentration ([Cai]) measured under the unstimulated (basal) condition and after the addition of 10⁻⁶ M Ang II in MCs.

<p>A: virgin group (white bars) and pregnant group (black bars). The data bars represent the mean±SEM in MCs isolated from six rats in each group. [Cai] after Ang II stimulation represents the peak value. * p<0.05 vs basal, ^# p<0.05 vs virgin. B: Representative traces showing the [Cai] before (basal) and after Ang II (10⁻⁶ M) addition in MCs isolated from virgin (left panel) and pregnant rats (right panel).</p

Photomicrograph of kidney tissue from virgin (left panel) and pregnant (right panel) rats.

<p>Representative LGR7 immunostaining (dark brown) in the cortex (A, 200× magnification), the cortex showing the apical membrane distribution of LGR7 in proximal tubules (B, 400× magnification), glomeruli (C, 400× magnification) and the negative control (D, omission of primary antibody, 200× magnification).</p

Representative LGR7 immunoassaying (dark brown) of MCs from virgin rats (A) and pregnant rats (B).

<p>The omission of primary antibody was the negative control (C) and actin labeling was used as a positive control (D). (200× magnification).</p

Quantitative real-time RT-PCR for AT1, AT2, and LGR7 in the renal cortex, the aorta, and the renal artery of virgin and pregnant rats.

<p>Individual samples were normalized to β-actin mRNA levels and data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p<0.05 vs virgin.</p

Quantitative real-time RT-PCR for the Ang II receptors (AT1 and AT2), the relaxin receptor (LGR7), and iNOS in MCs isolated from virgin and pregnant rats.

<p>Total RNA was isolated from pooled cells that were obtained from three or four culture flasks from each group. Individual samples were normalized to β-actin mRNA levels and the data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p<0.05 vs virgin.</p