Survival study and kidney injury in 129S2/SvPasCrl and C57BL/6J mice.

<p><b>A</b>, Survival proportions of mice from both strains before sacrifice at 16 weeks. 129S2/SvPasCrl mortality was significantly increased (p = 0.045) and necropsy revealed kidney dilation due to urolithiasis. <b>B</b>, Infrared cartography of 129S2/SvPasCrl kidney slices revealed the presence of focal cystine aggregates in renal tissues (cystine tubular casts). <b>C</b>, Renal function as assessed by enzymatic serum creatinine dosage was not significantly impaired in 129S1/SvPasCrl mice (n = 6) compared to C57BL/6J mice (n = 9) at 16 weeks (p = 0.11). <b>D–F</b>, Macrophage infiltrate in kidney tissues was assessed by morphometric analyses of F4-80 immunostaining and was increased in 129S2/SvPasCrl mice in comparison with C57BL/6J (n = 5/group, p = 0.046). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">Figures 3E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">3F</a> are representative of macrophage infiltrates in C57BL/6 and 129S1/SvPasCrl mice respectively (magnification x200+ zoom). <b>G</b>, Fibrosis assessed by sirius red morphometric analysis did not evidence significant fibrosis amount in both strains (percentage of fibrotic area, p = NS).</p

129S2/SvPasCrl crystalluria and aminoaciduria are similar to those of human patients with cystinuria.

<p><b>A–B</b>, Typical flat hexagonal cystine crystals were present in 129S2/SvPasCrl fresh urine. <b>C</b>, Ninety percent of 129S2/SvPasCrl mice presented crystalluria (p = 0.0001 vs C57BL/6J mice). <b>D</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice (n = 5) and C57BL/6J mice (n = 3). Dibasic aminoacids including cysteine (reduced form of cystine) were significantly higher in 129S2/SvPasCrl mice urine (p = 0.036 for each dibasic aminoacid) whereas other aminoacid renal excretion was similar in both strains. Some representative aminoacids are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g001" target="_blank">Figure 1D</a>. Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

129S2/SvPasCrl mouse is affected by cystine urolithiasis.

<p><b>A</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 16 weeks or dead before 16 weeks have been affected by urolithiasis whereas no C57BL/6J mice has been affected, p = 0.0007, n = 10/group. <b>B</b>, Bladder stones in a 129S2/SvPasCrl mouse. <b>C</b>, Spectrum of cystine was obtained by FTIR analysis of mouse stones. <b>D–F</b>, Scanning electron microscopy of a stone, at x58, x506, and x1070 magnification respectively, revealed the typical flat hexagonal structure of cystine crystals.</p

mRNA and protein expression of cystine transporters <i>Slc3a1</i>/rBAT and <i>Slc7a9</i>/b^0,+AT in kidney cortex from 129S2/SvPasCrl and C57bBL/6J mice.

<p><b>A–B</b>, Quantitative PCR: <i>Slc3a1</i> and <i>Slc7a9</i> mRNA expression was similar in both strains. <b>C</b>, Western Blot: b^0,+AT was expressed at a similar level in kidney cortex from both strains. <b>D</b>, Western Blot: rBAT was expressed in C57BL/6 mice but not in 129S2/SvPasCrl mice. <b>E–H</b>, Antibodies directed against the extracellular part of rBAT (Figures 4E and 4G) or against its intracellular part (Figures 4F and 4H) revealed the presence of rBAT at the brush border of proximal tubular cells in C57BL/6J mice (4E and 4F) but not in 129S2/SvPasCrl mice (4G and 4H).</p

Identification of a functional missense mutation in a highly conserved sequence of the <i>Slc3a1</i> gene in 129S2/SvPasCrl mouse.

