Rana Rustom

Whole-blood cultures from renal-transplant patients stimulated ex vivo show that the effects of cyclosporine on lymphocyte proliferation are related to P-glycoprotein expression.

Background. Cyclosporine (CsA) is a substrate for the MDR-1 gene product P-glycoprotein (P-gp). CsA efficacy may be modulated by lymphocyte P-gp expression levels. In this study, CsA inhibition of lymphocyte proliferation in whole-blood cultures ex vivo has been related to (1) lymphocyte P-gp expression and (2) the C3435T polymorphism in the MDR-1 gene, which has been reported to alter P-gp function. Methods. In 30 renal-transplant recipients taking CsA monotherapy, P-gp expression was measured by flow cytometry. Whole-blood samples were stimulated with purified protein derivative (PPD) and phytohemagglutinin (PHA). CsA resistance ex vivo was defined as less than 10% reduction in proliferation with either PPD or PHA at 2 hours compared with 0 hours. Results. CsA resistance was associated with greater P-gp expression using either PPD (median expression, resistant 1.89 vs. sensitive 0.96, P =0.02) or PHA (1.66 vs. 0.96, respectively, P =0.02). Whole-blood CsA levels in resistant and sensitive patients were similar. The C3435T polymorphism did not affect inhibition of proliferation by CsA (P >0.05 for all between genotype group comparisons). Conclusions. Our results indicate that lymphocyte P-gp expression determines the degree of inhibition of proliferation by CsA ex vivo; whether this also affects CsA effectiveness in vivo and therefore graft survival requires further study.

Characterisation of the expression of the Renin-Angiotensin system in primary and immortalised human renal proximal tubular cells.

Background/Aims: Angiotensin II (AngII) is pivotal in the pathogenesis of progressive kidney disease. We have recently shown that AngII induced an increase in markers of oxidative stress, adaptive responses and upregulated stress-related gene expression in immortalised human proximal tubular (HK-2) cells. However, these observed effects of AngII were not mediated solely via AngII type 1 receptor (ATR1). Both HK-2 cells and primary human renal proximal tubular cells (RPTEC) are useful tools to investigate the renin-angiotensin system (RAS), but data on the local expression of the RAS in these cells remain limited. We therefore characterised RAS expression in RPTEC and HK-2 cells. Methods: The mRNA and protein expression of RAS in RPTEC and HK-2 cells was examined by RT-PCR, Western blotting and immunoprecipitation. Results: In both cell lines, mRNA for angiotensin-converting enzyme (ACE) and mRNA and protein expression for angiotensinogen, renin, ACE2, ATR1 and ATR4 were detected. Candesartan, a specific ATR1 blocker, effectively blocked the expression of 80% of the stress-related genes that were upregulated in HK-2 cells following exposure to AngII. Con clusion: These data support a role for AngII in mediating oxidative stress via other receptor types stimulated by AngII and confirm that it is possible to investigate ATR4 pathways of potential injury in RPTEC.

Oxidative Stress in a Novel Model of Chronic Acidosis in LLC-PK1 Cells

Chronic metabolic acidosis occurs commonly in chronic renal failure (CRF). The proximal renal tubular cell is the site in the kidney of high oxidative metabolic activity and in CRF is associated with adaptive hypertrophy and hypermetabolism. We hypothesised that chronic acidosis may lead to increased generation of reactive oxygen species due to increased oxidative activity. We developed a novel model of chronic acidosis in LLC-PK1 cells and measured markers of oxidative stress and metabolism. Acidosis led to a reduction in cellular total glutathione and protein thiol content and an increase in glutathione peroxidase activity and NH3 generation. The expression of constitutively expressed heat stress protein (HSP) HSC70 and HSP60 increased at pH 7.0.

