Implementing the Kidney Health Initiative Surrogate Efficacy Endpoint in Patients With IgA Nephropathy (the PROTECT Trial)

There has been little progress in the developmentand regulatory approval of novel therapies forglomerular diseases. There are several reasons for thisdilemma, including safety and efficacy of tested therapies,the slowly progressive nature of glomerulardiseases, challenges with clinical trial design, and thetraditional endpoints required by regulatory agenciesfor drug labeling. This is compounded by the fact thatmost primary glomerular diseases are recognizedinternationally as rare diseases. The time required andfeasibility to conduct large-scale phase 3 clinical trialsto evaluate whether a new therapy improves kidneysurvival and decreases the development of end-stagekidney disease (ESKD) is prohibitive, particularlywhen that disease is rare. Even using doubling ofserum creatinine concentration, an accepted surrogateendpoint of ESKD, requires expensive trials withlengthy follow-up.</p

Transcriptome-Based Analysis of Kidney Gene Expression Changes Associated with Diabetes in OVE26 Mice, in the Presence and Absence of Losartan Treatment

<div><p>Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks), relative to non-diabetic control, in the presence and absence of short-term (seven-day) treatment with the angiotensin receptor blocker, losartan (n = 3–6 biological replicates per condition). We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (<i>p</i><1×10⁻⁸) with terms for oxidative stress and for endoplasmic reticulum stress/protein folding. Most of the individual gene products up- or down-regulated with diabetes were unaffected by losartan treatment; however, of the gene products dysregulated in diabetes and influenced by losartan treatment, the vast majority of changes were in the direction of amelioration rather than exacerbation of the diabetic dysregulation. This group of losartan-protected genes associated strongly with annotation terms for endoplasmic reticulum stress, heat shock proteins, and chaperone function, but not oxidative stress; therefore, the losartan-unaffected genes suggest avenues for additional therapeutic opportunity in diabetes. Interestingly, the gene product most highly upregulated by diabetes (>52-fold), encoded by the cationic amino acid transporter <i>Slc7a12</i>, and the gene product most highly downregulated by diabetes (>99%) – encoded by the “pseudogene” <i>Gm6300</i> – are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential.</p></div

“Switching” expression from Gm6300 to Slc7a12 with induction of diabetes.

<p>RNA-Seq expression data for <i>Slc7a12</i> and for the “pseudogene” <i>Gm6300</i>, mapped on the mm9 murine genome assembly. The <i>Gm6300</i> pseudogene shares homology with <i>Slc7a12</i> and is ∼125 kb upstream of <i>Slc7a12</i> on chr3 (<b>A</b>); they appear to be paralogs. <b>B</b>. RNA-Seq expression data (green) for <i>Gm6300</i> are shown for Control condition (representative sample Ot3451). Also shown are mm9 (chromosome 3) genomic coordinates, and clone AK143764 (the only known cDNA or EST matching the RefSeq <i>Gm6300</i> gene; see Results). <b>C</b>. Depicted are the expressed regions of <i>Slc7a12</i> (in green; between chr3∶14,480,680–14,508,475) from a representative tracing of RNA-Seq transcripts from diabetic mouse kidney (sample Ot3458). Also shown is the exonic structure of the canonical <i>Slc7a12</i> reference sequence (RefSeq). For <b>B</b> and <b>C</b>, note the alignment of expressed transcripts with the predicted exons. Not shown, there was essentially no <i>Slc7a12</i> expression under Control conditions, and no <i>Gm6300</i> expression under diabetic conditions (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096987#pone-0096987-g006" target="_blank">Figure 6</a>). For the canonical gene sequences AK143764 and <i>Slc7a12</i>, exons are indicated by blue boxes, introns by an intervening blue line; CDS (in contrast to UTR) is designated by the taller blue boxes. Intron-spanning real-time PCR primer pairs (two primer/probe sets per gene, designated “_1” and “_2”) are diagrammed and are used for generating data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096987#pone-0096987-g006" target="_blank">Figure 6</a>. The scale bar denotes 1 kb for <b>B</b> and <b>C</b>. Exons are labeled 1 through 6 (<b>B</b>) and 1 through 4 (<b>C</b>), based upon mapped RNA-Seq reads.</p

Diabetic changes in renal gene expression and the effect of losartan treatment.

<p><b>A</b>. Effect of losartan treatment upon diabetes-associated changes in renal gene expression. Number of changes (UPregulation and DOWNregulation) in renal gene expression with diabetes, relative to control, grouped by the impact of losartan treatment. For most diabetic gene changes, there was no effect of losartan (SAME w/LOS; gray bars). Of the diabetic gene changes affected by losartan treatment, nearly all ameliorated the diabetic change (BETTER w/LOS; white bars) whereas very few exacerbated the change (WORSE w/LOS; black bars). <b>B</b>. Effect of diabetes upon gene expression changes associated with losartan treatment. Number of changes (UPregulation and DOWNregulation) in renal gene expression with losartan-treated diabetes (w/LOS), relative to untreated diabetes, assessed based upon the effect of diabetes alone. Most of the genes regulated by losartan were unaffected by diabetes alone (gray bars). These represent potential off-target effects of losartan treatment. However, of the genes regulated positively or negatively by losartan and also up- or down-regulated in diabetes, the vast majority of the losartan-associated changes ameliorated (white DOWN bar in UP column, and black UP bar in DOWN column) rather than exacerbated the change.</p

Representative renal histological findings from control FVB mice and from untreated and treated diabetic OVE26 mice.

