Aldosterone metabolism in cultures of rat renal cortical and medullary cells.

The metabolism of 4-(14)C-d-aldosterone (at 3 nM) was studied in the primary target organ of the hormone, in renal cortical and medullary cell cultures obtained from Wistar rats. Larger amounts of aldosterone were metabolized in medullary cells than in cortical cells, as measured by a decreased 4-(14)C-d-aldosterone radioactivity concentration (26+/-9% and 12+/-7% of the initial aldosterone added, respectively (n=5, p<0.05)). The 14C radiometabolites of aldosterone in both cultures co-chromatographed with 5alpha dihydro- (DHA) and 3alpha,3beta tetrahydroaldosterone (THA). Aldosterone metabolism was totally inhibited by a mineralocorticoid receptor antagonist canrenoat (Soldactone) (at 10(-5) to 10(-3) M), while the glucocorticoid receptor antagonist RU 38486 (Roussel UCLAF) (at 10(-5) to 10(-4) M) had no effect. Thus, the study confirmed that, in rat kidney, aldosterone can be converted to its reduced metabolites by a metabolism which is inhibited by a mineralocorticoid receptor antagonist. This indicated that the metabolism might play some role in modulation of the intracellular response to aldosterone in the kidney.

Interaction between proximal tubular cells and allogenic leukocytes mediated by interleukin-1 and prostaglandin.

The present study investigates the interaction between proximal tubular cells (TC) and peripheral blood mononuclear cells (PBMC) with respect to interleukin-1 (Il-1) and prostaglandin E2 (PGE2) in co-culture experiments. Stimulator cells were proliferating human TC propagated in culture and characterized by expression of ICAM-1 and MHC-class II in response to cytokines. The presence of TC reduced spontaneous growth of PBMC by 42%, with a concomittant 50% reduction of Il-1 release and a 6-fold increase of PGE2 release in the growth medium. The growth reduction was partly counteracted by addition of II-1 and indomethacin. Addition of II-1 stimulated the PGE2 release from PBMC and TC by about 75% and 500%, respectively. Growth of TC cultured alone was inhibited by II-1 in a dose-dependent manner, with maximum effect at 10 U/ml, whereas PGE2 showed no effect. These results suggest that TC have the capacity to interact with PBMC, and this potentially tissue-protective mechanism induced by TC may have clinical implications.

Acute effects of FK506 and cyclosporine A on cultured human proximal tubular cells.

The effects were studied of FK506 and cyclosporine A on human proximal tubular cells. An in vitro assay was used with cultured renal cells which displayed characteristics of proximal tubular cells. Cell growth was measured by [3H]thymidine uptake. FK506 inhibited cell growth by about 50% at 1 micrograms/ml but cells were not killed as shown by the dye exclusion test. The growth rate of cells recovered to control levels after removal of FK506 from the cultures. FK506 and cyclosporine A showed an additive effect on tubular cell growth reduction. This effect was similar at equivalent doses of FK506 and cyclosporine A, whereas in cultures of leukocytes, the half maximal response to mitogen was obtained at 0.01 micrograms/ml FK506 and 0.5 micrograms/ml cyclosporine A, respectively. Immunocytochemistry demonstrated that FK506 reduced the expression of interferon-gamma induced major histocompatibility complex (MHC) class I on the proximal tubular cells, but had no effect on the expression of the MHC class II antigens or the intercellular adhesion molecule (ICAM-1) on the cultured cells.

Human proximal tubular cells modulate allogen responses of leukocytes in vitro.

The interaction between proximal tubular cells and leukocytes was examined. In co-cultures of tubular cells and allogenic leukocytes, tubular cells expressed MHC antigens and ICAM-1. A small number of leukocytes adhered to tubular cells and induced cytotoxic damage. Thus, 65% of the tubular cells were viable after co-culturing with allogenic leukocytes. These cells had low alloreactive capacity, which was not due to lack of interleukin-1 or interferon-gamma. The presence of tubular cells modulated the immune response of leukocytes by reducing the effect of mitogen by 80%, allogens by 65% and interleukin-2 by 40%. A soluble factor released in the co-cultures was a likely mediator, since addition of supernatants from co-cultures suppressed mitogen responses by 27% compared to leukocytes cultured alone. This mediator might be prostaglandin, because addition of indomethacin to co-cultures increased the growth response of leukocytes.

Metabolism of the aminoterminal propeptide of type III procollagen in cultures of human proximal tubular cells.

