A roadmap for the genetic analysis of renal aging

Several studies show evidence for the genetic basis of renal disease, which renders some individuals more prone than others to accelerated renal aging. Studying the genetics of renal aging can help us to identify genes involved in this process and to unravel the underlying pathways. First, this opinion article will give an overview of the phenotypes that can be observed in age-related kidney disease. Accurate phenotyping is essential in performing genetic analysis. For kidney aging, this could include both functional and structural changes. Subsequently, this article reviews the studies that report on candidate genes associated with renal aging in humans and mice. Several loci or candidate genes have been found associated with kidney disease, but identification of the specific genetic variants involved has proven to be difficult. CUBN, UMOD, and SHROOM3 were identified by human GWAS as being associated with albuminuria, kidney function, and chronic kidney disease (CKD). These are promising examples of genes that could be involved in renal aging, and were further mechanistically evaluated in animal models. Eventually, we will provide approaches for performing genetic analysis. We should leverage the power of mouse models, as testing in humans is limited. Mouse and other animal models can be used to explain the underlying biological mechanisms of genes and loci identified by human GWAS. Furthermore, mouse models can be used to identify genetic variants associated with age-associated histological changes, of which Far2, Wisp2, and Esrrg are examples. A new outbred mouse population with high genetic diversity will facilitate the identification of genes associated with renal aging by enabling high-resolution genetic mapping while also allowing the control of environmental factors, and by enabling access to renal tissues at specific time points for histology, proteomics, and gene expression

MyD88 SIGNALING THE ROLE OF MyD88 SIGNALING IN CHRONIC DYSFUNCTION OF THE TRANSPLANTED KIDNEY

Absence of MyD88 signaling reduces chronic allograft damage Kidney transplantation is the best treatment for end-stage kidney disease, however chronic allograft dysfunction remains the major barrier to long-term allograft survival. Toll-like receptors (TLRs) play an important role in innate immunity but also provide a link between innate and adaptive immunity. MyD88 is a key TLR signal adaptor. In this study, we aimed to determine whether MyD88 signaling is required for the development of chronic kidney allograft rejection using MyD88-/- mice in a fully MHC mismatched murine model of kidney allograft rejection. Kidney transplants were performed: BALB/c (H2d) to C57BL/6 (H2b) (WT, n=20), BALB/c.MyD88-/- to C57BL/6.MyD88-/- (MyD88-/-, n=9) as allografts and C57BL/6 to C57BL/6 (isografts, n=5). Mice underwent bilateral native nephrectomy, thus survival was dependent on graft function. Survival was observed to day 100 post-transplant and samples were harvested at day 100 for examination by histology, immunohistochemistry and quantitative RT-PCR. Data are expressed as mean ± SD and analysed by ANOVA. A p-value <0.05 was considered statistically significant. 12 out of 20 WT allografts were rejected with a mean graft survival of 49.5 days. In contrast, 7 out of 9 MyD88-/- allografts survived for >100 days (p<0.05). WT allografts developed kidney dysfunction with increased serum creatinine (51.1±28.0 vs. 14.4±6.0 & 13±3.9μmol/L, p<0.01) and proteinuria (0.93±0.18 vs. 0.51±0.24 & 0.43±0.07, p<0.05) as compared to MyD88-/- allografts and isografts. WT recipients developed histological evidence of chronic allograft damage which was less severe in MyD88-/- recipients, as measured by reduced interstitial fibrosis and tubular atrophy (2.0±1.0 vs. 0.23±0.1, p<0.001), glomerulosclerosis (48.5±24.4 vs. 12.3±5.5, p<0.01), αSMA expression (7.95±4.7 vs. 1.13±1.13, p<0.01), collagen accumulation (29.9±13.6 vs. 9.2±5.8, p<0.01), CD68+ cell infiltration (18.97±8.5 vs. 5.15±1.2, p<0.01) and CD11c+ infiltration (7.99±4.93 vs. 2.38±1.54, p<0.05). Compared to WT allografts, MyD88-/- allografts expressed less TH1 cytokine: IFN(p<0.01), pro-inflammatory cytokines: TNFα, IL-1β and IL-6 (p<0.05), chemokines: MCP1 and Mig (p<0.05) and fibrosis-related genes: TGFβ, TIMP1 and MMP2 (p<0.05). Absence of MyD88 signaling promoted kidney allograft acceptance, with reduced chronic allograft damage, in a fully MHC-mismatched murine model of kidney allograft rejection.

