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INCREASED EXPRESSION OF HLA-DR ANTIGENS ON RENAL TUBULAR CELLS IN RENAL TRANSPLANTS: RELEVANCE TO THE REJECTION RESPONSE

Whether the expression of DR antigens is altered in cadaver renal transplants was examined by the use of monoclonal antibodies to the non-polymorphic region of the DR molecule and an indirect immunoperoxidase stain. Expression of DR antigens increased considerably on renal tubular cells in all 25 biopsy specimens which showed severe cellular rejection, but in only 4 of 14 biopsy specimens with no or minimum evidence of rejection. 2 of these 4 specimens were from patients recently treated for severe rejection and the other 2 subsequently lost their grafts from chronic rejection. DR antigens were also expressed on the cell surface of isolated tubular cells aspirated from transplanted kidneys with acute cellular rejection but not on tubular cells in normal kidneys or aspirates from kidneys without rejection. Biopsy specimens with increased DR expression in tubules usually had an interstitial T cell infiltrate. Expression of DR antigens on tubular cells was not related to HLA-DR incompatibility between donor and host, or to the type of immunosuppressive therapy given. The expression of DR antigens on renal tubular cells may be induced by the infiltrating activated T cells or be a consequence of tubular regeneration following rejection or ischaemic damage. The increased expression of DR antigens on renal tubular cells during rejection makes these cells potential targets for delayed type hypersensitivity responses, which are only effective against DR antigen bearing cells.

Expression of HLA antigens on renal tubular cells in culture. I: Evidence that mixed lymphocyte culture supernatants and gamma interferon increase both class I and class II HLA antigens

The altered expression of HLA antigens on tissues during rejection is of potential importance to both the initiation and effector arms of the alloimmune response. Associated with rejection episodes, renal tubular cell expression of HLA DR (class II) antigens has been observed to be markedly increased. In this study an in vitro assay using cultured renal tubular cells from normal human kidneys was developed. Monoclonal antibodies to detect HLA DR and HLA A, B, C (class I) molecules were used to identify these antigens on cultured tubular cells either by indirect immunofluorescence stains and flow cytometric analysis or by immunoperoxidase stains with cytological analysis. With these techniques normal cultured cells had little membrane or cytoplasmic HLA DR but did have membrane HLA A, B, C. Culture of renal tubular cells with either snpernatants from mixed lymphocyte cultures or purified gamma interferon induced expression of HLA DR, with maximum expression occnring between day 4 and day 6. Increased HLA A, B, C expression was also observed. This increased expression of HLA antigens was shown not to be dependent upon increased cell division of the cultured cells, and removal of gamma interferon from the cultures resulted in a decay in both HLA DR and A, B, C expression within days. HLA expression was inhibited by addition of protein synthesis inhibitor cycloheximide, but not by the addition of puromycin, mitomycin C, or actinomycin D—which suggested that tubular cells had transcribed m RNA for both HLA DR and A, B, C antigens. Expression of HLA antigens was not inhibited by cyclosporine, corticosteroids, or azathioprine. These studies demonstrate that the increased expression of HLA antigens seen in renal allografts at the time of rejection episodes can be explained by the release of lymphokines, especially gamma interferon, by infiltrating cells, and that reduced HLA DR expression of transplanted kidneys in patients treated with cyclosporine is not due to the drug directly inhibiting HLA expression by graft cells.

Identification of the cellular subpopulations infiltrating rejecting cadaver renal allografts: Preponderance of the T4 subset of T cells

In the rejection response against renal allografts, the relative importance of helper/inducer T cells mediating a delayed-type hypersensitivity response and of T cells with direct cytotoxicity has not been defined. These subpopulations were identified with commercially available monoclonal antibodies and an indirect immunoperoxidase technique in 31 renal biopsies from patients undergoing acute rejection episodes and in 9 rejected nephrectomy specimens. T lymphocytes were the predominant cell population in all biopsies and in 8 of 9 nephrectomies. The T4 helper/inducer subset was equal to, or greater than, the T8 cytotoxic/suppressor subset in 28 of the 31 biopsies and in the 8 nephrectomy specimens that had histological evidence of cellular rejection. T4 lymphocytes were found predominantly in large areas of cellular infiltrate. T8 lymphocytes had a more diffuse interstitial distribution and were a minority of the cells in the large areas of cellular infiltration. These results show that helper/inducer T lymphocytes are often more frequent than cytotoxic/suppressor cells in acute renal allograft rejection in humans and they suggest that helper/inducer T cells may play an important role in the mediation of graft destruction.

Cyclosporin a inhibits protein kinase C activity: A contributing mechanism in the development of nephrotoxicity?

