Chronic interstitial nephritis in agricultural communities is a toxin-induced proximal tubular nephropathy.

Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity.

Hepatic symptoms and histology in 13 patients with a Zellweger spectrum disorder.

Patients with a Zellweger spectrum disorder (ZSD) have a defect in the assembly or maintenance of peroxisomes, leading to a multisystem disease with variable outcome. Liver disease is an important feature in patients with severe and milder phenotypes and a frequent cause of death. However, the course and histology of liver disease in ZSD patients are ill-defined. We reviewed the hepatic symptoms and histological findings of 13 patients with a ZSD in which one or several liver biopsies have been performed (patient age 0.2-39 years). All patients had at least some histological liver abnormalities, ranging from minor fibrosis to cirrhosis. Five patients demonstrated significant disease progression with liver failure and early death. In others, liver-related symptoms were absent, although some still silently developed cirrhosis. Patients with peroxisomal mosaicism had a better prognosis. In addition, we show that patients are at risk to develop a hepatocellular carcinoma (HCC), as one patient developed a HCC at the age of 36 years and one patient a precancerous lesion at the age of 18 years. Thus, regular examination to detect fibrosis or cirrhosis should be included in the standard care of ZSD patients. In case of advanced fibrosis/cirrhosis expert consultation and HCC screening should be initiated. This study further delineates the spectrum and significance of liver involvement in ZSDs.

Severe hepatopathy and neurological deterioration after start of valproate treatment in a 6-year-old child with mitochondrial tryptophanyl-tRNA synthetase deficiency.

BACKGROUND: The first subjects with deficiency of mitochondrial tryptophanyl-tRNA synthetase (WARS2) were reported in 2017. Their clinical characteristics can be subdivided into three phenotypes (neonatal phenotype, severe infantile onset phenotype, Parkinson-like phenotype). RESULTS: Here, we report on a subject who presented with early developmental delay, motor weakness and intellectual disability and who was considered during several years as having a non-progressive encephalopathy. At the age of six years, she had an epileptic seizure which was treated with sodium valproate. In the months after treatment was started, she developed acute liver failure and severe progressive encephalopathy. Although valproate was discontinued, she died six months later. Spectrophotometric analysis of the oxidative phosphorylation complexes in liver revealed a deficient activity of complex III and low normal activities of the complexes I and IV. Activity staining in the BN-PAGE gel confirmed the low activities of complex I, III and IV and, in addition, showed the presence of a subcomplex of complex V. Histochemically, a mosaic pattern was seen in hepatocytes after cytochrome c oxidase staining. Using Whole Exome Sequencing two known pathogenic variants were detected in WARS2 (c.797delC, p.Pro266ArgfsTer10/ c.938 A > T, p.Lys313Met). CONCLUSION: This is the first report of severe hepatopathy in a subject with WARS2 deficiency. The hepatopathy occurred soon after start of sodium valproate treatment. In the literature, valproate-induced hepatotoxicity was reported in the subjects with pathogenic mutations in POLG and TWNK. This case report illustrates that the course of the disease in the subjects with a mitochondrial defect can be non-progressive during several years. The subject reported here was first diagnosed as having cerebral palsy. Only after a mitochondriotoxic medication was started, the disease became progressive, and the diagnosis of a mitochondrial defect was made.

Inborn errors of metabolism in the biosynthesis and remodelling of phospholipids.

Since the proposal to define a separate subgroup of inborn errors of metabolism involved in the biosynthesis and remodelling of phospholipids, sphingolipids and long chain fatty acids in 2013, this group is rapidly expanding. This review focuses on the disorders involved in the biosynthesis of phospholipids. Phospholipids are involved in uncountable cellular processes, e.g. as structural components of membranes, by taking part in vesicle and mitochondrial fusion and fission or signal transduction. Here we provide an overview on both pathophysiology and the extremely heterogeneous clinical presentations of the disorders reported so far (Sengers syndrome (due to mutations in AGK), MEGDEL syndrome (or SERAC defect, SERAC1), Barth syndrome (or TAZ defect, TAZ), congenital muscular dystrophy due to CHKB deficiency (CHKB). Boucher-Neuhäuser/Gordon Holmes syndrome (PNPLA6), PHARC syndrome (ABHD12), hereditary spastic paraplegia type 28, 54 and 56 (HSP28, DDHD1; HSP54, DDHD2; HSP56, CYP2U1), Lenz Majewski syndrome (PTDSS1), spondylometaphyseal dysplasia with cone-rod dystrophy (PCYT1A), atypical haemolytic-uremic syndrome due to DGKE deficiency (DGKE).

Polymerase gamma deficiency (POLG): clinical course in a child with a two stage evolution from infantile myocerebrohepatopathy spectrum to an Alpers syndrome and neuropathological findings of Leigh's encephalopathy.

