Lieske, John C.

Biologic nanoparticles (NPs) ‐ protein/mineral complexes isolatable from human calcified, but not uncalcified arteries and from plasma ‐ propagate and accumulate calcium phosphate when in cell culture conditions. IV injection of NPs into animals results in vascular calcification at sites of endothelial injury. Our aim was to determine if NPs promote mineralization by localizing to vascular smooth muscle cells (VSMCs), then sequestering calcium from their environment. Porcine VSMCs were cultured in 24‐well plates to sub‐confluency (day 0). 45 Ca (1 μCi/well) to label deposited mineral was added to media containing vehicle (control), NPs (10 or 100 nephelometric turbidity units; NTU; average diameter 210 nm), or 2 mM Pi (positive‐control). After 15 days, VSMCs formed multicellular nodules. VSMCs in control media were negative for calcification by von Kossa staining; whereas micron‐sized to smaller particles stained positive for calcium in nodules from NP‐treated and Pi‐treated cells. Cells treated identically were demineralized with 0.6N HCl and the liberated 45 Ca was quantified by scintillation counting. CPM/well (x10 3 ) were 1.5 in controls and 4.6, 28.2 and 64.7 in cells treated with 10 NTU NPs, Pi, and 100 NTU NPs, respectively. NPs localize to VSMCs in vitro and promote mineralization through sequestration of Ca from the environment. (Supported by NIH grant HL88988, and Mayo Clinic).

Background Kidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl − for oxalate (Ox). Thiosulfate (S 2 O 3 ) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S 2 O 3 and TA on crystal formation. Methods S 2 O 3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis ‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S 2 O 3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT. Results dPrestin transports S 2 O 3 but not TA, yet both inhibited Ox transport (K i (S 2 O 3 )~10mM; K i (TA)~100μM). S 2 O 3 competitively inhibits while TA blocks dPrestin. MT treated with S 2 O 3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S 2 O 3 or TA also showed ↓ CaOx in MTs. Conclusions S 2 O 3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[ DK83007, DK92408]

Calcifying nanoparticles (NPs) have been implicated in many diseases including kidney stones and vascular calcification. The chemical composition of these NPs is unknown and advances in understanding their contribution to disease etiology has been hampered by the lack of specific antibodies. Therefore, a monoclonal antibody (MoAb) was developed using decalcified NPs isolated from a human kidney stone as an antigen. After clone screening, a monoclonal antibody was produced that recognized an approximately 125kDa band in the kidney stone NP isolate. Immunoprecipitation of NP lysate followed by proteomics was used to further characterize the protein(s) recognized by the antibody. Consecutive tissue sections from a human atherosclerotic plaque were also stained with the MoAb, which recognized punctuate structures at the media/plaque border and around cholesterol clefts. The staining pattern was similar to that observed with a commercially available antibody in our previous studies. Therefore, we have developed a monoclonal antibody against human kidney stone‐derived NPs that recognizes structures in human atherosclerotic plaques. This MoAB has great potential for use in NP detection in other diverse human tissues and in experimental protocols. Supported by NIH HL88988 and Division of Experimental Surgery, Mayo Clinic.