<p>Shown are PAS- (<b>A, C, E</b>) and trichrome- (<b>B, D, F</b>) stained sections from control FVB mice (<b>A, B</b>) from vehicle-treated diabetic OVE26 mice (<b>C, D</b>), and from losartan-treated OVE26 mice (<b>E, F</b>). In contrast to non-diabetic mice which did nor show any renal pathology, diabetic mice displayed diffuse mesangial expansion (<b>C, E; arrow</b>). Tubulointerstitial fibrosis was not detectable at this stage of DM; however, trichrome blue stain was detected in the interstitial space in OVE26 mice (e.g., <b>D, arrowhead</b>), suggesting early accumulation of extracellular matrix material.</p

<i>Summary of gene expression comparisons and corresponding functional annotations</i> (see text for details of individual analyses).

<p>A brief synopsis of the annotation findings is shown in the last column. For these analyses, dysregulation encompasses both up- and down-regulation of expression (see text).</p><p>“S-” refers to the Supplementary Table number (i<b>n <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096987#pone.0096987.s001" target="_blank">File S1</a></b>) depicting the list of genes or results of functional annotation.</p

Effect of experimental conditions upon water-balance genes.

<p>A. A module of mammalian water balance genes was affected under one or more experimental conditions in the present model. Depicted genes are instrumental in water handling in the distal nephron, or in central osmosensing. Data are represented as FPKM (i.e., expression normalized within-sample). Depending upon gene product, data are scaled to relatively low (left panel) or high (right panel) maximal expression. Several diabetes-associated changes are unaffected by losartan treatment (<i>Aqp4</i>, <i>Aqp3</i>, <i>Sgk1</i>), presumably owing to an inability of losartan to correct osmotic derangement associated with hyperglycemia. In contrast, the diabetes-associated down-regulation in renal <i>Trpv4</i> expression is partially reversed with losartan treatment. <i>P</i>-values for the depicted differences (relative to CTL or to DM) are shown in the right panel and are keyed to numbers over the significance symbols. <b>B</b> through <b>E</b>. Immunoblots of protein expression (AQP2, AQP3, AQP4, TRPV4, and SGK1) in whole-kidney lysates prepared from FVB mice (“CTL”) and from losartan-treated (“DM+Rx”) or vehicle-treated (“DM”) OVE26 diabetic mice. Densitometry data, normalized to actin expression level, are shown graphically below the immunoblots for each protein. Significance levels are as follows: *p<0.5 and †p<0.01 relative to CTL (FVB) mice; #p<0.05 and ‡p<0.01 relative to untreated OVE26 diabetic mice (DM).</p

Candidate diabetes-associated gene products in the OVE26 model.

<p>Owing to the high significance threshold arising from the multiple comparisons-informed transcriptome-wide statistical approach, a number of expected diabetes candidate genes did not achieve significance at the transcriptome level in the present model, and were not represented in the functional annotation-based analysis. Raw expression data were queried for fifteen candidate genes and tested for nominal significance (P<0.05) via <i>t</i>-test (see text). Eight of the 15 gene products – particularly members of the laminin gene family – were upregulated in the OVE26 kidneys (relative to control kidneys). <i>P</i>-values for the depicted differences (relative to CTL or to DM) are shown in the right panel and are keyed to numbers over the significance symbols. <i>S100a4</i> is also known as <i>Fsp1</i>, and <i>Ptgs2</i> (prostaglandin-endoperoxide synthase 2, or prostaglandin G/H synthase and cyclooxygenase) is also known as cyclooxygenase-2 (COX-2).</p

All genes exhibiting significantly DOWNregulated (>75%) renal expression with diabetes (OVE26) relative to control (FVB) (n = 453).

<p>All genes exhibiting significantly DOWNregulated (>75%) renal expression with diabetes (OVE26) relative to control (FVB) (n = 453).</p

Gene expression changes by condition.

<p>Shown are statistically significant changes in gene expression between pairs of experimental conditions (CTL, non-diabetic state; DM, diabetic; DM/LOS, diabetes + losartan treatment). The <b>area</b> of the circle representing each condition is proportional to the total number of significant expression changes involving that condition (e.g., 453+185+342+157 = 1137 total for CTL). The <b>width</b> (thickness) of each arrow is proportional to the number of changes represented, and the <b>direction</b> of the arrow points to the condition in which expression is greater. For example, there were 453 gene products for which renal mRNA expression was greater under control conditions than in the diabetic state (or, re-stated, 453 gene products for which expression was lower in the diabetic state than in the non-diabetic state). The smallest number of differences was seen in the DM <i>vs</i>. DM/LOS dyad and the greatest number in the CTL <i>vs.</i> DM dyad.</p