Early infiltrating macrophage subtype correlates with late-stage phenotypic outcome in a mouse model of hepatorenal fibrocystic disease.

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88Orpk mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88Orpk livers (Ly6clo in C57BL/6 vs Ly6chi in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88Orpk mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88Orpk livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88Orpk mice on a CCR2-/- background. Unexpectedly, the loss of Ly6chi macrophages, which were strongly enriched in BALB/c Ift88Orpk mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.

Tissue-Resident Macrophages Promote Renal Cystic Disease.

BACKGROUND: Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12-14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. METHODS: We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. RESULTS: Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11clo) to R2a (CD11chi) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. CONCLUSIONS: These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.

Heterozygous Pkhd1C642* mice develop cystic liver disease and proximal tubule ectasia that mimics radiographic signs of medullary sponge kidney.

Heterozygosity for human polycystic kidney and hepatic disease 1 ( PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1C642* mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1G617fs and PKHD1G644*). Mouse heterozygotes or homozygotes for the Pkhd1C642* mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1C642* heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1C642* homozygotes. Interestingly, aged female Pkhd1C642* heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1C642* mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population.

Truncating PKHD1 and PKD2 mutations alter energy metabolism.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Genetic and Informatic Analyses Implicate Kif12 as a Candidate Gene within the Mpkd2 Locus That Modulates Renal Cystic Disease Severity in the Cys1cpk Mouse.

We have previously mapped the interval on Chromosome 4 for a major polycystic kidney disease modifier (Mpkd) of the B6(Cg)-Cys1cpk/J mouse model of recessive polycystic kidney disease (PKD). Informatic analyses predicted that this interval contains at least three individual renal cystic disease severity-modulating loci (Mpkd1-3). In the current study, we provide further validation of these predicted effects using a congenic mouse line carrying the entire CAST/EiJ (CAST)-derived Mpkd1-3 interval on the C57BL/6J background. We have also generated a derivative congenic line with a refined CAST-derived Mpkd1-2 interval and demonstrated its dominantly-acting disease-modulating effects (e.g., 4.2-fold increase in total cyst area; p<0.001). The relative strength of these effects allowed the use of recombinants from these crosses to fine map the Mpkd2 effects to a <14 Mbp interval that contains 92 RefSeq sequences. One of them corresponds to the previously described positional Mpkd2 candidate gene, Kif12. Among the positional Mpkd2 candidates, only expression of Kif12 correlates strongly with the expression pattern of Cys1 across multiple anatomical nephron structures and developmental time points. Also, we demonstrate that Kif12 encodes a primary cilium-associated protein. Together, these data provide genetic and informatic validation of the predicted renal cystic disease-modulating effects of Mpkd1-3 loci and implicate Kif12 as the candidate locus for Mpkd2.

Kidney injury accelerates cystogenesis via pathways modulated by heme oxygenase and complement.

AKI accelerates cystogenesis. Because cystogenic mutations induce strong transcriptional responses similar to those seen after AKI, these responses may accelerate the progression of cystic renal disease. Here, we modulated the severity of the AKI-like response in Cys1(cpk/cpk) mice, a model that mimics autosomal recessive polycystic kidney disease. Specifically, we induced or inhibited activity of the renoprotective enzyme heme oxygenase (HO) and determined the effects on renal cystogenesis. We found that induction of HO attenuated both renal injury and the rate of cystogenesis, whereas inhibition of HO promoted cystogenesis. HO activity mediated the response of NFκB, which is a hallmark transcriptional feature common to both cystogenesis and AKI. Among the HO-modulated effects we measured, expression of complement component 3 (C3) strongly correlated with cystogenesis, a functionally relevant association as suggested by Cys1(cpk/cpk) mice with genetically induced C3 deficiency. Because both C3 deficiency and HO induction reduce cyst number and cyst areas, these two factors define an injury-stimulated cystogenic pathway that may provide therapeutic targets to slow the formation of new renal cysts and the growth of existing cysts.

Renal CD14 expression correlates with the progression of cystic kidney disease.