RENAL TUBULAR PEPTIDE CATABOLISM IN CHRONIC VASCULAR REJECTION

Chronic vascular rejection (CR) is the commonest cause of renal transplant loss, with few clues to etiology, but proteinuria is a common feature. In diseased native kidneys, proteinuria and progression to failure are linked. We proposed a pathogenic role for this excess protein at a tubular level in kidney diseases of dissimilar origin. We demonstrated in both nephrotic patients with normal function and in those with failing kidneys increased renal tubular catabolisman and turnover rates of a peptide marker, Aprotinin (Apr), linked to increased ammonia excretion and tubular injury. These potentially injurious processes were suppressed by reducing proteinuria with Lisinopril. Do similar mechanisms of renal injury and such a linkage also occur in proteinuric transplanted patients with CR, and if so, is Lisinopril then of beneficial value? We now examine these aspects in 11 patients with moderate/severe renal impairment (51CrEDTA clearance 26.2 ± 3.3 mL/min/1.73m2), proteinuria (6.1 ± 1.5 g/24 h) and biopsy proven CR. Lisinopril (10–40 mg) was given daily for 2 months in 7 patients. Four others were given oral sodium bicarbonate (Na HCO3) for 2 months before adding Lisinopril. Renal tubular catabolism of intravenous 99mTc-Apr (Apr* 0.5 mg, 80 MBq), was measured before and after Lisinopril by γ-ray renal imaging and urinary radioactivity of the free radiolabel over 26 h. Fractional degradation was calculated from these data. Total 24 h urinary N-acetyl-β-glucoaminidase (NAG) and ammonia excretion in fresh timed urine collections were also measured every two weeks from two months before treatment. After Lisinopril proteinuria fell significantly (from 7.8 ± 2.2 to 3.4 ± 1.9 g/24 h, p < 0.05). This was associated with a reduction in metabolism of Apr* over 26 h (from 0.5 ± 0.05 to 0.3 ± 0.005% dose/h, p < 0.02), and in fractional degradation (from 0.04 ± 0.009 to 0.02 ± 0.005/h, p <0.01). Urinary ammonia fell, but surprisingly not significantly and this was explained by the increased clinical acidosis after Lisinopril, (plasma bicarbonate fell from 19.1 ± 0.7 to 17.4 ± 0.8 mmol/L, p <0.01), an original observation. Total urinary NAG did fall significantly from a median of 2108 (range 1044–3816) to 1008 (76–2147) μmol/L, p < 0.05. There was no significant change in blood pressure or in measurements of glomerular hemodynamics. In the 4 patients who were given Na HCO3 before adding Lisinopril, both acidosis (and hyperkalemia) were reversed and neither recurred after adding Lisinopril. These observations in proteinuric transplanted patients after Lisinopril treatment have not been previously described.

Successful pregnancy in a patient with Landesman's Group C autosomal dominant polycystic kidney disease.

Background A female with autosomal dominant polycystic kidney disease was followed up over the course of four pregnancies. Her first three pregnancies were unsuccessful. Her fourth pregnancy resulted in a live birth, but at what expense?Investigations The diagnosis of autosomal dominant polycystic kidney disease was confirmed by ultrasound imaging. Physical examination, blood pressure measurement, and urine and blood analyses were performed at each follow-up visit.Diagnosis Deterioration of renal function following multiple complicated pregnancies.Management Attention to blood pressure and proteinuria delayed initiation of dialysis, but effects of the number of pregnancies took their toll. The patient was started on hemodialysis and underwent renal transplantation.

Urinary Monocyte Chemoattractant Protein-1 (MCP-1) in Renal Transplant Recipients: Implications in Proteinuric Patients

Background: After the first year of transplantation chronic allograft nephropathy is the most important cause of renal graft loss and hypertension and proteinuria occur commonly. In native nephropathies, proteinuria and progression to renal failure are linked and renal tubulo-interstitial fibrosis determines prognosis. Monocyte chemoattractant protein-1 (MCP-1) is a powerful chemokine promoting tubulo-interstitial fibrosis but data are limited in a transplant context. Hence this observational cross-sectional study. Methods: The MAP, 24h urinary creatinine clearance, proteinuria and MCP-1 were measured in 81 renal transplant pa- tients (43 with chronic allograft nephropathy). Most patients were on calcineurin-inhibitor based immunosuppression. Re- gression analysis was applied and comparisons made with 64 patients with native nephropathies and comparable function. Results: One fifth (18/81) of all renal transplant patients had less than optimally controlled hypertension. Proteinuria was heaviest in non-transplanted patients (average 3.0 g/24h, 0.1-12.2) and the ciclosporin-treated transplant patients (1.2 g/24h, 0.02-6.4). Proteinuria and MCP-1 were positively correlated in all patients (r 0.54, p<0.0001) and r 0.84, p<0.0001 in 19 transplant patients receiving angiotensin-converting enzyme inhibitors. MCP-1 levels were highest in non-transplant and ciclosporin-treated patients; geometric mean (SE), 412.4(1.16) and 314.5(1.21) pg/24h respectively. MCP-1 levels were unrelated to age, MAP, creatinine clearance, or blood ciclosporin or tacrolimus levels. Conclusions: Urinary MCP-1 may be a useful non-invasive marker of chronic graft dysfunction in transplant patients fa- cilitating monitoring of progression and response to treatment even in proteinuric patients.