Genetic analysis of mesangial matrix expansion in aging mice and identification of Far2 as a candidate gene

Aging of the kidney is associated with renal damage, in particular mesangial matrix expansion (MME). Identifying the genes involved in this process will help to unravel the mechanisms of aging and aid in the design of novel therapeutic modalities aimed at prevention and regression. In this study, structural changes in glomeruli of 24 inbred mouse strains were characterized in male mice at 6, 12, and 20 months of age. Haplotype association mapping was used to determine genetic loci associated with the presence of MME at 20 months. This analysis identified a significant association with a 200-kb haplotype block on chromosome 6 containing Far2. Sequencing revealed that mouse strains with MME contain a 9-bp sequence in the 5\u27 untranslated region of Far2 that is absent in most of the strains without MME. Real-time PCR showed a two-fold increase in the expression of Far2 in the kidneys of strains with the insert, and subsequent experiments performed in vitro with luciferase reporter vectors showed that this sequence difference causes differential expression of Far2. Overexpression of Far2 in a mouse mesangial cell line induced upregulation of platelet activating factor and the fibrotic marker TGF-β. This upregulation of MME-promoting factors may result, in part, from the FAR2-catalyzed reduction of fatty acyl-coenzyme A to fatty alcohols, which are possible precursors of platelet activating factor. Overall, these data suggest the identification of a novel pathway involved in renal aging that may yield therapeutic targets for reducing MME. J Am Soc Nephrol 2013 Dec; 24(12):1995-200

Identification of novel genes associated with renal tertiary lymphoid organ formation in aging mice.

A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10-137) in male mice; Ctnnbip1 (P = 6.42×10-267) and Tnfrsf8 (P = 5.42×10-245) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation. PLoS One 2014 Mar 17; 9(3):e91850

Genetic analysis of intracapillary glomerular lipoprotein deposits in aging mice

Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes. Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0-4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping. Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3. By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponse

Genome wide HAM results.

<p>Genome-wide scan for intracapillary glomerular deposits. A peak reaching the significant threshold of <i>P</i><10⁻⁶ is seen on Chr 1 (A). The haplotype block of six SNPs between rs48848476 and rs46020199 is associated with glomerular deposits. The region spanning 1 Mb on each site from this area includes the genes <i>Spata17, Gpatch2, Esrrg, Ush2a,</i> and <i>Kctd3</i> (B).</p

Lymphocytes in renal TLOs express WISP2 and CTNNBIP1, but not TNFRSF8.

<p>(A) H&E staining on a kidney section from a 20 month old male LP/J mouse with perivascular infiltrates (TLOs). Magnification: 20x (left panel) and 400x (right panel). (B) Immunohistochemistry revealed expression of WISP2 (left panel) and CTNNBIP1 (middle panel), but not TNFRSF8 (right panel). Magnification: 400x. v: vein.</p

Mean perivascular cell cluster number and cumulative cluster size identified in male and female mice of the various mouse strains at the age of 20 months.

<p>*Cluster #: number of perivascular cell clusters present per renal cross-section.</p><p>**“0”: relative cluster size <0.15; “1”: relative cluster size >0.15.</p>§<p>strains included in the kinetics analyses: 6, 12 and 20 months.</p>†<p>strains without TLOs included in lymphatics and liver infiltration analyses.</p>‡<p>strains with TLOs included in lymphatics and liver infiltration analyses.</p><p>n: number of kidneys/mice analyzed; nd: not determined.</p