John G. Simpson

The Expression of Connexin 43 in Human Kidney and Cultured Renal Cells

Gap junctions enable intercellular communication and play an important role in a variety of vital cellular functions including differentiation and the control of growth. These junctions are formed by a hexameric of proteins known as connexins. We investigated the distribution of the connexin 43 (Cx43) gap junction protein in renal cells and human kidney using the alkaline phosphatase anti-alkaline phosphatase immunohistochemical technique with a monoclonal antibody directed against the cytoplasmic domain of this antigen. Strong staining was demonstrated on the vascular endothelium, the smooth muscle of larger vessels and on glomerular epithelial cells. In addition, Cx43 was expressed on proximal tubular cells, glomerular endothelial cells and occasional cells infiltrating the interstitium. In areas of tubular atrophy there was increased staining for Cx43. Using reverse transcription-polymerase chain reaction we have also demonstrated that cultured human and rat mesangial cells and human proximal tubular cells express Cx43 messenger RNA. In summary, we have described for the first time the distribution of Cx43 in human kidney and cultured renal cells.

A toxicological study in rats receiving immunotherapeutic doses of cyclosporin A.

SUMMARY Cyclosporin A (Cy A, 25 or 50 mg.kg-1/48 hr) administered during the course of the response, markedly suppressed graft-versus-host (GVH) reactivity in the rat, as assessed by the 7-day popliteal lymph node weight assay. Serum biochemical studies revealed small, but statistically significant increases in serum urea levels at both doses of Cy A and, at 50 mg.kg-1/48 hr, reduction in total serum protein, alkaline phosphatase, serum transaminase, and iron. In an additional experiment, Cy A (50 mg.kg-1/48 hr) was administered to rats over 4 weeks. Serum biochemical and hematological investigations were conducted at weekly intervals and at 28 days tissue (liver, kidney, spleen, lymph nodes, and small intestine) was taken for histological and ultrastructural examination. Glomerular function, monitored by creatinine and urea clearance was unaffected by Cy A treatment, but serum urea and creatinine levels were elevated. Hepatic function was not affected and hematological, histological, and ultrastructural observations, apart from evidence of hepatic fatty change, did not differ from those in vehicle-treated controls.

Upregulation and co-localization of connexin43 and cellular adhesion molecules in inflammatory renal disease

Connexin43 (Cx43) is a major component of gap junctions. These are widely distributed in the human kidney and are thought to be involved in the inflammatory response and in the regulation of cell growth. Cellular adhesion molecules (CAMs) are also thought to be important in these processes, where they possibly facilitate gap junction formation. The aims of the current study were to define for the first time the expression of Cx43 in inflammatory glomerulonephritis and to compare the localization of this connexin with that of the intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin. Human renal biopsies and control sections of normal human kidney were stained using the alkaline phosphatase/anti‐alkaline phosphatase immunohistochemical technique, demonstrating that Cx43 was strongly expressed on inflammatory cells, on damaged tubular cells, and on interstitial cells. This pattern of expression was paralleled closely by that of ICAM‐1 and, to a lesser extent, by that of VCAM‐1. Cx43 is therefore primarily implicated in tubulointerstitial inflammation.© 1997 John Wiley & Sons, Ltd.

Amelioration of cyclosporin-induced nephrotoxicity in rats by induction of hepatic drug metabolism

The aim of this study was to determine the effect of altered hepatic drug metabolism on the nephrotoxic and immunosuppressive properties of cyclosporin A (CsA) in the rat. From a consideration of the structures of those CsA metabolites identified so far, it seemed probable that the metabolism of CsA would occur at the hepatic cytochrome P-450 (cyt P-450) enzyme system. CsA (50 mg/kg/24 hr) administered orally for 14 days resulted in significant increases in both serum urea concentration and urinary N-acetyl-beta-D-glucosaminidase activity, accompanied by renal proximal tubular vacuolation. The concomitant administration of either Aroclor 1254 (25 mg/kg/24 hr, i.p.) or phenobarbitone (PB) (40 mg/kg/24 hr, i.p.) but not 3-methylcholanthrene (3-MC) (15 mg/kg/72 hr, i.p.) resulted in abolition of the nephrotoxicity, assessed both biochemically and histologically, whilst the suppressive effect on the humoral response to SRBC was unaltered. Phenobarbitone also significantly decreased serum CsA concentrations. These results suggest that the PB-inducible set of cyt P-450 isoenzymes may be responsible or partly responsible for hepatic CsA metabolism.

Serum cyclosporin levels, hepatic drug metabolism and renal tubulotoxicity

The present study was designed to examine inter-relationships between serum cyclosporin (CsA) levels, hepatic drug metabolising enzyme activity and CsA induced nephrotoxicity. CsA (25 mg/kg p.o.) was administered daily to male Sprague-Dawley rats: groups of animals were killed on days 0, 4, 7, 10 and 14 and thereafter at weekly intervals over the 7-week course of the experiment. Nephrotoxicity was evaluated by measuring tubular enzymuria and by light microscopy and serum CsA levels (parent drug plus certain metabolites) were determined by radioimmunoassay. The hepatic microsomal mono-oxygenase enzyme system was monitored by measurement of cytochrome P-450, aminopyrine N-demethylase and NADPH-cytochrome c reductase. Nephrotoxicity appeared within 4 days of starting treatment and continued for 4 weeks. Between weeks 4 and 6 there was a period of complete remission followed by the return of renal damage. Aminopyrine N-demethylase activity fell during the first 4 weeks. During the period of remission, however, N-demethylase activity rose to a point significantly higher than pretreatment values and serum CsA levels fell to their lowest concentration. With relapse, hepatic N-demethylase activity again fell below normal and serum drug levels rose to their pre-remission values. From the third week onward, changes in NADPH-cytochrome c reductase activity paralleled those in N-demethylase activity. The hepatic microsomal concentration of cytochrome P-450 did not, however, change significantly during the 7-week period of CsA treatment. Our results suggest that the spontaneous remission of CsA-induced nephrotoxicity is due to a reduction in circulating drug levels caused by increased hepatic CsA metabolism.

