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. Author manuscript; available in PMC: 2009 Jun 25.
Published in final edited form as: Aging Cell. 2009 Apr;8(2):192–200. doi: 10.1111/j.1474-9726.2009.00463.x

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Denis Susa 1,4, James R Mitchell 2,4,5,6, Marielle Verweij 1, Marieke van de Ven 2, Henk Roest 1, Sandra van den Engel 1, Ingeborg Bajema 3, Kirsten Mangundap 1, Jan N M IJzermans 1, Jan H J Hoeijmakers 2, Ron W F de Bruin 1,6
PMCID: PMC2701740  NIHMSID: NIHMS114257  PMID: 19338497

Abstract

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo. Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wildtype animals to surgically-induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure-related mortality was significantly reduced in Csb-/- mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome-associated gene, Csa. Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.

Keywords: DNA repair, aging, ischemia reperfusion injury, oxidative stress, hormesis, progeria

Introduction

The case for the involvement of oxidative damage to macromolecules in the etiology of aging, aging-related disorders and so-called “premature aging” disorders (here referred to as segmental progerias) is compelling. The free radical theory as originally proposed by Harman (Harman 1956) and extended in recent years (Beckman and Ames 1998) posits that reactive oxygen species (ROS), byproducts of endogenous cellular metabolism, inflict damage on lipids, protein and DNA. Despite defense systems to prevent or repair such damage, oxidized macromolecules accumulate over time in vivo and are thought to underlie both normal and pathological aging. When combined with congenital defects in certain of these defense systems, for example genome stability mechanisms, endogenous oxidative damage may also contribute to the symptoms of segmental progerias, which display some but not all of the characteristics of normal aging in accelerated or exacerbated forms (Martin and Oshima 2000).

Nucleotide excision DNA repair (NER) is one such defense system that removes a range of damages, including helix distorting lesions induced either by exogenous ultraviolet radiation (e.g. pyrimidine dimers) or endogenous oxidative radicals (e.g. cyclopurines) (Brooks et al. 2000). Two basic modes of lesion recognition have been defined. In the first, lesions occurring anywhere in the genome are recognized by damage-binding proteins such as the XPC-HR23A/B-CEN1 complex. This triggers the assembly of the multiprotein NER machinery which functions via a cut and patch mechanism to remove the damage and fill the remaining single-strand gap (Hoeijmakers 2001). In the second mode of recognition, transcription-coupled (TC)-NER, lesions that block an elongating RNA polymerase trigger assembly of the NER machinery in a process which depends on the chromatin remodeling protein CSB (Citterio et al. 2000) and a ubiquitin ligase complex containing the TC-NER specific protein CSA (Groisman et al. 2003; Fousteri et al. 2006).

Defects in CSA or CSB can give rise to Cockayne syndrome, a severe neurodevelopmental disease marked by photosensitivity (but curiously without skin cancer predisposition), dysmyelination within the central nervous system, progressive sensineuronal hearing loss, retinal degeneration and cachectic dwarfism resulting in an aged appearance (Nance and Berry 1992). Mouse models lacking Csa or Csb function recapitulate some progeroid characteristics of the human disease, including cachexia and progressive loss of photoreceptor cells, but with a normal lifespan and an overall milder phenotype than in humans (van der Horst et al. 1997; van der Horst et al. 2002; Dolle et al. 2006). In support of the role of oxidative stress hypersensitivity in Cockayne syndrome etiology, an increase in photoreceptor cell loss following whole-body ionizing radiation was observed in both Csa-/- and Csb-/- mice (Gorgels et al. 2006). Thus, at least particular CS cell types appear hypersensitive to the effects of oxidative DNA damage, a property that may underlie tissue-specific disease phenotypes.

