Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method.

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

Nanobacteria: controversial pathogens in nephrolithiasis and polycystic kidney disease.

Nanobacteria are unconventional agents 100-fold smaller than common bacteria that can replicate apatite-forming units. Nanobacteria are powerful mediators of biogenic apatite nucleation (crystal form of calcium phosphate) and crystal growth under conditions simulating blood and urine. Apatite is found in the central nidus of most kidney stones and in mineral plaques (Randall's plaques) in renal papilla. The direct injection of nanobacteria into rat kidneys resulted in stone formation in the nanobacteria-injected kidney during one month follow-up, but not in the control kidney injected with vehicle. After intravenous administration in rats and rabbits, nanobacteria are rapidly excreted from the blood into the urine, as a major elimination route, and damage renal collecting tubuli. Nanobacteria are cytotoxic to fibroblasts in vitro. Human kidney cyst fluids contain nanobacteria. Nanobacteria thus appear to be potential provocateurs and initiators of kidney stones, tubular damage, and kidney cyst formation. It is hypothesized that nanobacteria are the initial nidi on which kidney stone is built up, at a rate dependent on the supersaturation status of the urine. Those individuals having both nanobacteria and diminished defences against stone formation (i.e. genetic factors, diet and drinking habits) could be at high risk. Kidney cyst formation is hypothesized to involve nanobacteria-induced tubular damage and defective tissue regeneration yielding cyst formation, the extent of which is dependent on genetic vulnerability.

Endotoxin and nanobacteria in polycystic kidney disease.

BACKGROUND: Microbes have been suspected as provocateurs of polycystic kidney disease (PKD), but attempts to isolate viable organisms have failed. Bacterial endotoxin is the most often reported microbial product found in PKD fluids. We assessed potential microbial origins of endotoxin in cyst fluids from 13 PKD patients and urines of PKD and control individuals. METHODS: Fluids were probed for endotoxin and nanobacteria, a new bacterium, by the differential Limulus Amebocyte Lysate assay (dLAL), genus-specific antilipopolysaccharide (LPS) antibodies, monoclonal antibodies to nanobacteria, and hyperimmune serum to Bartonella henselae (HS-Bh). Selected specimens were also assessed by transmission electron microscopy (TEM) and nanobacterial culture methods. RESULTS: LPS or its antigenic metabolites were found in more than 75% of cyst fluids tested. Nanobacteria were cultured from 11 of 13 PKD kidneys, visualized in 8 of 8 kidneys by TEM, and immunodetected in all 13 PKD kidneys. By immunodetection, nanobacterial antigens were found in urine from 7 of 7 PKD males, 1 of 7 PKD females, 3 of 10 normal males, and 1 of 10 normal females. "Nanobacterium sanguineum" was dLAL positive and cross-reactive with antichlamydial LPS and HS-Bh. Some cyst fluids were also positive for LPS antigens from Escherichia coli, Bacteroides fragilis and/or Chlamydia, and HS-Bh, as were liver cyst fluids from one patient. Tetracycline and citrate inhibited nanobacterial growth in vitro. CONCLUSION: Nanobacteria or its antigens were present in PKD kidney, liver, and urine. The identification of candidate microbial pathogens is the first step in ascertaining their contribution, if any, to human disease.

Phenotypic mapping of human mesothelial cells.

In recent years it has become clear that the mesothelium plays a prominent homeostatic role in the peritoneum, and can be profoundly altered in disease and during peritoneal dialysis. The cell-surface phenotype of the mesothelial cell has not been thoroughly investigated. This study begins to identify cell surface molecules which may be important in mesothelial functions such as adhesion and interaction with cells of the immune system. The expression of adhesion structures on mesothelial cells such as CD44, the beta integrin chain CD29, the beta 3 integrin chain CD61 and alpha chains CD49 alpha (alpha 1), CD49b (alpha 2), CD49c (alpha 3), CD49e (alpha 5), and CD51 (alpha v) is described. In addition, a wide range of novel molecules including CD90, CD105, CD140b, CD142, CD147, CD151, CD157, CD165, and CD166 are identified. The role and function of such molecules in mesothelial biology and their significance for peritoneal dialysis is discussed.