Cyclosporin A modifies many intracellular functions in a variety of different cells. This study investigated the potential interaction between cyclosporin A and protein kinase C, as a possible mechanism for the development of nephrotoxicity. The activity of protein kinase C, in the cytosol of renal epithelial cells, was shown to be significantly inhibited in a dose-dependent manner by CSA. Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (phorbol ester) in rat mesangial cells in culture leads to an increase in PGE2 release. Phorbol ester stimulated PGE2 release was significantly inhibited by cyclosporin A. These results would suggest that intracellular site of action of cyclosporin A, in producing alterations in intracellular function and toxicity, may be at the level of protein kinase C.

CALCIUM BALANCE IN PREGNANCY

Abstract Metabolic-balance studies were performed on seven patients in the last trimester of pregnancy. All had normal diets containing approximately 1 g. calcium and three had a supplement of 1 g. calcium per day. All were in positive balance and five had hypercalciuria; however, the amount of calcium retained was only equivalent to that incorporated in the fetus in patients taking the calcium supplement. These results suggest that the calcium intake during pregnancy should be 2 g, rather than 1 g. per day.

Effect of angiotensin II on baroreceptor reflex control of heart rate in conscious baboons.

Studies of the baroreceptor-heart rate reflex were performed in four conscious, unrestrained male baboons to determine whether changes in circulating angiotensin II within the physiological range are associated with alterations in baroreceptor reflex sensitivity. With the animals on a high sodium intake, studies were performed before and during graded angiotensin II infusion (10 and 20 ng/kg/min). To separate effects on baroreceptor reflex function mediated by angiotensin II-induced increases in arterial pressure, these studies were repeated on a different day with simultaneous glyceryl trinitrate infusion to prevent increases in pressure during angiotensin II infusion. With the animals on a low sodium intake, studies were performed before and after angiotensin converting enzyme inhibition with captopril (1 and 5 mg/kg). These studies were also repeated on a separate day during simultaneous phenylephrine infusion to prevent a decrease in pressure with captopril. Reduction in sodium intake had no significant effect on arterial pressure, heart rate, or plasma volume, although arterial plasma angiotensin II concentration and renin activity were significantly increased (p less than 0.01). Infusion of angiotensin II produced a significant reduction in baroreceptor reflex sensitivity (p less than 0.01), and converting enzyme inhibition produced a significant increase (p less than 0.05). These effects accompanied significant increases and decreases in arterial angiotensin II concentration, respectively (p less than 0.01), but were independent of angiotensin II-related changes in arterial pressure. The data indicate that physiological variations in circulating angiotensin II have a direct effect on sensitivity of the baroreceptor-heart rate reflex.

Diagnosis of renal allograft rejection by analysis of fine-needle aspiration biopsy specimens with immunostains and simple cytology.

Fine-needle aspiration biopsy specimens of renal transplants were analysed by means of commercially available monoclonal antibodies and an immunoperoxidase stain. Three cellular features associated with acute cellular rejection were identified--heavy infiltrates of activated T cells or large mononuclear cells strongly expressing HLA-DR antigens, and HLA-DR expression by renal tubular cells. A combination of semiquantitative scores for these features correctly identified rejection in 32 of 34 cases, with no false positives in cases of cyclosporin nephrotoxicity or stable graft function.

Cyclosporin A-induced increases in renin storage and release.

The effect of Cyclosporin A (CyA) on renin storage and release in rats was investigated. Renin release from incubated renal cortical slices was increased in rats treated with 25 or 50 mg/kg/day of CyA for 3 to 7 days. This was associated with an increase in renal cortical renin content, which was also increased over the same time period. Plasma renin activity was similarly elevated in these rats. It is possible that these increases in renin are related to the nephrotoxicity of CyA.

EFFECT OF ANTIHYPERTENSIVE DRUGS ON NEONATAL BLOOD PRESSURE

1. This study evaluates the perinatal outcome of infants born to ninety‐five mothers with hypertension in pregnancy whose blood pressure was treated in a double blind trial comparing clonidine hydrochloride (C) and α‐methyldopa (A).

Cyclosporin A and Renal Prostaglandin Biosynthesis

The effect of cyclosporin A (CyA) treatment of rats on prostaglandin synthesis in the renal cortex was studied. Renal cortical slices were prepared from control and CyA-treated rats and the release of prostaglandins into the medium during incubation at 37 degrees was measured. Rats killed 4 hours after receiving 100 mg/kg CyA orally showed no changes in renal slice release of PGE2, PGF2 alpha, 6-keto-PGF1 alpha or thromboxane B2. The slice release of 6-keto-PGF1 alpha was tested after 5 days of CyA treatment and again there was no difference from control rats. The effect of CyA added to slice incubations in vitro was examined: CyA had no effect on PGE2 or 6-keto-PGF1 alpha release in the presence or absence of angiotensin II. Under all of these experimental conditions there was evidence of CyA-induced stimulation of the renin-angiotensin system. Indomethacin treatment did not inhibit CyA-mediated accumulation of renin in the renal cortex. The results suggest that renal prostaglandins do not play a role in CyA-stimulated renin storage or release, or in CyA nephrotoxicity.