Contrary to the notion of hypersensitivity to oxidative stress in Cockayne syndrome is the observation that mouse models of Cockayne syndrome and related NER-deficient segmental progerias (NER progerias) display characteristics of hypopituitary dwarf mutants or dietary restricted wildtype mice (Wijnhoven et al. 2005; Niedernhofer et al. 2006; van de Ven et al. 2006; van der Pluijm et al. 2006; Schumacher et al. 2008). In dwarf and dietary restricted mice, these phenotypes, including hypoglycemia, reduced body weight and temperature, hypoglycemia, hypoinsulinemia and reduced serum IGF-1, are correlated not only with extended longevity but also resistance to acute oxidative stress (Bartke and Brown-Borg 2004). In progeroid NER mice, these phenotypes have been interpreted as an adaptive response to unrepaired endogenous DNA damage engaged to protect from further oxidative stress injury (van de Ven et al. 2007). One important prediction of this interpretation is that mice should be resistant rather than hypersensitive to acute oxidative stress.

Ischemia reperfusion injury is a complex insult initiated by loss of blood flow to an organ. During the ischemic period, the lack of molecular oxygen as an electron acceptor in oxidative phosphorylation prevents ATP generation and compromises processes with high energy demand, such as maintenance of ion gradients across intracellular membranes. Reinitiation of oxygenated blood flow, or reperfusion, results in inappropriate activation of cellular oxidases and ROS generation, which can affect not only the reperfused organ but distant sites in the body as well (Kelly 2003). Following reperfusion, a maladaptive inflammatory response mediated in part by the release of ROS from neutrophils infiltrating the tissue causes further injury (Friedewald and Rabb 2004). In the kidney, ischemia reperfusion injury is associated with cell death primarily in the stripe between the cortex and medulla consisting mainly of tubular epithelial cells via necrosis or apoptosis, depending on the severity of the insult (Padanilam 2003). Recovery and a return to proper kidney function depends on regeneration of the tubular epithelial cells and remodeling of the renal tubules, a process which takes place in the days and weeks following the injury.

We used surgically-induced renal ischemia reperfusion injury to test whether mouse models of Cockayne syndrome are susceptible to acute oxidative stress, as predicted by photoreceptor sensitivity to ionizing radiation, or protected from it, as predicted by physiological similarities between other short-lived progeroid NER mice and long-lived, stress resistant mice. Here we report resistance to renal ischemia reperfusion injury in mouse models of Cockayne syndrome.

Results

Warm ischemia was induced in the left kidney of male wildtype and Csb-/- (subsequently referred to as WT and CSB, respectively) mice for 37 minutes by clamping the renal artery/vein with a nontraumatic surgical clamp. The 37 minute time point was chosen based on the results of preliminary experiments in which ischemia times were titrated between 30 and 45 minutes. Following clamp release, the undamaged right kidney was removed so that animal survival depended on the function of the damaged kidney; mice entirely lacking kidney function build up toxic compounds in their blood and die within 2-4 days. Survival of CSB mice was significantly higher than WT (Fig. 1A). On post-operative day (POD) 7, survival was 56% in CSB mice but only 30% in the WT group (log rank score p<0.0033; n=24 CSB, 40 WT).

Figure 1.

Figure 1

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CSB mice are better protected against warm renal ischemia reperfusion injury than WT.

A. Kaplan-Meier survival curves of CSB (n=24) and WT (n=40) animals; a log rank score of p<0.0033 indicates a highly significant difference between the genotypes.

B, C. Kidney function following IRI as determined by serum levels of urea (B) and creatinine (C). Statistically significant differences in magnitude observed at individual timepoints between genotypes following injury are indicated by asterisks (** p<0.01, * p<0.05). Groups receiving a mock treatment that did not involve ischemia are indicated.

