Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1-7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.     

Renal interstitial fluid ATP responses to arterial pressure and tubuloglomerular feedback activation during calcium channel blockade

A close relationship between changes in renal interstitial fluid (RIF) ATP concentrations and renal autoregulatory or tubuloglomerular feedback (TGF)-dependent changes in renal vascular resistance (RVR) has been demonstrated, but it has not been determined whether the changes in RIF ATP are a consequence or the cause of the changes in RVR. The present study was performed in anesthetized dogs to assess the changes in RIF ATP following changes in renal arterial pressure (RAP) or stimulation of the TGF mechanism under conditions where changes in RVR were prevented by nifedipine, a calcium channel blocker. RIF ATP levels were measured by using microdialysis probes. Intra-arterial infusion of nifedipine (0.36 microg x kg(-1) x min(-1)) increased renal blood flow (RBF: from 4.49 +/- 0.27 to 5.34 +/- 0.39 ml x min(-1) x g(-1)) and glomerular filtration rate (GFR: from 0.84 +/- 0.07 to 1.09 +/- 0.11 ml x min(-1) x g(-1)). Under conditions of nifedipine infusion, autoregulatory adjustments in RBF, GFR, and RVR were not observed during stepwise reductions in RAP within the autoregulatory range (from 135 +/- 7 to 76 +/- 1 mmHg, n = 7). Furthermore, stimulation of the TGF mechanism with intra-arterial infusion of acetazolamide (100 microg x kg(-1) x min(-1)) did not alter RBF, GFR, and RVR (n = 7). During treatment with nifedipine, RIF ATP levels were significantly decreased in response to reductions in RAP (10.7 +/- 0.7, 5.8 +/- 0.7 and 2.8 +/- 0.3 nmol/l at 135 +/- 7, 101 +/- 4, and 76 +/- 1 mmHg, n = 7) and increased by acetazolamide infusion (from 8.8 +/- 0.8 to 17.0 +/- 1.8 nmol/l, n = 7). These results are similar to those that occurred in dogs not treated with nifedipine and thus demonstrate that the changes in RIF ATP can occur in the absence of autoregulatory or TGF-mediated changes in RVR. The data provide further support to the hypothesis that RIF ATP contributes to adjustments in RVR associated with renal autoregulation and changes in activity of the TGF mechanism.     

Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 micromol/l CO solution, or 15 micromol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l N(omega)-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 micromol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 micromol/l CORM-2 or with CO (10 micromol/l) significantly dilated AAs (22.0 +/- 0.9 and 22.8 +/- 0.9 vs. 18.3 +/- 0.9 microm, n = 5, P < 0.05; and 26.0 +/- 1.4 vs. 18.8 +/- 0.7 microm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 +/- 0.7 vs. 17.2 +/- 0.6 microm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 +/- 1.2 microm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 +/- 1.6 microm, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 +/- 0.4 vs. 18.8 +/- 0.4 microm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 +/- 0.7 microm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.     

Select de novo Gene and Protein Expression During Renal Epithelial Cell Culture in Rotating Wall Vessels is Shear Stress Dependent

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types. Received: 30 June 1998/Revised: 30 November 1998     

Tubuloglomerular feedback-dependent influence of angiotensin II on the kidney in rats

To examine the modulatory role of angiotensin II on the tubuloglomerular feedback (TGF) mechanism, TGF responses were assessed during control conditions, converting enzyme inhibition (CEI; MK 422, 0.6 mg/kg.hr) and during continued CEI with the replacement of angiotensin II. TGF responses were assessed from stop flow pressure (SFP) feedback responses obtained during step increases in the late proximal perfusion rate from 0-40 nl/min. SFP values in the absence of perfusion were used to estimate glomerular pressure (GP) under conditions where the influence of the TGF mechanism should be at a minimum. During CEI, the arterial pressure decreased from 124 +/- 3 to 106 +/- 3 mmHg and the estimated GP decreased from 53 +/- 1.4 to 49 +/- 0.8 mmHg. There was a marked attenuation in the magnitude of SFP feedback responses from 11.0 +/- 1.3 to 2.7 +/- 0.6 mmHg. TGF feedback responses, however, were restored towards normal during superimposed angiotensin II infusion (7.7 +/- 0.9 mmHg). These results indicate that converting enzyme inhibition decreases the effects of angiotensin II on the kidney through TGF dependent mechanism.