Degradation of the intact form of the aminoterminal propeptide of type III procollagen (PIIINP) has been established in the liver, whereas the col 1 domain of PIIINP is extracted by the kidneys. We used native human PIIINP and col 1 domain of PIIINP to investigate the degradation of PIIINP in cultures of human proximal tubular cells. Normal renal tissue was obtained from the healthy part of kidneys surgically removed and from biopsies from a total of 10 patients. The degradation was characterized by incubation of [125I]-PIIINP followed by gel filtration. We found that in physiological concentrations (4.4 micrograms l-1 and 11.9 micrograms l-1 intact PIIINP was almost totally degraded, but not col 1 domain. High concentrations of PIIINP (20-50 micrograms l-1) had a non-linear, non-monoexponential degradation over time, which suggests several steps. Gel filtration of [125I]-PIIINP after 1 h, 3 h, 6 h and 24 h of incubation confirmed the observation by showing the rapid formation of a high-molecular-weight fraction, followed by the slower formation of a low-molecular-weight fraction. The high-molecular-weight fraction was PIIINP immunoreactive, but not the low-molecular-weight fraction. We conclude that cultures of human proximal tubular cells degrade intact human PIIINP by the formation of high- and low-molecular-weight fractions. Earlier findings that extraction of the PIIINP col 1 domain takes place in the kidneys, cannot be explained by degradation by the proximal tubular cells.

Expression of the intercellular adhesion molecule-1 (ICAM-1) in human renal allografts and cultured human tubular cells.

The in vivo distribution of the intercellular adhesion molecule ICAM-1 was investigated in renal tissue specimens obtained from 17 renal allotransplanted patients, nine normal kidneys (controls), seven native kidneys, nine patients with mesangioproliferative glomerulonephritis, and nine patients with active extracapillary glomerulonephritis. Biopsies from patients with signs of acute rejection showed a significant increase in ICAM-1 expression in the tubular epithelium (P less than 0.05). In normal kidneys (controls) ICAM-1 expression was found in endothelial cells; additional expression in the tubular epithelial cells was induced in patients with extracapillary glomerulonephritis. The in vitro expression of ICAM-1 was examined in cultured human tubular cells after stimulation with gamma-interferon and interleukin-1, treatment with cyclosporin and/or verapamil and coculture with allogenic mononuclear cells. An increased ICAM-1 expression was demonstrated by coculture with allogenic mononuclear cells and after stimulation with gamma-interferon and/or interleukin-1. Cyclosporin or verapamil induced no changes. Our results give support to the hypothesis that ICAM-1 upregulation is important in immune interactions such as allograft rejection. Furthermore, the in vitro model indicates that ICAM-1 expression is regulated by gamma-interferon and interleukin-1 produced by activated T lymphocytes and macrophages.

Production of prostaglandin E2 by macrophages in uraemia.

Production of prostaglandin E2 (PGE2), interleukin-2, and response to lectin stimulation were examined in peripheral blood mononuclear cell cultures from 46 patients on haemodialysis, 20 patients on continuous peritoneal dialysis, 18 non-dialysed patients with mild to moderate renal failure, and 52 control subjects. Production of PGE2 and responses to phytohaemagglutinin (PHA) were decreased in cell cultures from patients on dialysis. Similarly, production of interleukin-2 was significantly reduced in patient groups as compared to control subjects, but there was no relationship between production of PGE2 and responses to PHA or production of interleukin-2. Addition of exogenous indomethacin did not return the response to normal and the patient cultures were not more sensitive to exogenous PGE2 than the control cultures. Thus, release of PGE2 does not account for the impaired in vitro response of uraemic lymphocytes.

Human renal biopsies as source of cells for glomerular and tubular cell cultures.

Glomerular and tubular cells were obtained from normal and pathological human renal biopsies. Single nephron structures were isolated by microdissection for culture. Proximal and distal tubular cells were cultivated for 5-6 weeks (three passages), whereas outgrowth of glomerular cells was sparse and after three weeks infiltrated by mesangial cells. The morphology of cultures obtained from pathological tissue was comparable with the morphology of normal cells, although cultures were more often overgrown by fibroblasts. In culture, both proximal and distal tubular cells retained physiological responses characteristic of their origin. Epidermal growth factor induced growth of proximal tubular cells. The proximal tubular cells were furthermore characterized by cAMP response to parathyroid hormone (PTH) stimulation. The distal tubular cells showed cAMP response to both PTH and vasopressin stimulation. Twelve of 17 cultures obtained from patients with no tubular injuries showed cAMP response to PTH stimulation compared with 2 of 9 cultures from renal tissue with tubular injuries.

Release and effect of prostaglandin E2 in cocultures of lymphocytes and human tubular cells.

Prostaglandin E2 reduced the expression of HLA-ABC as well as HLA-DR antigens in cultured human tubular cells, when cells were stimulated by gamma-interferon. The PGE2 release was low in tubular cell cultures, but highly increased by coculture of tubular cells and allogeneic lymphocytes, compared to cultures of unstimulated lymphocytes, or to cocultures with autologous lymphocytes. Cocultures between non-adherent mononuclear cells and tubular cells produced low amounts of PGE2, whereas cocultures of adherent monocytes and tubular cells produced equal as much PGE2 as cultures made with stimulated peripheral blood leucocytes. The adherent cells were dominated by macrophages. The proliferative response of lymphocytes in cocultures with tubular cells was low, and was increased by indomethacin. The results indicated, that presence of monocytes adherent to the tubular cells in cocultures, down-regulate the proliferative response of allogeneic lymphocytes to the tubular cells, which may in part attribute to an inhibitory effect of PGE2.