Abstract Background and Aims Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder characterized by hepatic overproduction of oxalate. Excretion of oxalate via the kidneys leads to recurrent kidney stones, nephrocalcinosis, progressive kidney disease, and multiorgan damage from systemic oxalosis. Lumasiran, a subcutaneously administered RNAi therapeutic indicated for the treatment of PH1 in all age groups, has multiple ongoing phase 3 studies including ILLUMINATE-A, which enrolled 39 patients ≥6 years of age with eGFR ≥30 mL/min/1.73m2, and ILLUMINATE-B, which enrolled 18 patients <6 years of age with eGFR >45 mL/min/1.73m2 if ≥12 months of age or normal serum creatinine if <12 months of age. Here we present a comparison of the efficacy and safety of lumasiran in children versus adults with PH1 using pooled data from these two phase 3 studies of lumasiran. Method Efficacy and safety data from ILLUMINATE-A and ILLUMINATE-B, including urinary oxalate, plasma oxalate, eGFR, and adverse events were pooled and assessed by age <18 (N=40), or ≥18 years old (N=17). The analysis included all available data from 57 patients with PH1, ages 4 months to 60 years, who completed the initial 6 months of treatment with lumasiran. Results During the initial 6 months of treatment with lumasiran, patients with PH1 had a rapid and sustained decrease in urinary oxalate. The overall mean (SEM) percent reduction in urinary oxalate:creatinine ratios as measured in random spot urine samples from baseline to Month 6 was 63.1% (2.6) across all ages (N=54). A similar time course and magnitude of reduction was seen in patients <18 years at 64.5% (3.3) (N=38), and ≥18 years old at 59.8% (4.4) (N=16). The oxalate reductions observed in random spot urine samples were comparable to those observed from 24-hour urine collections. The overall mean (SEM) percent reduction in urinary oxalate excretion from 24-hour collections was 63.8% (2.6) across all ages (N=40), 63.2% (3.5) in the <18 group (N=23) and 64.8% (3.9) in the ≥18 group (N=17). The overall mean (SEM) percent reduction in plasma oxalate from baseline to Month 6 was 39.5% (3.7) across all ages (N=44), with similar reductions of 39.4% (4.6) and 39.7% (6.5) in patients <18 (N=30) and ≥18 (N=14) years of age, respectively. eGFR was calculated for all patients ≥12 months of age and remained stable in both age groups during the 6 months of treatment with lumasiran. The 57 patients had a cumulative exposure of 27.1 patient-years and 227 doses were given. Adverse events were reported in 86% of all patients, 88% of patients <18, and 82% of patients ≥18 years of age. All adverse events were graded as mild or moderate in severity by the investigator. One patient had a serious adverse event of viral infection that was not related to lumasiran. In all patients, the most common adverse events related to lumasiran were mild, transient, injection site reactions, experienced by 30% of all patients, 23% of patients <18 and 47% of patients ≥18 years of age. No treatment interruptions or discontinuations related to lumasiran or deaths occurred. Conclusion Lumasiran reduced urinary and plasma oxalate to a similar degree in pediatric and adult patients with PH1 enrolled in the Phase 3 studies ILLUMINATE-A and ILLUMINATE-B. Reductions in urinary oxalate were similar between random spot urine samples and valid 24-hour urine collections. Overall safety was comparable between pediatric and adult patients.

Abstract Background and Aims Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder characterized by hepatic oxalate overproduction. Excess oxalate is excreted by the kidneys, leading to recurrent kidney stones, nephrocalcinosis, and progressive kidney disease. Patients with PH1 generally experience a slow decline in kidney function over time that can be punctuated by abrupt worsening precipitated by infection, obstructing stones, or dehydration. Approximately half of patients with PH1 progress to kidney failure by early adulthood, and nearly all by 60 years of age. Lumasiran is an RNAi therapeutic indicated for the treatment of PH1 in all age groups. In clinical trials, treatment with lumasiran resulted in substantial reductions in urinary and plasma oxalate in pediatric and adult patients, with an acceptable safety profile. This analysis evaluates the change in kidney function of patients with PH1 with an eGFR of ≥30 mL/min/1.73m2 enrolled in clinical trials of lumasiran. Method We analyzed kidney function from 75 patients with PH1, age ≥12 months old, eGFR ≥30 mL/min/1.73m2, enrolled in 3 clinical trials of lumasiran (Phase 2 open-label extension, and Phase 3 ILLUMINATE-A and ILLUMINATE-B). In these trials, eGFR was calculated with the Modification of Diet in Renal Disease (MDRD) Study equation in adults or the bedside Schwartz equation in children. The effect of lumasiran on eGFR was assessed by baseline eGFR subgroup: ≥90, <90, 60 to <90, 45 to <60, and 30 to <45 mL/min/1.73m2. Results Of 75 patients available for analysis, 46 were treated with lumasiran from the start of the study through the Month 12 visit. eGFR remained stable in all eGFR subgroups. Patients with eGFR ≥90 mL/min/1.73m2 at baseline (N=16) demonstrated fluctuations in mean values by timepoint, with a mean change (95% CI) from baseline of -1 (-8, 6) mL/min/1.73m2 at Month 12. In patients with eGFR <90 mL/min/1.73m2 (N=30), no change in mean eGFR from baseline was observed at Month 12; the mean change (95% CI) was 0 (-3, 3). When evaluating subgroups with impaired kidney function, variations in the mean change were observed at Month 12 due to small sample sizes. For patients with eGFR 60 to <90 mL/min/1.73m2 (N=22), the mean change (95% CI) was -2 (-5, 1). For patients with eGFR 45 to <60 mL/min/1.73m2 (N=5), the mean change (95% CI) was 3 (-8, 14). For patients with eGFR 30 to <45 mL/min/1.73m2 (N=3), the mean change (95% CI) was 9 (7, 11). Conclusion Patients with PH1 had stable kidney function over time with lumasiran treatment, regardless of kidney function at baseline. Given the progressive kidney function decline that is characteristic of PH1, the eGFR stability observed during 12 months of treatment with lumasiran is encouraging. Kidney function will continue to be monitored for the duration of the lumasiran clinical trials.

