Spironolactone suppresses inflammation and prevents L-NAME-induced renal injury in rats.

Chronic inhibition of nitric oxide synthase by N(omega)-nitro- L-arginine methyl ester (L-NAME) causes progressive renal injury with systemic hypertension and interstitial macrophage infiltration. We have previously shown that there is local activation of the renin-angiotensin-aldosterone system in the renal cortex as a major pathogenic feature of macrophage infiltration. In this study, we measured the effects of the aldosterone antagonist, spironolactone, on renal injury in L-NAME-treated male Wistar rats. After 12 weeks of L-NAME-treatment, rats had increased systolic blood pressure, urinary protein excretion, and serum creatinine and histological analysis showed glomerulosclerosis, interstitial fibrosis, and macrophage infiltration. Treatment with spironolactone significantly prevented these renal changes, whereas treatment with hydralazine had no effect. The cortical expression of osteopontin was significantly elevated in L-NAME-treated rats, and expression of its mRNA significantly correlated with the number of infiltrating macrophages and degree of interstitial fibrosis. Spironolactone treatment markedly suppressed osteopontin expression. Our results suggest that reduced nitric oxide bioavailability caused renal inflammation and fibrosis through an aldosterone receptor-dependent mechanism associated with osteopontin expression independent of its systemic hemodynamic effects.

Involvement of p53-transactivated Puma in cisplatin-induced renal tubular cell death.

AIMS: The tumor suppressor protein p53 plays a critical role as a determinant of cell survival when cells are exposed to various toxic stresses, by preventing growth arrest, replication of damaged DNA, and apoptosis. A novel p53-dependent proapoptotic gene, Puma (p53 upregulated modulator of apoptosis) is thought to participate in this process. Recently, p53 was reported to play an essential role in cisplatin-induced renal tubular cell (RTC) death. The objective of the present study was to elucidate the roles of p53 and Puma in cisplatin-induced RTC death. MAIN METHODS: We examined the in vivo expression of p53 and Puma-alpha in the kidney and evaluated the modification of Puma-alpha expression and RTC death by in vitro treatment with pifithrin-alpha (PFT-alpha), a specific p53 inhibitor, or Puma-alpha-specific small interfering RNA (siRNA). KEY FINDINGS: While no immunoreactivity for anti-p53- and anti-Puma-alpha antibody was detected in the control rat kidney, de novo expression of p53 and Puma-alpha was identified in the proximal tubular cells of the outer medulla at 6 h after cisplatin injection. Upregulation of these proteins preceded severe RTC injury. In vitro experiments revealed that PFT-alpha inhibited upregulation of Puma-alpha, and inhibition of Puma-alpha, either by PFT-alpha or by Puma-alpha-specific siRNA, decreased RTC death induced by 24-h cisplatin exposure. SIGNIFICANCE: Our results indicated that p53 activation mediated cisplatin-induced RTC death through upregulation of Puma, and suggested that inhibition of p53 and Puma is beneficial for the prevention and treatment of cisplatin-induced acute renal failure.

The possibility of a valuable resource of circulating DNA for single nucleotide polymorphisms genotyping: the application of a rapid and simple polymerase chain reaction with melting curve analysis for methyltetrahydrofolate reductase polymorphisms.

Circulating DNA from plasma is easily stored and is a valuable resource to access genetic information indispensable to modern hematology. The aim of the present project was to evaluate the integrity of circulating DNA and to investigate whether such DNA is practically tolerable for genotyping single nucleotide polymorphisms (SNP) of methyltetrahydrofolate reductase (MTHFR). We first established a protocol combined with polymerase chain reaction (PCR) and melting curve analysis (MCA) based on the different melting temperatures of heteroduplex amplicons. This method was simple and rapid, requiring 3 hours without any complex manipulation, and allowed for a reliable test and diagnostic validity. The median of the circulating DNA density in 240 donors was 33.5 ng/mL. The DNA consisted of fragments with approxiately 100 to 500 base pairs. Such DNA fragments were acceptable for quantifying the housekeeping genes of - globin using a real-time PCR method and also for genotyping the MTHFR SNP using the method of PCR with MCA. Circulating DNA from storage plasma is acceptable for genetic tests, but it is necessary to note the integrity of DNA.