Fig 2: HG-induced chromatin remodelling is reversed by miR-93 overexpression.(a) Experimental approach employed for DNase-Seq. (b) Hierarchical clustering analysis of ΔDHS scores for NG, HG+ NT mimic, and HG+ miR-93 mimics. Dashed red lines surround regions that demonstrate a reversal in HG-induced changes in hypersensitivity, following miR-93 overexpression. P value <0.05 for all comparisons. (c–g) Examples of chromatin accessibility that highlight differences in DNase hypersensitivity at the (c) Ctgf, (d) Fn1, (e) Serpine1 (f) Rock1 and (g) Wt1 loci. As reference, DHS signal tracks from the Encode project are depicted in pink24. Each data track shows tag density from DNase-Seq assays from podocytes treated with NG, HG+NT mimics or HG+miR-93 mimics (h) Gene expression analysis of the aforementioned genes. All values normalized to internal Gapdh controls. (i) Volcano plot highlighting differentially regulated genes by miR-93. (j) mRNA tag density at exons 13–18 and 3′-UTR of murine Msk2 in podocytes transfected with miR-93 mimics or NT controls. (k) MSK2 expression values from patients with DN obtained from publicly available datasets from Nephroseq (nephroseq.org)19. Data expressed as log2 median-centered intensity values. (l) Linear regression analysis of relative Msk2 mRNA levels from patients in (k) correlated with estimated glomerular filtration rate values obtained from Nephroseq. (m) Top, western blot and densitometric quantification of total Msk2 expression levels in podocytes isolated from 24-week-old db/m and db/db mice, (n=5 mice/group). (n) Top, western blot of total Msk2 expression levels from whole cell lysates in podocytes cultured under NG or HG conditions. Bottom, immunofluorescence micrographs from podocytes cultured in NG or HG conditions. Scale bar, 10 μm. (o) Quantification of nuclear fluorescent intensity of Msk2 expression in podocytes cultured under NG or HG conditions. (p) Evolutionary conservation of the miR-93 seed site within the 3′-UTR of Msk2. Mmu: Mus musculus, Hsa: Homo sapiens, Ptr: Pan troglodytes, Mml: Macaca multta, Str: Spermophilus tridecemlineatus, Rno: Rattus norvegicus, Ssc: Sus scrofa, Bta: Bos taurus. Data expressed as mean±s.e.m. ns: no significance, *P<0.05, **P<0.01, ***P<0.001. Student's t-test was employed for comparisons between two groups, one-way analysis of variance with Tukey's post test for multiple comparisons was used for groups of three or more.

Fig 3: Targeting Msk2 in vivo prevents progression of DN.(a) Left, representative western blot analysis for Msk2 levels in podocytes isolated from non-diabetic (db/m) (n=4), db/db+shCtl (n=3), and db/db+shMsk2 (n=5) mice treated with shMsk2. Right, densitometric quantification of western blots to quantify Msk2 levels. (b) ACR of non-diabetic (db/m), db/db+shCtl (n=3) and db/db+shMsk2 (n=5). (c) Representative images of TEM micrographs, PAS staining and WT1 staining from shMsk2-treated mice. Scale bars, 0.5 μM (first column) or 50 μM (second and third columns). (d–h) Quantification of (d) mesangial matrix expansion, (e) WT1+ cells, (f) GBM thickness, (g) Foot process density and (h) Foot process width in shMsk2-treated mice and controls. (i) Representative immunofluorescence micrographs of kidney sections from shMsk2-treated mice and controls from a, stained with anti-synaptopodin and anti-H3S10P. Sections were counterstained with DAPI. Merged images are meant to highlight podocyte-specific staining and insets are meant to highlight podocyte-specific localization of H3S10P. Scale bars, 50 μM. (j) qPCR analysis of podocyte-specific genes, Wt1, podocin and nephrin from podocytes isolated from shMsk2-treated mice and controls. All expression values were normalized to Gapdh internal controls. (k) Schematic describing mode of action and general design of LNA-modified long RNA gapmers (l) Top, western blot analysis of podocytes transfected with Msk2 or Scramble, negative control gapmers with antibodies directed against Msk2, Msk1 and actin. Bottom, study design used for the administration of Msk2 or Scramble, negative control gapmers in db/db mice. (m) Western blot analysis of kidney cortices from db/m and db/db mice administered gapmers directed against Msk2 or Scramble, negative control gapmers, all values normalized to actin (n=5 mice/group). (n) ACR of non-diabetic (db/m), db/db mice administered gapmers directed against Msk2 or Scramble, negative control gapmers (n=5 mice/group). (o) ACR of 24-week-old db/m;Msk2+/+ (n=3), db/m;Msk2−/− (n=5), db/db;Msk2+/+ (n=6), and db/db;Msk2−/− (n=6) mice. Data expressed as mean±s.e.m. ns: no significance, *P<0.05, **P<0.01, ***P<0.001. One-way analysis of variance with Tukey's post test for multiple comparisons was used for groups of three or more.