Monocyte and macrophage markers are among the most highly overexpressed genes in cpk mouse kidneys with severely progressive renal cystic disease. We show here that one of these markers, CD14, is abnormally transcribed, activated and shed in cystic kidneys. However, these abnormalities were not associated with an increased number of interstitial CD14-positive mononuclear cells. Instead, we found that most non-cystic and cystic renal tubular epithelia were CD14-positive; even distal nephron-derived principal cells. Cd14 was significantly overexpressed in the kidneys of 5-day-old cpk mice and further increased as the disease progressed. In the cpk model with variable rates of cystic kidney enlargement (due to an intercross of two distinct genetic backgrounds), Cd14 expression positively correlated with kidney volume, exceeding the correlation with MCP-1, an established marker of autosomal-dominant polycystic kidney disease (ADPKD). In 16 patients with ADPKD, the baseline urinary CD14 level showed some tendency to correlate with the 2-year change in total kidney volume; however, the tendency was not statistically significant. But the association was significant when the analysis was confined to males. Clearly more studies need to be done to evaluate the utility of CD14 as a marker for outcomes in ADPKD.

Validation of endogenous internal real-time PCR controls in renal tissues.

BACKGROUND: Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. METHODS: To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. RESULTS: Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. CONCLUSION: A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity.

CD8 T-cell immune phenotype of successful aging.

The nonagenarian population by definition represents individuals who have demonstrated success in aging. We determined the status of CD8(+) T-cell senescence in nonagenarians by analyzing the expression of CD28 and Fas (CD95), and analyzing activation and activation-induced cell death (AICD). Peripheral blood mononuclear cells (PBMCs) were isolated from three groups of subjects: adults (20-64-year-old), older adults (65-89-year-old), and nonagenarians (>or=90-year-old). PBMCs were stimulated with phytohemagglutinin (PHA) (10 microg/ml). The cells were labeled with conjugated antibodies specific for CD4, CD8, CD28, CD45RO, and Fas, and were analyzed by FACS((R)). There was a strong negative correlation of the percentage of CD28(+)Fas(-) CD8(+) T-cells with the age of each individual prior to stimulation in vitro (R(2)=0.76, p<0.0001). Compared to other biomarkers (CD28(-), CD28(-)CD45RO(+), and Fas(+)) that have been associated with CD8(+) T-cell aging, the loss of the CD28(+)Fas(-) CD8(+) T-cell population exhibited the strongest correlation with the individual's chronologic age. After stimulation with PHA, there was a decrease in the percentage of CD8(+) T-cells from individual >or=65-year-old that expresses both CD28(+) and Fas(+) at day 3. Surprisingly, the AICD response of CD8(+) T-cells at day 7 in the nonagenarians was higher than that in the other two groups. These results suggest that successful aging does not prevent development of the senescent phenotype of unstimulated CD8(+) T cells, but is associated with preservation of CD8 T cell functions including activation and AICD. Increased AICD may result in enhanced rejuvenation capacity of T cells and limit the impact of aging on T cell function in nonagenarians.

Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis.

OBJECTIVE: To determine novel genes regulated by tumor necrosis factor alpha (TNFalpha) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIkappaB-dominant negative (AdIkappaB-DN) (n = 3) or with control AdTet expressing the reverse tetracycline trans-activator (n = 3). The cells were stimulated for 3 hours with TNFalpha, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarray-identified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. RESULTS: Blocking of NF-kappaB by AdIkappaB-DN was associated with a down-modulation of antiapoptosis genes, including BIRC-3, and several novel genes, including GG2-1, a TNFalpha-inducible FLIP-like gene. Other families of genes that were significantly down-regulated by AdIkappaB-DN included cytokines/chemokines (interleukin-1beta [IL-1beta], IL-8, IL-15, and RANTES), adhesion molecule (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), and unique genes that have not previously been reported to be regulated by TNFalpha in RA. Inhibition of the GG2-1 gene using the siRNA technique resulted in significantly enhanced apoptosis, decreased proliferation, and decreased production of MMP-1 in TNFalpha-stimulated RASFs. CONCLUSION: These studies provide a comprehensive analysis of genes that are differentially regulated by TNFalpha signaling and NF-kappaB nuclear translocation in RASFs and demonstrate methods for confirming the expression and functional significance of such genes.