Background and objectives In the rare disease primary hyperoxaluria type 1, overproduction of oxalate by the liver causes kidney stones, nephrocalcinosis, kidney failure, and systemic oxalosis. Lumasiran, an RNA interference therapeutic, suppresses glycolate oxidase, reducing hepatic oxalate production. The objective of this first-in-human, randomized, placebo-controlled trial was to evaluate the safety, pharmacokinetic, and pharmacodynamic profiles of lumasiran in healthy participants and patients with primary hyperoxaluria type 1. Design, setting, participants, & measurements This phase 1/2 study was conducted in two parts. In part A, healthy adults randomized 3:1 received a single subcutaneous dose of lumasiran or placebo in ascending dose groups (0.3–6 mg/kg). In part B, patients with primary hyperoxaluria type 1 randomized 3:1 received up to three doses of lumasiran or placebo in cohorts of 1 or 3 mg/kg monthly or 3 mg/kg quarterly. Patients initially assigned to placebo crossed over to lumasiran on day 85. The primary outcome was incidence of adverse events. Secondary outcomes included pharmacokinetic and pharmacodynamic parameters, including measures of oxalate in patients with primary hyperoxaluria type 1. Data were analyzed using descriptive statistics. Results Thirty-two healthy participants and 20 adult and pediatric patients with primary hyperoxaluria type 1 were enrolled. Lumasiran had an acceptable safety profile, with no serious adverse events or study discontinuations attributed to treatment. In part A, increases in mean plasma glycolate concentration, a measure of target engagement, were observed in healthy participants. In part B, patients with primary hyperoxaluria type 1 had a mean maximal reduction from baseline of 75% across dosing cohorts in 24-hour urinary oxalate excretion. All patients achieved urinary oxalate levels ≤1.5 times the upper limit of normal. Conclusions Lumasiran had an acceptable safety profile and reduced urinary oxalate excretion in all patients with primary hyperoxaluria type 1 to near-normal levels. Clinical Trial registry name and registration number: Study of Lumasiran in Healthy Adults and Patients with Primary Hyperoxaluria Type 1, NCT02706886

Abstract Background Primary hyperoxaluria (PH) type 3 (PH3) is caused by mutations in the hydroxy-oxo-glutarate aldolase 1 gene. PH3 patients often present with recurrent urinary stone disease in the first decade of life, but prior reports suggested PH3 may have a milder phenotype in adults. This study characterized clinical manifestations of PH3 across the decades of life in comparison with PH1 and PH2. Methods Clinical information was obtained from the Rare Kidney Stone Consortium PH Registry (PH1, n = 384; PH2, n = 51; PH3, n = 62). Results PH3 patients presented with symptoms at a median of 2.7 years old compared with PH1 (4.9 years) and PH2 (5.7 years) (P = 0.14). Nephrocalcinosis was present at diagnosis in 4 (7%) PH3 patients, while 55 (89%) had stones. Median urine oxalate excretion was lowest in PH3 patients compared with PH1 and PH2 (1.1 versus 1.6 and 1.5 mmol/day/1.73 m2, respectively, P < 0.001) while urine calcium was highest in PH3 (112 versus 51 and 98 mg/day/1.73 m2 in PH1 and PH2, respectively, P < 0.001). Stone events per decade of life were similar across the age span and the three PH types. At 40 years of age, 97% of PH3 patients had not progressed to end-stage kidney disease compared with 36% PH1 and 66% PH2 patients. Conclusions Patients with all forms of PH experience lifelong stone events, often beginning in childhood. Kidney failure is common in PH1 but rare in PH3. Longer-term follow-up of larger cohorts will be important for a more complete understanding of the PH3 phenotype.