Kidney function was determined by measuring the concentrations of urea and creatinine in the blood serum. High levels of these waste products indicate an inability of the kidney to remove them from the blood and thus correlate inversely with kidney function. Interestingly, pre-operative urea and creatinine values were slightly but significantly lower in CSB mice than in WT mice (urea 8.5 ± 1.3 mmol/L (CSB) vs 9.7 ± 1.2 mmol/L (WT), p=0.0007; creatinine 95 ± 17 umol/L (CSB) vs. 122 ± 34 umol/L (WT), p=0.002). Following ischemia-reperfusion injury, these markers of kidney function rose with similar kinetics, suggesting maximal dysfunction on POD 2 and a return to function beginning on POD 3. However, average values were significantly lower in CSB animals on POD 1 for urea and on PODs 1 and 2 for creatinine (Fig 1B, C), consistent with better survival in the CSB group.

We next analyzed cell death at various timepoints after the injury to look for differences that could explain the observed survival and functional benefits in CSB animals vs. WT controls. We used histology to score for acute tubular necrosis, the major form of cell death due to this type of injury (Fig. 2A). In both groups, acute tubular necrosis peaked on POD2; in the CSB group, average total cell death was lower at all time points, reaching statistical significance on PODs 1 and 2. The high mortality due to kidney dysfunction in the wildtype group prevented meaningful comparisons beyond POD 3.

Figure 2.

Figure 2

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Cell loss and regeneration following 37 min warm renal ischemia reperfusion injury.

A. Cell death as determined by H&E-stained paraffin sections on the indicated day following IR. Day 0 kidneys were contralateral kidneys harvested immediately after clamp release.

B-D. Regeneration as determined by mitotic index in H&E stained sections (B); relative number of cells expressing PCNA as determined by IHC (C); and total PCNA protein level as determined by immunoblotting on the indicated days following ischemia reperfusion injury (D). Statistically significant differences between CSB and WT on a given day are indicated by asterisks (** p<0.01, * p<0.05).

Following cell death and clearance of cellular debris by shedding into the lumen of the tubules, kidneys damaged by ischemia reperfusion injury may undergo a regenerative phase in which cellular proliferation can be observed. Since the survival benefit of the CSB mice was likely only partially explained by the lower cell death on PODs 1 and 2, we next asked whether regeneration of the kidney was enhanced. We used three different assays to gauge this parameter: histology, or the presence of mitotic figures in H&E stained sections; immunohistochemistry against the proliferating cell nuclear antigen (PCNA); and PCNA immunoblotting of total kidney homogenates to assess total levels of this protein. In both groups, mitotic figures were first seen on POD 3, with significantly higher scores in CSB mice (Fig. 2B; p = 0.025). We next looked at PCNA on the single-cell level by immunohistochemistry. The number of PCNA positive cells was significantly increased in the CSB group on POD 1 (Fig. 2C; p = 0.035), possibly indicative of its additional role in DNA repair. We further analyzed PCNA levels in total kidney homogenates by immunoblot. Although pre-operative PCNA levels were similar between groups, significantly higher levels of PCNA were observed on POD 3 in the CSB group (Fig. 2D; p = 0.042). Taken together, these results are consistent with enhanced proliferation leading to better regeneration and survival in CSB vs. WT animals.

Previously it was reported that wildtype C57BL/6 mice subjected to 30-50 minutes of warm ischemia to one kidney followed by contralateral nephrectomy had a one-week survival of approximately 80%, with no statistically significant differences between ischemia times (Megyesi et al. 2002). We consistently found mortality of approximately 70% due to kidney failure following 30-37 minutes warm ischemia (Fig 1A and data not shown), making it difficult to study the course of organ recovery beyond POD3. To circumvent this problem, we repeated our experiments in WT and CSB mice using 25 minutes of warm renal ischemia. With this amount of ischemic damage, survival was 100% in both WT and CSB groups (data not shown). However, 25 minutes of ischemia resulted in significantly less kidney dysfunction in mice lacking the CSB protein relative to WT controls at all timepoints following ischemic damage as measured by serum urea (Fig. 3A, top). A similar trend was seen for creatinine (Fig. 3A, bottom).

Figure 3.

Figure 3

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Kidney function, cell death and regeneration following 25 min warm renal ischemia reperfusion injury in CSB and WT animals.

