Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse

Abstract

Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP). The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA) and porphobilinogen (PBG) in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD)-deficient (AIP) mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males) and over 13 (females) in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.

Figure 1. Lack of glomerular, tubulointerstitial or vascular damage in acute intermittent porphyria mice after sustained urinary porphyrin precursors and porphyrin excretion induced by phenobarbital challenge.

The effect of repetitive acute attacks on renal function was assayed in four AIP male mice aged from 9 to 14 months. Seven consecutive phenobarbital challenges were administered at intervals of two weeks in order to maintain high hepatic production and high flow of both porphyrin precursors and porphyrin throughout the kidney. Four age-matched wild type male animals were used as controls. After the fourth consecutive dose of each phenobarbital challenge AIP mice developed a biochemical attack, characterized by A) high levels of urinary porphyrin precursors and B) porphyrin accumulation. C) Blood urea nitrogen (BUN) and individual pathological analysis of kidney necropsies. Histological analysis was performed in renal samples taken three days after the last phenobarbital challenge. Prognostic scores in histological analysis of renal biopsies were analysed by a trained pathologist (ES) as described in Methods. D) Light micrographs of kidney sections showed relatively innocuous tubular dilatation in one AIP animal. Another AIP animal showed diffuse cortical atrophy E), which was more prominent in the subcapsular region. Magnification ×200. The Wilcoxon matched pairs test was used for comparison. The null hypothesis was rejected when p≤0.05. WT, wild type; AIP, Acute Intermittent Porphyria.

Figure 2. Porphyrin precursor excretion in wild type and AIP mice suffering from different degrees of renal insufficiency.

The effect of five-sixth nephrectomy on urine excretion of aminolevulinate acid (ALA) and porphobilinogen (PBG) was compared with respect to urinary excretion in sham-operated mice. Urine levels of ALA and PBG were also measured in these animals after phenobarbital challenge that induces a biochemical attack of porphyria specifically in AIP mice. A) Urinary PBG excretion. B) Urinary ALA excretion. C) Urinary PBG/ALA ratio. The non-parametric Mann–Whitney U-test was used for comparison of two groups. Nx5/6, 2/3 nephrectomy of the left kidney and extirpation of the right kidney. The null hypothesis was rejected when p≤0.05. WT, wild type; AIP, Acute Intermittent Porphyria.

Figure 3. Expression profile of hepatic ALAS, ALAD and PBGD in female wild type and AIP mice suffering from different degrees of renal insufficiency.

A group of 4 wild type and 9 AIP female mice were subjected to 5/6 nephrectomy (5/6 Nx) while complete bilateral nephrectomy was performed in 6 wild type and 7 AIP mice. A group of 5 wild type and 6 AIP female animals were sham-operated. A) Quantitative real-time PCR analysis of ALAS mRNA from liver samples. The amount of each transcript was expressed according to the formula 2^{ΔCt(Actin)−ΔCt(gene)}, where ΔCt is the point at which the fluorescence rises appreciably above background fluorescence. B) ALAD and C) PBGD activities measured in the liver of mice taken at sacrifice. The non-parametric Mann–Whitney U-test was used for comparison of two groups. Nx5/6, 2/3 nephrectomy of the left kidney and extirpation of the right kidney; Nx 6/6, total nephrectomy. ALAS, hepatic aminolevulinate acid synthase; ALAD, aminolevulinate acid dehydratase; AIP, Acute Intermittent Porphyria.

Figure 4. Expression profile of hepatic ALAS, ALAD and PBGD in male wild type and AIP mice suffering from different degrees of renal insufficiency.

A group of 5 wild type and 8 AIP male animals were subjected to 5/6 nephrectomy (5/6 Nx) while complete bilateral nephrectomy was performed in 4 wild type and 6 AIP mice. A group of 6 wild type and 8 AIP male animals were sham-operated. A) Quantitative real-time PCR analysis of ALAS mRNA, B) enzyme ALAD and C) PBGD activities measured in the liver of wild type and AIP male animals. The non-parametric Mann–Whitney U-test was used for comparison of two groups. Nx5/6, 2/3 nephrectomy of the left kidney and extirpation of the right kidney; Nx 6/6, total nephrectomy. ALAS, hepatic aminolevulinate acid synthase; ALAD, aminolevulinate acid dehydratase; AIP, Acute Intermittent Porphyria.