Gene expression of 5-lipoxygenase and LTA4 hydrolase in renal tissue of nephrotic syndrome patients

Abstract

Leukotrienes (LT) of the 5-lipoxygenase pathway constitute a class of potent biological lipid mediators of inflammation implicated in the pathogenesis of different models of experimental glomerulonephritis. The key enzyme, 5-lipoxygenase (5-LO), catalyses oxygenation of arachidonic acid to generate the primary leukotriene LTA4. This LT, in turn, serves as a substrate for either LTA4 hydrolase, to form the potent chemoattractant LTB4, or LTC4 synthase, to produce the powerful vasoconstrictor LTC4. To investigate the potential role of LT in the pathogenesis of human glomerulonephritis with nephrotic syndrome, we examined the gene expression of 5-LO and LTA4 hydrolase in renal tissue of 21 adult patients with nephrotic syndrome and 11 controls. The patients consisted of 11 cases of membranous nephropathy (MN), seven focal and segmental glomerulosclerosis (FSGS), two non-IgA mesangial glomerulonephritis and one minimal change disease. Total RNA purified from renal tissue was reverse transcribed into cDNA and amplified with specific primers in a polymerase chain reaction (RT-PCR). Eight patients' renal tissue, four MN and four FSGS, co-expressed 5-LO and LTA4 hydrolase. In situ hybridization analysis revealed 5-LO expression and distribution limited to the interstitial cells surrounding the peritubular capillaries. Comparative clinical and immunohistological data showed that these eight patients had impaired renal function and interstitial changes that significantly correlated with 5-LO expression. These findings suggest that leukotrienes may play an important role in the pathogenesis of MN and FSGS. These results are also relevant to elucidating the pathophysiologic mechanisms which underlie progression to renal failure in these diseases.

Fig. 1

Representative expression profile in renal tissue detected by polymerase chain reaction (PCR). Cytoplasmic β-actin gene (lane 1); CD3, T lymphocyte marker (lane 2); CD14, macrophage marker (lane 3); CD19, B lymphocyte marker (lane 4); LTA₄ hydrolase (lane 5); and 5-lipoxygenase (5-LO) (lane 6). Scanned computer image of silver-stained 12.5% Phastgel containing 30 cycles amplified PCR products of target genes. MW, molecular marker of φX174 RF DNA/Hae III digest.

Fig. 2

Examples of distributions of CD3⁺ and CD14⁺ immunohistochemical staining in frozen sections of a specimen from a nephrotic syndrome patient with membranous nephropathy (no. 6 of Tables 1 and 2). (a) CD3⁺ interstitial lymphocytes (arrows). (b) CD14⁺ interstitial mononuclear cells (arrows).

Fig. 3

Representative example of immunohistochemistry analysis of 5-lipoxygenase (5-LO) distribution in a frozen section of a specimen from a nephrotic syndrome patient with membranous nephropathy (no. 6 of Tables 1 and 2) showing positive interstitial peritubular spindle-shaped cells (b), and (a) positive interstitial mononuclear cells (arrows).

Fig. 4

Representative example of in situ hybridization analysis of 5-lipoxygenase (5-LO) gene distribution in a specimen from a nephrotic syndrome patient with membranous nephropathy (no. 5 of Tables 1 and 2). Focal staining surrounding peritubular capillary vessels related to interstitial cells is evident (a). An anti-digoxigenin antibody conjugated with alkaline phosphatase and NBT/BCIP as chromogen were used as detecting system (see Patients and Methods) (×400). Normal tissue is shown for comparison (b).

Fig. 5

Comparison of clinical data in patients according to 5-lipoxygenase (5-LO) expression. Statistical significance determined by t-test for effective renal plasma flow in (a) (P = 0.01) and glomerular filtration rate in (c) (P = 0.004). Association of proteinuria in (b) (P = NS, Z = 1.23) and level of serum creatinine in (d) (P = 0.01) with 5-LO expression analysed by Mann–Whitney rank sum test. ERPF, Effective renal plasma flow; GFR, glomerular filtration rate; sCr, serum creatinine; 5-LO⁺ and 5-LO⁻, five-lipoxygenase-positive and negative cases. *Significant (P ≤ 0.01) compared with 5-LO⁻ cases.