A. Kidney function of the indicated genotypes following IRI or mock treatment as determined by concentration of serum urea (top) or creatinine (bottom). Statistically significant differences between CSB and WT on a given day are indicated by asterisks (** p<0.01, * p<0.05). B-E. Regeneration as indicated by mitotic figures in H&E stained sections (B) and immunohistochemistry against PCNA (C), Ki67 (D), and p21 (E) as indicated. Statistically significant differences between CSB and WT on a given day are indicated by asterisks (** p<0.01, * p<0.05).

Analysis of total cell death by histology revealed a significant elevation above preoperative values in CSB and WT (although less than after 37 min warm ischemia) on all PODs examined, but no significant differences between genotypes (data not shown). Despite similar low amounts of cell death between groups, we observed large differences in proliferation following 25 min warm renal ischemia. As with 37 min ischemia, proliferation was elevated in CSB vs. WT mice. Regeneration scored by mitotic figures in histological sections was first seen in wildtype mice on POD 3, while regeneration was already significantly elevated relative to baseline in CSB mice on PODs 2 and 3 (Fig. 3B; p=0.035 and p=0.021, respectively). The number of PCNA positive cells on PODs 1 and 2 was also significantly increased in the CSB group (Fig. 3C; p = 0.005 and p = 0.002, respectively). Because PCNA is involved in DNA repair as well as proliferation, we stained sections for an additional proliferative marker, Ki-67, which is expressed during all active phases of the cell cycle (G1, S, G2 and mitosis) but is absent from resting cells (G0) (Scholzen and Gerdes 2000). Ki-67 positive cells were also increased in CSB mice, with a maximum on POD 2 (Fig. 3D; p = 0.016).

The cyclin-dependant kinase inhibitor p21, which binds stoichiometrically to PCNA and inhibits its action in DNA replication, is a key regulator of proliferation following renal ischemic injury. Mice lacking p21 display hyperproliferation and greatly reduced survival (Megyesi et al. 2002). We tested p21 protein levels by immunohistochemistry (Fig. 3E). Consistent with increased proliferation in CSB relative to wildtype on PODs 1 and 2, the number of p21 positive cells was significantly reduced in CSB mice (POD 2, p = 0.016). Interestingly, p21 staining was equal in wildtype and CSB on POD3, coincident with a decline in the first wave of proliferation following injury.

We next turned to potential mechanisms of protection against renal ischemia reperfusion injury specific to CSB animals. Addition of antioxidants is a proven way to ameliorate the effects of this injury (Devarajan 2005). Furthermore, antioxidant capacity is increased in multiple organs in both genetic dwarfism and dietary restriction, a property thought to contribute to stress resistance and increased longevity in these mice (Bartke and Brown-Borg 2004). We thus looked for evidence of increased antioxidant capacity on the level of gene expression. We used quantitative RT-PCR over a time course before and after renal ischemia reperfusion injury to analyze antioxidant defense capabilities as represented by the steady state levels of candidate mRNAs including superoxide dismutase 1, glutathione reductase and hemoxygenase 1 (Fig 4A and data not shown). At baseline, steady state amounts of these mRNAs were not significantly different between WT and CSB groups. Similarly, over a 24 hour period following 25 or 37 minutes warm renal ischemia, some of these genes (e.g. hemoxygenase-1) were significantly upregulated above baseline but none were consistently, significantly differentially regulated between CSB and WT groups (Fig. 4A and data not shown).

Figure 4.

Figure 4

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Mechanisms of reduced susceptibility to renal ischemia reperfusion injury in CSB vs WT mice.

A. Time course of hemeoxygenase-1 (HO-1), P-selectin and ICAM-1 mRNA expression as indicated in the kidney following 37 min renal ischemia reperfusion injury using quantitative real time PCR. Statistically significant differences between genotypes at a given time point day are indicated by an asterisk (* p<0.05).