AIMS: Description of the clinical course in a child compound heterozygous for POLG1 mutations, neuropathology findings and results of dietary treatment based on fasting avoidance and long chain triglycerides (LCT) restriction. RESULTS: At 3(1/2) months of age the patient presented with severe hypoglycemia, hyperlactatemia, moderate ketosis and hepatic failure. Fasting hypoglycemia occurred 8 h after meals. The hypoglycemia did not respond to glucagon. She was supplemented with IV glucose and/or frequent feedings, but developed liver insufficiency which was reversed by long-chain triglyceride (LCT) restriction. Alpha-foeto-protein (AFP) levels were elevated and returned to low values after dietary treatment. Liver biopsy displayed cirrhosis, bile ductular proliferation, steatosis, isolated complex IV defect in part of the liver mitochondria, and mitochondrial DNA depletion (27% of control values). Two heterozygous mutations (p. [Ala467Thr] + p. [Gly848Ser]) were found in the POLG1 gene. At 3 years of age she progressively developed refractory mixed type seizures including a focal component and psychomotor regression which fulfilled the criteria of Alpers syndrome (AS) although the initial presentation was compatible with infantile myocerebrohepatopathy spectrum (MCHS). She died at 5 years of age of respiratory insufficiency. Neuropathologic investigation revealed lesions in the right striatal area and the inferior colliculi typical for Leigh's encephalopathy. CONCLUSION: The present patient showed an evolution from infantile MCHS to AS, and dietary treatment seemed to slow the progression of liver failure. In spite of the late clinical features of AS, it extends the neuropathological spectrum of AS and polymerase gamma deficiency (POLG) to Leigh syndrome lesions.

Mitochondrial mosaics in the liver of 3 infants with mtDNA defects.

BACKGROUND: In muscle cytochrome oxidase (COX) negative fibers (mitochondrial mosaics) have often been visualized. METHODS: COX activity staining of liver for light and electron microscopy, muscle stains, blue native gel electrophoresis and activity assays of respiratory chain proteins, their immunolocalisation, mitochondrial and nuclear DNA analysis. RESULTS: Three unrelated infants showed a mitochondrial mosaic in the liver after staining for COX activity, i.e. hepatocytes with strongly reactive mitochondria were found adjacent to cells with many negative, or barely reactive, mitochondria. Deficiency was most severe in the patient diagnosed with Pearson syndrome. Ragged-red fibers were absent in muscle biopsies of all patients. Enzyme biochemistry was not diagnostic in muscle, fibroblasts and lymphocytes. Blue native gel electrophoresis of liver tissue, but not of muscle, demonstrated a decreased activity of complex IV; in both muscle and liver subcomplexes of complex V were seen. Immunocytochemistry of complex IV confirmed the mosaic pattern in two livers, but not in fibroblasts. MRI of the brain revealed severe white matter cavitation in the Pearson case, but only slight cortical atrophy in the Alpers-Huttenlocher patient, and a normal image in the 3rd. MtDNA in leucocytes showed a common deletion in 50% of the mtDNA molecules of the Pearson patient. In the patient diagnosed with Alpers-Huttenlocher syndrome, mtDNA was depleted for 60% in muscle. In the 3rd patient muscular and hepatic mtDNA was depleted for more than 70%. Mutations in the nuclear encoded gene of POLG were subsequently found in both the 2nd and 3rd patients. CONCLUSION: Histoenzymatic COX staining of a liver biopsy is fast and yields crucial data about the pathogenesis; it indicates whether mtDNA should be assayed. Each time a mitochondrial disorder is suspected and muscle data are non-diagnostic, a liver biopsy should be recommended. Mosaics are probably more frequent than observed until now. A novel pathogenic mutation in POLG is reported.Tentative explanations for the mitochondrial mosaics are, in one patient, unequal partition of mutated mitochondria during mitoses, and in two others, an interaction between products of several genes required for mtDNA maintenance.

Hepatocellular transport and gastrointestinal absorption of lanthanum in chronic renal failure.

Lanthanum carbonate is a new phosphate binder that is poorly absorbed from the gastrointestinal tract and eliminated largely by the liver. After oral treatment, we and others had noticed 2-3 fold higher lanthanum levels in the livers of rats with chronic renal failure compared to rats with normal renal function. Here we studied the kinetics and tissue distribution, absorption, and subcellular localization of lanthanum in the liver using transmission electron microscopy, electron energy loss spectrometry, and X-ray fluorescence. We found that in the liver lanthanum was located in lysosomes and in the biliary canal but not in any other cellular organelles. This suggests that lanthanum is transported and eliminated by the liver via a transcellular, endosomal-lysosomal-biliary canicular transport route. Feeding rats with chronic renal failure orally with lanthanum resulted in a doubling of the liver levels compared to rats with normal renal function, but the serum levels were similar in both animal groups. These levels plateaued after 6 weeks at a concentration below 3 microg/g in both groups. When lanthanum was administered intravenously, thereby bypassing the gastrointestinal tract-portal vein pathway, no difference in liver levels was found between rats with and without renal failure. This suggests that there is an increased gastrointestinal permeability or absorption of oral lanthanum in uremia. Lanthanum levels in the brain and heart fluctuated near its detection limit with long-term treatment (20 weeks) having no effect on organ weight, liver enzyme activities, or liver histology. We suggest that the kinetics of lanthanum in the liver are consistent with a transcellular transport pathway, with higher levels in the liver of uremic rats due to higher intestinal absorption.