<p><b>A</b>, Mutation of G by A nucleotide at position 1232 in 129S2/SvPasCrl mouse (c.1232G>A). <b>B</b>, In position 383, the glutamine is substituted by a lysine in the extracellular part of rBAT protein in 129S2/SvPasCrl mouse. The sequence is highly conserved among mammal species. <b>C</b>, 129S1/Sv mice from the Jackson laboratory express rBAT at the brush border of proximal tubular cells. <b>D</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 10 weeks were affected by urolithiasis whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.047). <b>E</b>, One hundred percent of 129S2/SvPasCrl mice presented cystine crystals in urine whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.008). <b>F</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice and 129S2/SvPas mice (n = 5/group). Cysteine (reduced form of cystine) was significantly higher in 129S2/SvPasCrl mice urine (p = 0.008). Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

Efficacy and safety of sparsentan versus irbesartan in patients with IgA nephropathy (PROTECT): 2-year results from a randomised, active-controlled, phase 3 trial

Background Sparsentan, a novel, non-immunosuppressive, single-molecule, dual endothelin angiotensin receptor antagonist, significantly reduced proteinuria versus irbesartan, an angiotensin II receptor blocker, at 36 weeks (primary endpoint) in patients with immunoglobulin A nephropathy in the phase 3 PROTECT trial's previously reported interim analysis. Here, we report kidney function and outcomes over 110 weeks from the double-blind final analysis. Methods PROTECT, a double-blind, randomised, active-controlled, phase 3 study, was done across 134 clinical practice sites in 18 countries throughout the Americas, Asia, and Europe. Patients aged 18 years or older with biopsy-proven primary IgA nephropathy and proteinuria of at least 1·0 g per day despite maximised renin–angiotensin system inhibition for at least 12 weeks were randomly assigned (1:1) to receive sparsentan (target dose 400 mg oral sparsentan once daily) or irbesartan (target dose 300 mg oral irbesartan once daily) based on a permuted-block randomisation method. The primary endpoint was proteinuria change between treatment groups at 36 weeks. Secondary endpoints included rate of change (slope) of the estimated glomerular filtration rate (eGFR), changes in proteinuria, a composite of kidney failure (confirmed 40% eGFR reduction, end-stage kidney disease, or all-cause mortality), and safety and tolerability up to 110 weeks from randomisation. Secondary efficacy outcomes were assessed in the full analysis set and safety was assessed in the safety set, both of which were defined as all patients who were randomly assigned and received at least one dose of randomly assigned study drug. This trial is registered with ClinicalTrials.gov, NCT03762850. Findings Between Dec 20, 2018, and May 26, 2021, 203 patients were randomly assigned to the sparsentan group and 203 to the irbesartan group. One patient from each group did not receive the study drug and was excluded from the efficacy and safety analyses (282 [70%] of 404 included patients were male and 272 [67%] were White) . Patients in the sparsentan group had a slower rate of eGFR decline than those in the irbesartan group. eGFR chronic 2-year slope (weeks 6–110) was −2·7 mL/min per 1·73 m2 per year versus −3·8 mL/min per 1·73 m2 per year (difference 1·1 mL/min per 1·73 m2 per year, 95% CI 0·1 to 2·1; p=0·037); total 2-year slope (day 1–week 110) was −2·9 mL/min per 1·73 m2 per year versus −3·9 mL/min per 1·73 m2 per year (difference 1·0 mL/min per 1·73 m2 per year, 95% CI −0·03 to 1·94; p=0·058). The significant reduction in proteinuria at 36 weeks with sparsentan was maintained throughout the study period; at 110 weeks, proteinuria, as determined by the change from baseline in urine protein-to-creatinine ratio, was 40% lower in the sparsentan group than in the irbesartan group (−42·8%, 95% CI −49·8 to −35·0, with sparsentan versus −4·4%, −15·8 to 8·7, with irbesartan; geometric least-squares mean ratio 0·60, 95% CI 0·50 to 0·72). The composite kidney failure endpoint was reached by 18 (9%) of 202 patients in the sparsentan group versus 26 (13%) of 202 patients in the irbesartan group (relative risk 0·7, 95% CI 0·4 to 1·2). Treatment-emergent adverse events were well balanced between sparsentan and irbesartan, with no new safety signals. Interpretation Over 110 weeks, treatment with sparsentan versus maximally titrated irbesartan in patients with IgA nephropathy resulted in significant reductions in proteinuria and preservation of kidney function.</p