Abstract-Hypertension induced by long-term infusion of angiotensin II (Ang II) is associated with augmented intrarenalAng II levels to a greater extent than can be explained on the basis of the circulating Ang II levels. Although part of this augmentation is due to AT 1 receptor-dependent internalization, the intracellular compartments involved in this Ang II accumulation remain unknown. In the present study, we sought to determine whether Ang II trafficking into renal cortical endosomes is increased during Ang II hypertension, and if so, whether the AT 1 receptor antagonist, candesartan, prevents this accumulation. Compared with controls (nϭ12; 114Ϯ2 mm Hg), Ang II-infused rats (nϭ12; 80 ng/kg/min, SC, for 13 days) developed hypertension with systolic blood pressure rising to 185Ϯ4 mm Hg by Day 12. In Ang II hypertensive rats, plasma renin activity was suppressed, whereas plasma and kidney Ang II levels were increased by 3-fold (348Ϯ58 versus 119Ϯ16 fmol/mL) and 2-fold (399Ϯ39 versus 186Ϯ26 fmol/g). Intracellular endosomal Ang II levels were increased by more than 10-fold (1100Ϯ283 versus 71Ϯ12 fmol/mg protein), whereas intermicrovillar cleft Ang II levels were increased by more than 2-fold (88Ϯ22 versus 37Ϯ7 fmol/mg protein T he importance of angiotensin II (Ang II) in the development and maintenance of hypertension is well documented in several animal models of experimental hypertension, such as two-kidney, one-clip renal hypertension (2K1C), the Ren-2 gene transgenic rats, and the Ang IIinfused model. [1][2][3][4] The Ang II-dependent increases in arterial blood pressure are commonly associated with higher levels of circulating and intrarenal Ang II levels, structural abnormalities, and functional derangements in the kidney. [1][2][3][4][5][6] One important feature in the renin-angiotensin system profile observed in these rats is that renal Ang II levels are greater than can be explained on the basis of circulating Ang II and suppressed renin expression. 2,4,[7][8][9] This suggests that angiotensin peptides continue to be generated intrarenally via a renin-independent pathway, or that circulating Ang II accumulates in one or more compartments within the kidney. 4,9 Previous studies have shown that intrarenal Ang II levels are increased in the contralateral nonclip kidney of 2K1C hypertensive rats and in kidneys of Ang II-infused rats and Ren-2 transgenic rats. 2,4,[7][8][9] Because blockade of the AT 1 receptor with losartan normalizes blood pressure and prevents augmentation of intrarenal Ang II levels, this enhanced uptake of Ang II within the kidney appears to be mediated by the AT 1 receptor. 9,10 These results suggest that Ang II levels are augmented in intracellular compartments by an AT 1 receptormediated internalization mechanism and are protected from degradation to some extent.Recent studies have localized angiotensin peptides in renal endosomes and intermicrovillar clefts, thus implicating them as potential sites for intracellular accumulation in the kid-
Three-dimensional suspension culture is a gravity-limited phenomenon. The balancing forces necessary to keep the aggregates in suspension increase directly with aggregate size. This leads to a self-propagating cycle of cell damage by balancing forces. Cell culture in microgravity avoids this trade-off. We determined which genes mediate three-dimensional culture of cell and tissue aggregates in the low-shear stress, low-turbulent environment of actual microgravity. Primary cultures of human renal cortical cells were flown on the space shuttle. Cells grown in microgravity and ground-based controls were grown for 6 days and fixed. RNA was extracted, and automated gene array analysis of the expression of 10, 000 genes was performed. A select group of genes were regulated in microgravity. These 1,632 genes were independent of known shear stress response element-dependent genes and heat shock proteins. Specific transcription factors underwent large changes in microgravity including the Wilms' tumor zinc finger protein, and the vitamin D receptor. A specific group of genes, under the control of defined transcription factors, mediate three-dimensional suspension culture under microgravity conditions.
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NF-kappaB mediates many features of urinary bladder inflammation induced by LPS. The NF-kappaB cascade is an important target for anti-inflammatory management of cystitis.
The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.
Therapeutic studies with cross‐transplant cross‐transfusion were begun in 1966 and have included 56 patients with melanoma, 2 with renal cell carcinoma, 3 with osteogenic sarcoma, 2 with colon cancer, and 1 with synovial sarcoma. Minced fragments of viable tumor were implanted subcutaneously into ABO‐Rh matched pairs with similar tumor on days 1 and 10, followed on the 14th day by 3–10 daily cross‐transfusions of plasma and white blood cells. Subsequently, intradermal implants of viable tumor cells grown in tissue culture have been used, some with added BCG. Complete responses have occurred in 3 patients with melanoma lasting 12, 46, and 51 months; the latter 2 remain alive and well. Partial response occurred in 6 others. Three melanoma patients with a poor prognosis were immunized prophylactically following primary surgical treatment. These 3 developed antibodies (shown by flourescence) against their tumor and remained free of disease—1 for 2 years and 2 for 1 year. Complications have mainly resulted from transfusion incompatibility. Transfer factor is now being used to avoid this problem. Two patients died following the seventh transfusion from complications probably related to treatment.
Twenty-three cases of laryngeal papilloma have been treated and followed for four years utilizing laser excision and laser excision/immunotherapy. Fourteen cases responded well to laser excision alone, nine did not and immunotherapy was instituted as adjunctive treatment. To date, immunotherapy has contributed little to the relief in this group of refractory cases.
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