High Isl1 levels disrupt β cell maturation of transdifferentiated liver cells.

<p>Adult human liver cells were treated by the direct “hierarchical” sequential infection order (C) and supplemented by <i>Ad-CMV-Isl1</i> (1 or 100MOI) at the 3rd day (C+Isl1). <b>(A)</b> Quantitative Real-Time PCR analyses for insulin gene expression levels. CT values were normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean ± SE compared to control virus treated cells. *P<0.05, n>6 in 3 independent experiments preformed in cells isolated from different donors. <b>(B)</b> Insulin (and or pro-insulin) secretion was measured by static incubation of the cells for 15 min at 2 and 17.5 mM glucose in KRB. **P<0.01, compared to the direct “hierarchical” sequential infection order (C). n>6 in 3 independent experiments preformed in cells isolated from different donors. <b>(C)</b> Quantitative Real-Time PCR analyses for the gene expression levels of SLC2A2. CT values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean ±SE compared to control virus treated cells. *P<0.05, n>6 in 3 independent experiments preformed in cells isolated from different donors.</p

Combined expression of pTFs promotes transdifferentiation efficiency, however only their sequential expression increases β-cell maturation.

<p>Cultured adult human liver cells were infected with <i>Ad-CMV-Pdx1</i> (1000 MOI), <i>Ad-CMV-Pax-4</i> (100 MOI) and <i>Ad-CMV-Mafa</i> (10 MOI) together or in a sequential manner as summarized in (A) (treatments B–E) or with control virus (<i>Ad-CMV-β-gal</i>, 1000 MOI, treatment A), and analyzed for their pancreatic differentiation six days later. (B) Immunofluorescence staining of treated human liver cells for insulin (red). Nuclei were stained with DAPI (blue), original magnification X20. The percent of insulin positive cells were calculated by counting at least 1000 positive cells from at least two independent experiments preformed in cells isolated from different donors. Insulin (and or pro-insulin) (C) and c-peptide (D) secretion were measured by static incubation of the cells for 15 min at 2 and 17.5 mM glucose in KRB. *<i>p<</i>0.05, **<i>p<</i>0.01, compared to control virus treated cells; n≥12 in 5 independent experiments preformed in cells isolated from different donors. (E) Quantitative Real-Time PCR analysis for gene expression of endogenous pTFs. CT values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean±SE of the relative expression versus control virus treated cells. *<i>p<0.05</i>, n≥8 in 4 independent experiments preformed in cells isolated from different donors. The arrow points to the specific decrease in Isl1 expression level under the C-protocol, sequential and direct hierarchical administration of pTFs.</p

Schematic representation of the proposed sequential and hierarchical control of multiple pancreatic transcription factors (pTFs) induced liver to pancreas transdifferentiation.

<p>Liver to pancreas transdifferentiation is a fast and sequential process which is temporally controlled in a hierarchical manner. Three transcription factors promote the transdifferentiation efficiency such that many more cells produce pancreatic hormones (B compared to A). However, to increase both transdifferentiation efficiency and the cells maturation along the β cell lineage (maturation means pro-insulin processing and its glucose regulated secretion), the 3 pTFs which control distinct stages of pancreas organogenesis should be sequentially supplied in a direct hierarchical mode (C).</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