Sphingosine and sphinganine levels in human mesothelial cells in vitro as a potential index of signal transduction pathways impacted by microbes and osmolality.

Sphingolipids are emerging as important regulators of mammalian cell biology. In this study, the contents of six separate preparations of human omental mesothelial cells in vitro were examined for free sphingosine and sphinganine, and for the total levels of these sphingoid bases in ceramide-containing sphingolipids. Two high-performance liquid chromatography (HPLC) methods for determination of sphingoid base levels in cultured cells were compared. The rapid-HPLC method was found to yield the highest recovery of internal standard. Mesothelial cells initially isolated by collagenase digestion of the omentum were found to have higher free- and total-sphingoid base levels than cells isolated by trypsin-EDTA digestion. Use of sphingoid base levels to gain insights into the status of cellular nutrition, inflammation, programmed cell death, exposure to microbial toxins, cytokines, and growth factors within the peritoneum will require a systematic description of sphingolipids in normal, diseased, and dialyzed mesothelium.

Polycystic kidney disease: an unrecognized emerging infectious disease?

Polycystic kidney disease (PKD) is one of the most common genetic diseases in humans. We contend that it may be an emerging infectious disease and/or microbial toxicosis in a vulnerable human subpopulation. Use of a differential activation protocol for the Limulus amebocyte lysate (LAL) assay showed bacterial endotoxin and fungal (1-->3)-beta-D-glucans in cyst fluids from human kidneys with PKD. Fatty acid analysis of cyst fluid confirmed the presence of 3-hydroxy fatty acids characteristic of endotoxin. Tissue and cyst fluid from three PKD patients were examined for fungal components. Serologic tests showed Fusarium, Aspergillus, and Candida antigens. IgE, but not IgG, reactive with Fusarium and Candida were also detected in cyst fluid. Fungal DNA was detected in kidney tissue and cyst fluid from these three PKD patients, but not in healthy human kidney tissue. We examine the intertwined nature of the actions of endotoxin and fungal components, sphingolipid biology in PKD, the structure of PKD gene products, infections, and integrity of gut function to establish a mechanistic hypothesis for microbial provocation of human cystic disease. Proof of this hypothesis will require identification of the microbes and microbial components involved and multifaceted studies of PKD cell biology.

Multiple drug classes and hyperosmolarity alter binding of muscarinic drugs to mesothelial cells in vitro.

Mesothelial cells in vitro exhibited binding sites for L-quinuclidinyl[phenyl-4-3H]-benzilate ([3H]-QNB), but not [3H]-N-methylscopolamine (NMS), a cell-impermeable ligand. [3H]-QNB binding demonstrated a biphasic pattern of binding in living cells: a maximum after 15 min at 37 degrees C was followed by a decrease out to 90 min. [3H]-QNB binding was blocked by increasing concentrations of atropine; WIN35428 and GBR12909, dopamine transport inhibitors also decreased binding. Pretreatment of cells for 18 hours with atropine, QNB, or WIN35428 resulted in enhanced [3H]-QNB binding, but coexposure to cycloheximide blocked this increase. Hyperosmolarity caused by NaCl or mannitol decreased binding of [3H]-QNB to living cells. Thus rabbit peritoneal mesothelial cells possess binding sites for [3H]-QNB that are influenced by other drugs and osmolarity.

The biology of the mesothelium during peritoneal dialysis.

Substantial derangements of mesothelial biology are observed during experimental simulations of dialysis conditions, inferred from the content of human dialysis effluent and visualized by microscopy of human mesothelial biopsies. Can osmotically active solutions be made biocompatible with the osmoregulatory system of the mesothelium? Can the contributions of the mesothelium to host defenses against inflammation and/or infection be supported during CAPD? Do underlying metabolic derangements present in various kidney diseases and end-stage renal disease, regardless of cause, require customized CAPD protocols and solutions? Use of dialysis solutions less directly toxic to the mesothelium is a necessary step toward some day manipulating peritoneal biology by pharmacological and therapeutic modalities.