Membranous nephropathy and granulomatous interstitial nephritis in sarcoidosis.

A 56-year-old woman developed nephrotic syndrome in association with pulmonary sarcoidosis. Renal biopsy revealed both granulomatous interstitial nephritis and membranous nephropathy. Treatment with steroids resulted in a decrease in proteinuria and there was no deterioration in renal function over a subsequent period of 10 months. This case provides further evidence that secondary membranous nephropathy associated with sarcoidosis should be treated with steroids.

Enhancement of high dose cyclosporin A toxicity by frusemide

Adult Sprague-Dawley rats were given cyclosporin A (CyA), frusemide (Fr) or both drugs daily for 14 days. The doses of CyA (50 mg/kg) and Fr (5 mg/kg) were approximately 3-6 times and twice respectively those used in man. Fr on its own produced a diuresis lasting approximately 3 hr. This was characterized by a 10-fold increase in urine flow rate, a 40-fold increase in the rate of sodium excretion, and by 2- and 4-fold increases in urea and creatinine clearance rates, respectively. In addition, there was a doubling in urinary N-acetyl-beta-D-glucosaminidase (NAG) activity. After 4 days of combination treatment with CyA and Fr, the diuretic-induced increases in urine flow rate, sodium excretion and urinary NAG activity were similar to those following frusemide alone. However, urea and creatinine clearances did not increase during the diuresis. Fr itself did not impair renal function, but rats receiving only CyA did show elevations in serum urea and creatinine, with reductions in clearance rates, which progressed with time. There was also an increase in NAG enzymuria. When the two drugs were exhibited together, renal function was more severely impaired. All animals given CyA showed proximal renal tubular cell vacuolation: in half the damage was confined to the straight segment, while the rest showed additional severe convoluted segment change. Renal function was most abnormal in those rats in which both segments were affected. All animals given both drugs showed both straight and convoluted tubular abnormalities and a 2-fold increase in serum CyA levels. CyA-induced disturbances in hepatic function and lymphoid tissue atrophy were unaffected by the addition of Fr, nor did Fr affect the immunosuppressive action of CyA.

Scleroderma, D-penicillamine treatment, and progressive renal failure associated with positive antimyeloperoxidase antineutrophil cytoplasmic antibodies

Progressive renal failure in patients with scleroderma is a sinister development that is usually attributed to impaired renal blood flow. In some exceptional cases, the underlying pathology is a crescentic glomerulonephritis, which has been associated with positive antineutrophil cytoplasmic antibodies, and in particular antimyeloperoxidase antibodies. The prognosis in such cases has been very poor. We report such a patient whose renal function has improved and stabilized on immunosuppressive therapy.

The influence of enalapril or spironolactone on experimental cyclosporin nephrotoxicity

Adult Sprague-Dawley rats treated daily for 14 days with 50 mg/kg cyclosporin A (CsA) exhibited nephrotoxicity, characterized by reduced glomerular filtration rate, decreased urinary sodium and potassium flow, tubular enzymuria and proximal tubular structural damage. Elevations in plasma renin activity (PRA) were observed on day 4, but returned to normal within 7 days. Co-treatment of animals for the 14 day period with enalapril (8 mg/kg/day), a potent inhibitor of angiotensin converting enzyme (ACE), or spironolactone (25 mg/kg/day), the distal tubular antagonist of aldosterone, reduced the nephrotoxicity, although PRA remained elevated. Neither enalapril nor spironolactone affected circulating CsA levels. These data suggest that the action of aldosterone on the distal tubule may be important in the pathogenesis of CsA nephrotoxicity.

Comparisons of Cutaneous Microvessels from Spontaneously Hypertensive, Normotensive Wistar-Kyoto, and Normal Wistar Rats

Abstract Samples of abdominal skin from SHR, WKY, and NW rats were examined to determine: (a) if gross structural differences were apparent among the cutanous vascular beds of the three strains of rats, and (b) if the structural differences were generalized or localized to a specific level of the vascular tree. The most significant differences among the three strains of rats were at the level of the smallest (fourth-order) arterioles. The SHR had 30% fewer of the smallest arterioles than WKY and 38% fewer than NW. However, the smallest arterioles from SHR had diameters 22 and 26% larger than similar vessels from WKY and NW, respectively. Consequently, there were no significant differences in calculated resistance among vessels from the three strains of rats. No significant differences were found in the vessel branching angles among the three strains of rats either. It cannot be determined from these studies whether the genetic mechanism responsible for the differences in arteriolar numbers and diameters observed among these highly inbred strains of rats is related to the genetic mechanism responsible for hypertension in SHR.