Lieske, John C.

ABSTRACT Primary hyperoxaluria type 1 (PH1) is a rare and severe autosomal recessive disease of oxalate metabolism, resulting from a mutation in the AGXT gene that encodes the hepatic peroxisomal enzyme alanine–glyoxylate aminotransferase (AGT). Until recently, treatment of PH1 was supportive, consisting of intensive hyperhydration, use of crystallization inhibitors (citrate and neutral phosphorus), in a subset of responsive PH1 patients’ pharmacologic doses of vitamin B6 (pyridoxine), and kidney and liver transplantation when patients progressed to kidney failure. Treatment approaches have been similar for PH2 caused by mutations in hepatic glyoxylate reductase/hydroxypyruvate reductase (GR/HPR), although pyridoxine does not have any benefit in this group. PH3 is caused by mutations of mitochondrial 4-hydroxy-2-oxoglutarate aldolase (HOGA1) and was the most recently described. Kidney failure appears less common in PH3, although kidney stones occur as frequently as in PH1 and PH2. Oxalate metabolism in the liver is complex. Novel therapies based on RNA interference (RNAi) have recently emerged to modulate these pathways, designed to deplete substrate for enzymes upstream and decrease/avoid oxalate production. Two hepatic enzymes have been targeted to date in PH: glycolate oxidase (GO) with lumasiran and lactate dehydrogenase A (LDH-A) with nedosiran. Lumasiran was approved for the treatment of PH1 in 2020 by both the European Medicines Agency and the Food and Drug Administration, whilst clinical trials with nedosiran are ongoing. Results with the two RNAi therapies demonstrate a significant reduction of urinary oxalate excretion in PH1 patients, but long-term data on efficacy (preservation of kidney function, decreased stone events) and safety remain to be established. Nevertheless, the hepatically targeted RNAi approach represents a potential ‘game changer’ in the field of PH1, bringing hope to families and patients that they may be able to avoid liver and/or kidney transplantation in the future and suffer fewer stone events, perhaps with less strict therapeutic regimens. Pharmacological compounds directly inhibiting GO or LDH are also under development and could be of special interest in developing countries where RNAi therapies may not be readily available in the near future. Approaches to manipulate the intestinal microbiome with a goal to increase oxalate degradation or to stimulate secretion of oxalate into the intestine from plasma are also under development. Overall, we appear to be entering a new phase of PH treatment, with an array of promising approaches emerging that will need optimization and evaluation to establish long-term efficacy and safety.

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

Biologic nanoparticles (NPs) can be isolated from bovine serum, human kidney stones, and human arterial calcifications. In addition, it is possible to detect both antigen and antibody for NPs in human blood, suggesting that perhaps NPs represent a novel infectious agent participating in human disease. However, it is not known when and how persons might become exposed to NPs and if exposure produces sensitization to subsequent exposures. Experiments were designed to determine whether exposure to nanoparticles would induce an immune response in rabbits that can be detected by a delayed‐type hypersensitivity test. Rabbits were injected intravenously with bovine‐derived NPs (1ml of 1000NTU solution). Plasma was collected prior to injection and on days, 3, 7, 14, 21, 28, 35 post‐injection. At this time, NPs (1ml of 1000NTU solution) were injected subcutaneously in the ear of exposed and naïve rabbits. Areas of injection were observed daily and after three days, blood and ears were collected for analysis. Injection sites showed leukocyte infiltration only in ears of rabbits previously exposed to NPs. These data indicate that biologic NPs induce immune sensitization in rabbits.

Biologic nanoparticles (NPs) isolated from human arterial calcifications and kidney stones form an adherent film on the bottom of flasks when placed in standard cell culture conditions. Since NPs have certain features consistent with microorganisms, we hypothesized that antibiotics and/or RNAse might inhibit this biofilm formation. NPs isolated from calcified human arterial tissue were cultured onto glass coverslips in 24‐well plates containing DMEM with 10% fetal calf serum, plus either no treatment (control), RNase, tetracycline or gentamicin. At 1, 7 and 14 days after inoculation, coverslips were fixed, mounted, and analyzed using a CytoViva darkfield scope and KS‐400 imaging software. In control wells, the percent coverslip covered by biofilm increased by 120% from day 1 to day 14, compared to 47% and 35% in wells treated with RNase and gentamicin, respectively. Wells treated with tetracycline showed a 3% decrease in biofilm by day 14. Since RNase should not enter intact cells, these results indicate that extracellular RNA may play a role in biofilm formation by NPs. Although tetracycline can chelate calcium, it also inhibits ribosomal function, as does gentamicin. Therefore these results also suggest that intact ribosomal function is required for NP‐associated biofilm growth. These experiments suggest that biofilm formation by NPs consists of biological as well as physicochemical processes.

Biologic nanoparticles (NPs) isolated from human atheromas propagate and form an apatite shell when placed in cell‐culture conditions. These characteristics led us to hypothesize that NPs might derive from microorganisms. NPs isolated from human aortic aneurysms and maintained in DMEM, 10% fetal bovine serum at 37 o C increased both in density (measured in nephelometric turbidity units, NTU) from 1.6 to 107.1 and in diameter (from < 0.2 μm to 0.5‐1 μm) over the course of 12 days. Crystals present on the surface of NPs were found by electron microscopy/elemental analysis to be composed of Ca 2+ and P i . NPs adhered to the surface of the culture flasks, forming a biofilm strikingly similar to that seen in microbial communities. Confocal laser scanning microscopy showed that the biofilm stained positively with concanavalin‐A, a probe for mannose and glucose, both major constituents of microbial biofilm matrix exopolysaccharides. Further, the biofilm stained positively by Bac Light Green, a bacteria‐specific cell wall label, as well as the lipid membrane‐selective dye Vybrant CM‐DiI. These results reveal that calcifying NPs isolated from human atheromas share characteristics common to bacteria since they propagate when placed in cell culture conditions, form a biofilm containing polysaccharides, and stain with agents that recognize bacterial components. Supported by grants from the Fetzer Foundation and NIH HL88988.

The microbiologic nature of self‐replicating, calcifying entity nanoparticles (NPs) isolated from calcified human tissues, including blood vessels and kidney stones is debated because of the difficulty in discriminating minerals precipitated on microbes from minerals nucleated on macromolecules and cell membranes. To begin to resolve this controversy, calcifying bacteria of aquatic origin ( Caulobacter crescentus ) and NPs cultured from human samples were examined using a Scanning Transmission X‐ray Microscope (STXM). The monochromated soft X‐ray beam (150–2000 eV) produced by synchrotron radiation yields microscopic and spectroscopic information on both organic compounds and minerals at the 25 nm scale. High‐spatial and energy resolution Near‐Edge X‐ray Absorption Fine Structure (NEXAFS) spectra indicative of elemental speciation acquired at the C K‐edge and Ca L 2,3 ‐edge on a single‐cell scale from calcified C. crescentus displayed a unique spectral signature different from non‐biologic hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Preliminary NEXAFS measurements of calcium and carbon functional groups of cultured NPs revealed organics associated with hydroxyapatite minerals and in one case, spectroscopic signatures similar to those identified in C. crescentus . Using NEXAFS, it is possible to define a biochemical signature of proteins, polysaccharides, nucleic acids, and hydroxyapatite for cultured calcified bacteria suggesting that NPs isolated from human samples share spectroscopic characteristics with calcifying bacteria.