Alternatively Spliced Landscape of PPARγ mRNA in Podocytes Is Distinct from Adipose Tissue

Figure 1. Alternatively Spliced Variants and Isoforms of PPARγ in Podocytes. (A) The schematic depicts the exon and intron usage and the transcription and translation start and stop sites for various PPARγ variants. The major isoforms of PPARγ are isoforms γ1 and γ2, which encode for proteins containing 475 and 505 amino acids, respectively. The most common human annotated variants 1 (var1: NM_138712) and 2 (var2: NM_015869) encode isoforms γ1 and γ2 (depicted in the topmost PPARγ illustration). These two variants are transcribed by different promoter usage by RNA Polymerase II (depicted in tall arrows) and alternative splicing (depicted by exon skipping). PPARγ1 variant 1 includes exons A1 and A2, a start codon (ATG) on exon 1, and γ2 variant 2 excludes exons A1 and A2, but includes exon B and a start codon in the same exon B. Thus, γ1 protein product is 30 amino acids shorter than γ2. The less known and understood variants are represented below, which include Variants 3, 4, 11, 9 and tORF4/Var 10. These are depicted with A1 and A2 usage in the schematic to emphasize those likely to be expressed in podocytes, although their adipocyte counterparts with exon B usage at the 5’ UTR also…

Figure 2. Expression of PPARγ Splice Variants in Podocytes vs. Adipose Tissue. (A) Total RNA was isolated from glomeruli, liver, and WAT samples harvested from rats, and from cultured differentiated human podocytes. Expression of the RNA variants encoding PPARγ1 and γ2 (All variants), γ1 only (Var1), and γ2 only (Var 2) isoforms was measured by RT-PCR in isolated samples as well as commercially available human and rat whole kidney RNA. Rat total (all) γ [primer pair ex1 F, ex 2 R; 315 bp], rat γ1 [primer pair exA2 F, ex2 R; 380 bp], rat γ2 [primer pair exB F, ex2 R; 329 bp], rat Rpl6 [178 bp], human total (all) γ [primer pair Ex1 F, aR; 315 bp], human γ1 [primer pair cF, aR; 387 bp], human γ2 [primer pair bF, aR; 476 bp], Human RPL6 [370 bp]. (B) Several PPARγ mRNA AS variants were analyzed by RT-PCR in cultured differentiated human podocytes and commercially available human whole kidney RNA using the primer pairs drawn in Figure 1A and detailed in Supplementary Table S1. Var1 [primer pair cF, aR; 387 bp], Var3 [primer pair dF, aR; 415 bp], Var4 [primer pair eF, aR; 333 bp], Var11 [primer pair fF, gR; 242 bp], Var9 [primer pair hF, iR; 254 bp], tORF4 [primer pair hF, jR; 490 bp], Total…

Figure 3. PPARγ-Response Element (PPRE) Containing Genes in Podocytes vs. Adipocytes. (A) Normalized average log counts of untreated control human differentiated podocyte and adipocyte GEO datasets (GSE124622 and GSE129153, respectively) were cross-referenced against the verified target and predicted PPRE genes from the PPARgene database. (i) 1669 PPRE-containing genes were detectable in both podocyte and adipocyte datasets, 453 unique to podocytes and 77 unique to adipocytes. (ii) PPRE-containing genes classified by their confidence level. High-confidence (p > 0.8), median-confidence (0.8 ≥ p > 0.6), low-confidence category (0.6 ≥ p > 0.45). Genes with p value < 0.45 were predicted as negative. (B) Total RNA was isolated from cultured differentiated human podocytes and WAT harvested from rats. Expression of the podocyte marker genes (NPHS1 [Hu: 234 bp, Rat: 138 bp], SYNPO [Hu: 182 bp, Rat: 325 bp], and WT1 [Hu/Rat: 134 bp]) and adipose tissue genes (CD36 [Hu: 304 bp, Rat: 123 bp], ADIPOQ [Hu: 347 bp, Rat: 95 bp], ADIPSIN [Hu: 301 bp, Rat: 202 bp], and AP2 [Hu: 133 bp, Rat: 133 bp]), and housekeeping gene RPL6 [Hu: 370 bp, Rat: 178 bp] were analyzed. (i) Representative gels depicting the…

Figure 4. Illustration Describing the Roles of PPARγ1 and 2 Isoforms. Schematic of the suggested roles of PPARγ 1 and 2 forms in regulating podocyte/glomerular disease specific effects (right panel) vs. metabolic/systemic and adipogenic effects (left panel). PPARγ agonism plays a well-established role in the setting of treatment of Type II Diabetes, and treatment with its agonists, thiazolidinediones (TZDs), leads to increased (‘a’ pathway) insulin sensitivity and glycemic control [53,54]. However, these beneficial effects are also accompanied with adverse effects (‘a’ pathway), such as increased cholesterol, adipogenesis and decreased osteoblastogenesis [25]. TZDs or PPARγ agonists have now been demonstrated to reduce podocyte injury (‘b’ pathway) by enhancing their viability and cytoskeletal integrity and decreasing apoptosis [1,2,3,4,5]. A few pathways involved in this process include crosstalk with the glucocorticoid receptor (GR), decreased caspase-3 activity and decreased TRPC6 expression. Gain of PPARγ activity or its activation by TZDs (‘c’ pathway) has been shown to reduce proteinuria and glomerulosclerosis in various animal models of glomerular disease, such as 5/6 nephrectomy…