Acute inhibition of human renal tubular cell growth by cyclosporin A.

Human proximal tubular cells were isolated and grown in culture for three passages. The proliferation of these cells were inhibited by the immunosuppressive drug cyclosporin A in dose dependent manner in the range of 250 to 2000 ng/ml growth medium. The cultures with low cell density were more sensitive to cyclosporin A compared to the cultures with high density, measured by the incorporation of 3H-thymidine. The toxic effect of cyclosporin A on cells isolated from patients treated with cyclosporin A, did not differ from cells isolated from normal tissue. The calcium channel blocker, verapamil, reversed the inhibitory effect of low concentrations of cyclosporin A on cell proliferation. The electron microscopy showed that cells treated with cyclosporin A, had severe morphological alterations with rounded mitochondria and giant vesicles in the cytoplasma. The results support the hypothesis that the toxic effect of cyclosporin A may be mediated through an increased Ca++ influx into the proximal tubular cells.

Mitogen-induced lymphocyte transformation in four different serum-free media.

The proliferative responses of lymphocytes induced by the mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (ConA) were tested in four different serum-free lymphocyte culture media in which serum was replaced by commercially produced serum substitutes. Two of the serum-free lymphocytes cultures (SF-X and FEB-100) achieved proliferative responses to PHA and PWM comparable with those obtained in medium containing fetal calf serum, but only when the cell number was increased, whereas none of the lymphocyte cultures in serum-free medium showed adequate proliferative responses to ConA stimulation. The essential factors in serum-free media for optimal growth and mitogen stimulation of lymphocytes include insulin, transferrin, electrolytes, glutamine and a high cell concentration.

Effects of indomethacin on glomerular hemodynamics in experimental diabetes.

The glomerular hemodynamics were studied in streptozotocin diabetic rats 3 months after the induction and in age-matched normal animals. Indomethacin infusion failed to cause changes in glomerular hemodynamics in normal rats but produced striking effects in the diabetic rats: The afferent arteriolar hydraulic resistance increased substantially while the efferent resistance rose moderately causing large reductions in single nephron blood flow and in the hydraulic pressure in the glomerular capillaries. Consequently, single nephron glomerular filtration rate (SNGFR) was reduced significantly below normal. The ultrafiltration coefficient of the glomerular membrane was significantly lower in the diabetic than in the normal animals (2.8 versus 5.0 nl min-1 mm Hg-1) and remained unchanged during indomethacin infusion. Our results suggest that the prostaglandin system compensates for the changes in the glomerular hemodynamics induced by diabetes by reducing the arteriolar resistances to increase the glomerular capillary pressure making it possible to maintain normal values of SNGFR and nephron blood flow.

Release of prolactin, thyrotropin, and growth hormone in women with cyclical mastalgia and fibrocystic disease of the breast.

Prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH) response to metoclopramide and TRH was investigated in seven women with fibrocystic disease and cyclical mastalgia, in eight similar patients without mastalgia, and in six normal controls. The basal PRL level was significantly elevated in patients with cyclical mastalgia (P less than 0.025). PRL and TSH response to metoclopramide did not differ significantly between the three groups, indicating that decreased dopaminergic tone is not the cause of elevated basal PRL level in cyclical mastalgia. PRL and TSH response to TRH and the abscent GH response to both metoclopramide and TRH further indicate that the hypothalamicpituitary axis is not primarily disturbed in cyclical mastalgia. The basal GH level was elevated in patients with fibrocystic disease with or without mastalgia. The increased basal GH secretion is not believed to be directly involved in cyclical mastalgia, but may be of importance in fibrocystic disease.

Evaluation of the micropuncture determination of single nephron filtration fraction.

Spontaneous and aspirated blood samples were collected from the welling points of surface efferent arterioles in the rat kidney. The amount of tubular reabsorbate in each sample was calculated from measurements of the inulin concentration in the sample and systemic arterial plasma. Both spontaneous and aspirated samples contained substantial amounts of tubular reabsorbate (4% +/- 1% and 11% +/- 1%, respectively). Accordingly, the single nephron filtration fraction values derived from measurements of albumin concentrations alone were significantly lower than those calculated considering the addition of the amount of tubular reabsorbate in each sample (0.26 +/- 0.01 vs. 0.30 +/- 0.01 and 0.25 +/- 0.01 vs. 0.34 +/- 0.01 for the spontaneous and aspirated samples, respectively). Increasing the rate of blood collection from the welling point by aspiration was associated with a significant decrease in the hydraulic pressure in the same vessel. The efferent arteriolar blood samples were therefore probably contaminated with tubular reabsorbate as a result of retrograde flow from the peritubular capillaries. It was confirmed that single nephron filtration fractions derived from hematocrit measurements were significantly higher than those based on albumin concentration measurements, supporting that the hematocrit of afferent arteriolar blood of the superficial nephrons is higher than that of systemic arterial blood.