Renal Calcium Oxalate Deposits Induce a Pro-Atherosclerotic and Pro-Osteoporotic Response in Mice.

Urinary stone disease (USD) is increasing in adult and pediatric populations. Adult and pediatric studies have demonstrated decreased bone mineral density and increased fracture rates. USD has also been independently linked to increased rates of myocardial infarction and cerebral vascular accidents. Although USD is a multisystem disorder involving the kidneys, bone, and vasculature, the molecular mechanisms linking these three organs remain unknown. Calcium oxalate nephropathy was induced in C57BL/6J mice with intra-peritoneal (ip) injection of sodium glyoxolate. Half of each kidney underwent Pizzalato staining and half was snap frozen for RNA extraction. RT2 Profiler Mouse Atherosclerosis, Osteoporosis, and Calcium Signaling PCR Arrays (Qiagen) were performed. Only results that passed quality checks in PCR array reproducibility and genomic DNA contamination were included. Genes had to show at least fourfold differential expression and P < 0.01 to be considered significant. Atherosclerosis array showed upregulation of 19 genes by fourfold, 10 of which were ≥10-fold. All 19 had P ≤ 0.002. The Osteoporosis array showed fourfold upregulation of 10 genes, five showed >10-fold increase. All 10 have P ≤ 0.003. The calcium signaling array showed significant fourfold upregulation of 10 genes, four of which were ≥10-fold. All 10 have P ≤ 0.03. We have demonstrated that calcium oxalate nephropathy can induce upregulation of atherosclerotic, metabolic bone, and calcium homeostasis genes in a murine model. This may be and initial step in identifying the molecular mechanisms linking stone, bone, and cardiovascular disease. J. Cell. Biochem. 118: 2744-2751, 2017. © 2017 Wiley Periodicals, Inc.

Polymorphisms in α-Defensin-Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux.

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

The Interaction between Enterobacteriaceae and Calcium Oxalate Deposits.

BACKGROUND: The role of calcium oxalate crystals and deposits in UTI pathogenesis has not been established. The objectives of this study were to identify bacteria present in pediatric urolithiasis and, using in vitro and in vivo models, to determine the relevance of calcium oxalate deposits during experimental pyelonephritis. METHODS: Pediatric kidney stones and urine were collected and both cultured and sequenced for bacteria. Bacterial adhesion to calcium oxalate was compared. Murine kidney calcium oxalate deposits were induced by intraperitoneal glyoxalate injection and kidneys were transurethrally inoculated with uropathogenic Escherichia coli to induce pyelonephritis. RESULTS: E. coli of the family Enterobacteriaceae was identified in patients by calcium oxalate stone culture. Additionally Enterobacteriaceae DNA was sequenced from multiple calcium oxalate kidney stones. E. coli selectively aggregated on and around calcium oxalate monohydrate crystals. Mice inoculated with glyoxalate and uropathogenic E. coli had higher bacterial burdens, increased kidney calcium oxalate deposits and an increased kidney innate immune response compared to mice with only calcium oxalate deposits or only pyelonephritis. CONCLUSIONS: In a murine model, the presence of calcium oxalate deposits increases pyelonephritis risk, likely due to preferential aggregation of bacteria on and around calcium oxalate crystals. When both calcium oxalate deposits and uropathogenic bacteria were present, calcium oxalate deposit number increased along with renal gene transcription of inner stone core matrix proteins increased. Therefore renal calcium oxalate deposits may be a modifiable risk factor for infections of the kidney and urinary tract. Furthermore, bacteria may be present in calcium oxalate deposits and potentially contribute to calcium oxalate renal disease.

Pediatric Origins of Nephrolithiasis-Associated Atherosclerosis.

OBJECTIVES: To determine if nephrolithiasis-associated atherosclerosis has pediatric origins and to consider possible association between kidney stones and atherosclerosis-related proteins. STUDY DESIGN: We matched children aged 12-17 years with kidney stones and without kidney stones. Carotid artery intima-media thickness (cIMT) was measured by ultrasound. Participants' urine was investigated by enzyme-linked immunosorbent assay for the atherosclerosis-related proteins fibronectin 1, macrophage scavenger receptor 1, osteopontin, and vascular cell adhesion molecule 1 levels, and normalized to urine creatinine levels. RESULTS: Subjects with nephrolithiasis had higher cIMT in the right common carotid artery and overall mean measurement. Urine osteopontin and fibronectin 1 were significant predictors of cIMT. CONCLUSIONS: We have provided initial preliminary evidence that nephrolithiasis-associated atherosclerosis has pediatric origins and performed studies that begin to identify potential reasons for the association of nephrolithiasis and vascular disease.

Carbonic anhydrase 2 deficiency leads to increased pyelonephritis susceptibility.

Carbonic anhydrase 2 regulates acid-base homeostasis, and recent findings have indicated a correlation between cellular control of acid-base status and the innate defense of the kidney. Mice deficient in carbonic anhydrase 2 (Car2(-/-) mice) have metabolic acidosis, impaired urine acidification, and are deficient in normal intercalated cells. The objective of the present study was to evaluate the biological consequences of carbonic anhydrase 2 deficiency in a murine model of pyelonephritis. Infection susceptibility and transcription of bacterial response components in Car2(-/-) mice were compared with wild-type littermate controls. Car2(-/-) mice had increased kidney bacterial burdens along with decreased renal bacterial clearance after inoculation compared with wild-type mice. Standardization of the urine pH and serum HCO(3)(-) levels did not substantially alter kidney infection susceptibility between wild-type and Car2(-/-) mice; thus, factors other than acid-base status are responsible. Car2(-/-) mice had significantly increased neutrophil-gelatinase-associated lipocalin mRNA and protein and expression at baseline and a marked decreased ability to upregulate key bacterial response genes during pyelonephritis. Our findings provide in vivo evidence that supports a role for carbonic anhydrase 2 and intercalated cells in promoting renal bacterial clearance. Decreased carbonic anhydrase expression results in increased antimicrobial peptide production by cells other than renal intercalated cells, which is not sufficient to prevent infection after a bacterial challenge.