Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Hypoxia Inducible Factor 1-Alpha (HIF-1 Alpha) Is Induced during Reperfusion after Renal Ischemia and Is Critical for Proximal Tubule Cell Survival

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant

HIF-1α is transcriptionaly active <i>in vivo</i> promoting gene expression.

<p>PHD3, VEGF and EPO mRNAs levels were determined by qRT-PCR in total renal tissue lysates and 28 mRNA levels were used as internal control. Statistical significance was found in comparison to sham condition.</p

HIF-1α is required for proximal epithelial cell survival in response to oxygen and nutrient alterations.

<p>Quantification of cell death, by propidium iodide staining and flow cytometry in HK2 cells subjected to H/R and D/R, (a) previously transfected with specific HIF-1α siRNA (100 nM) and scramble (100 nM) or (b) treated with 10 mM YC-1 added during hypoxia (hyp) or during reoxygenation (reox). Statistical significance was found in comparison to scramble or control respectively, p≤0.05. (c) 100 nM of siRNA (sc-44225) efficiently prevents both HIF-1α inductions (hypoxia and R-3h). (d) 10 mM YC-1 inhibits HIF-1α inductions when added during hypoxia and reoxygenation or (e) added separately during hypoxia (+h) or reoxygenation (+r). Representative blots are shown and actin was used as control. (f) Akt activation in HK-2 cells subjected to H/R protocol, HK-2 cells treated with 10 mM YC-1 or 100 nM siRNA for HIF-1α, all estimated by western blot of 473Ser phosphorylation.</p

HIF-1α interference <i>in vivo</i>.

<p>(a) Scheme of siRNA and scramble treatments <i>in vivo.</i> SD Rats were injected with 100 nM of specific siRNA against HIF-1α or scramble through the tail vein at indicated times. (b) Percentage of HIF-1α interference estimated by qRT-PCR in total renal lysates from rats treated with HIF-1 α siRNA in comparison to rats treated with scramble in each condition.</p

HIF-1 α expression in an <i>in vitro</i> model mimicking I/R.

<p><b>Akt/mTOR signaling mediates HIF-1 α induction during reoxygenation.</b> (a) Scheme of <i>in vitro</i> protocol of Hypoxia/Reoxygenation (H/R) HK-2 cells were subjected to H/R protocol which includes oxygen and nutrients deprivation and replenishment. (b) HIF-1α protein expression was determined by western blot. Actin expression was used as loading control. Representative western blot are shown. (c) qRT-PCR analysis of HIF-1 α mRNA, expressed as mean ±SEM of HIF-1α levels using b-actin mRNA as internal control. No significant alterations on mRNA levels of HIF-1 α during H/R were found in four different experiments. (d) Activation of Akt estimated by western blot of 473Ser phosporylation in HK2 cells subjected to H/R. Total Akt expression was used as control. (e) Effect of LY294002 on HIF-1 α inductions: 50 mM LY294002 applied during hypoxia (+h) did not have any effect but during reoxygenation (+r) it reduced HIF-1α expression. Representative western blots are shown. (f) Control of Akt inhibition by LY294002. 50 mM of LY294002 efficiently inhibits Akt phosphorylation when added during reoxygenation (+r). (g) Effect on the HIF-1α inductions of 20 nM rapamycin applied during reoxygenation, estimated by western blot. Phosphorylation of p70S6K was used as control of rapamycin efficiency.</p

HIF-1 α is induced unexpectedly during reperfusion in rat kidney, with normal oxygen levels in renal parenchyma.

<p>(a) Immunohistochemitry to determine HIF-1 α expression in paraffin-embedded renal tissue sections from SD rats during I/R. Ischemia of 45 min and different times of reperfusion: 24 hours or 3, 5 or 7 days (R-24h, R-3d, R-5d, R-7d). HIF-1 α is detected in the nucleus of proximal tubule cells after ischemia and in reperfusion (3-5-7days). Magnification: ×400 (b) Immunostaining for pimonidazol-protein and HIF-1α adducts in renal tissue sections of rats during I/R. Ischemia of 45 min and 5 days of reperfusion. Notice positive pimonidazole immunostaining exclusively after ischemia. Magnification: ×200.</p

Primer sequences used for qRT-PCR <i>in vivo</i> and <i>in vitro.</i>

<p>Primer sequences used for qRT-PCR <i>in vivo</i> and <i>in vitro.</i></p

Human postransplant Renal Biopsies features.

<p>ATN: acute tubular necrosis estimation; CyA: cyclosporine A; MMF: mophetil-mycophenolate. Biopsies which representative images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033258#pone-0033258-g009" target="_blank">figure 9</a> are marked with asterisks.</p