Intra-familial clinical heterogeneity: absence of genotype-phenotype correlation in primary hyperoxaluria type 1 in Israel.

BACKGROUND/AIMS: Primary hyperoxaluria type 1 (PH1) is caused by the deficiency of the liver enzyme alanine:glyoxylate aminotransferase which results in increased synthesis and excretion of oxalate. The clinical manifestations of PH1 are heterogeneous with respect to the age of onset and rate of progression. The aim of this study was to investigate possible relationships between a given genotype, the biochemical profile and the clinical phenotype. METHODS: We conducted a study of 56 patients from 22 families with PH1 from Israel. The clinical and biochemical data were compiled and the genotype was determined for each family. RESULTS: The prevalent phenotype was of early onset with progression to end-stage renal disease during the first decade of life. Fifteen PH1-causing mutations were detected in 21 families: 10 were first described in this patient population. Marked intra-familial clinical heterogeneity was noted, meaning that there was no correlation between a given genotype and the phenotype. CONCLUSIONS: The clinical course of patients with PH1 is not dictated primarily by its genotype. Other genetic and/or environmental factors play a role in determining the ultimate phenotype.

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator.

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARalpha-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARalpha independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

No evidence for involvement of SDHD in neuroblastoma pathogenesis.

BACKGROUND: Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. METHODS: SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. RESULTS: Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. CONCLUSIONS: Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.

Human peroxisomal disorders.

Peroxisomes are single membrane-bound cell organelles performing numerous metabolic functions. The present article aims to give an overview of our current knowledge about inherited peroxisomal disorders in which these organelles are lacking or one or more of their functions are impaired. They are multiorgan disorders and the nervous system is implicated in most. After a summary of the historical names and categories, each having distinct symptoms and prognosis, microscopic pathology is reviewed in detail. Data from the literature are added to experience in the authors' laboratory with 167 liver biopsy and autopsy samples from peroxisomal patients, and with a smaller number of chorion samples for prenatal diagnosis, adrenal-, kidney-, and brain samples. Various light and electron microscopic methods are used including enzyme- and immunocytochemistry, polarizing microscopy, and morphometry. Together with other laboratory investigations and clinical data, this approach continues to contribute to the diagnosis and further characterization of peroxisomal disorders, and the discovery of novel variants. When liver specimens are examined, three main groups including 9 novel variants (33 patients) are distinguished: (1) absence or (2) presence of peroxisomes, and (3) mosaic distribution of cells with and without peroxisomes (10 patients). Renal microcysts, polarizing trilamellar inclusions, and insoluble lipid in macrophages in liver, adrenal cortex, brain, and in interstitial cells of kidney are also valuable for classification. On a genetic basis, complementation of fibroblasts has classified peroxisome biogenesis disorders into 12 complementation groups. Peroxisome biogenesis genes (PEX), knock-out-mice, and induction of redundant genes are briefly reviewed, including some recent results with 4-phenylbutyrate. Finally, regulation of peroxisome expression during development and in cell cultures, and by physiological factors is discussed.

Inhibition of cytokine production by the herbicide atrazine. Search for nuclear receptor targets.

The hematological toxicity of the commonly used triazine herbicides is a cause for concern. In a search for molecular targets of these compounds, as their effects paralleled those seen with dexamethasone (DEX), we first looked for interaction with the glucocorticoid receptor. In contrast to the effects on proliferation and cytokine production of DEX, those induced by atrazine were not prevented by the glucocorticoid antagonist RU486. Also, whereas DEX was able to inhibit the promoter activity of genes regulated by NF-kappaB, atrazine failed to do so. We next looked for interaction with members of the peroxisome proliferator-activated receptor (PPAR) family. No peroxisome proliferation was observed in the liver or kidneys of mice treated with atrazine. Moreover, no PPAR-mediated induction of promoter activity was seen on targets of PPARalpha, PPARgamma, or PPARdelta. Similarly, neither atrazine nor simazine were able to stimulate RORalpha-mediated promoter activity. Finally, no binding of atrazine to the AR was observed. In conclusion, the effects of atrazine-type herbicides most probably do not result from interaction with the above-mentioned nuclear receptors.