B. Improved glucose clearance and insulin sensitivity in CSB vs. WT mice as indicated. Whole blood glucose levels at the indicated timepoints following intraperitoneal injection of insulin (E; n=11 WT, 7 CSB) or glucose (D, n= 9 WT, 5 CSB) into overnight fasted animals. Statistically significant differences between genotypes at a given time point day are indicated by asterisks (** p<0.01, * p<0.05).

Another proven way to reduce the effects of ischemia reperfusion injury is to prevent the inflammatory response that follows tissue injury, for example by neutralization of cellular adhesion molecules that serve to recruit neutrophils to the site of tissue injury by genetic or antibody-based methods (Takada et al. 1997). We examined the expression of P-selectin and ICAM-1, endothelial adhesion molecules that are upregulated upon tissue injury and whose expression correlates negatively with survival and functional outcomes. We observed a significant increase in both markers following renal IRI above baseline levels; this increase, however, was less in the CSB animals (Fig. 4A). An area-under-the-curve analysis revealed significant differences in expression of both P-selectin (p=0.02) and ICAM-1 (p=0.05) following renal ischemia reperfusion injury, consistent with less damage subsequent to the reperfusion and a better outcome.

Finally, enhanced sensitivity to the effects of insulin on glucose metabolism is a property shared by dwarf and dietary restricted mice that had been proposed to underlie longevity benefits observed in these models (Bartke and Brown-Borg 2004). We chose to analyze glucose metabolism by performing both glucose tolerance and insulin sensitivity tests in overnight fasted CSB and WT mice. We found a significant increase in the ability of CSB animals to clear glucose from the circulation in response to a bolus injection of insulin or glucose, respectively (Fig. 4B). It should be noted that despite the slight but significant elevation of fasting glucose levels observed at baseline in CSB animals subject to the glucose tolerance test, no significant difference was observed in the insulin tolerance test between CSB and WT (p=0.34) nor in the combined data set (p=0.41). This lack of a significant difference is consistent with previous reports of fed glucose levels of single mutant CS and XPCS mice at postnatal day 15 prior to weaning, while in more severe XPA-deficient double mutant animals hypoglycemia was observed (van de Ven et al. 2006; van der Pluijm et al. 2006)

Cockayne syndrome can also be caused by defects in the CSA gene. In mice, Csa-/- (subsequently referred to as CSA) mice are nearly indistinguishable from CSB mice (van der Horst et al. 2002). We tested the resistance of CSA mice to renal ischemia reperfusion injury and found evidence of protection against both 25 and 37 min warm ischemia in CSA mice vs. WT controls as at the level of kidney function (Fig. 5A).

Figure 5.

Figure 5

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Protection from renal ischemia reperfusion correlates with genetic defects in TC-NER and extends to CSB females.

A. Functional response of TC-NER deficient CSA vs. WT animals to 25 and 37 min warm IRI according to serum urea (top) and creatinine (bottom) values on the indicated days after IRI. Statistically significant differences between genotypes at a given time point day are indicated by asterisks (** p<0.01, * p<0.05).

B. Functional response of WT vs. CSB females to 37 min warm renal IRI as measured by serum urea (top) and creatinine (bottom) values prior to and 24 hours after injury.

No sex bias has been previously reported for Cockayne syndrome (Nance and Berry 1992)or for mouse models of this disease. To see whether the protective effects we observed are specific to males, we tested CSB females for resistance to 37 minutes of warm renal ischemia. Female mice are in general less susceptible to this injury than males (Kher et al. 2005), and as expected we observed less evidence of kidney dysfunction and inflammation following 37 min warm ischemia in females than in males. However, CSB females displayed evidence of increased resistance to injury, including significantly lower urea and creatinine in the serum 24 hours after reperfusion (Fig. 5B).

Discussion

Although the genetic lesions causative of Cockayne syndrome are known, the molecular defect(s) leading to the observed symptoms remains unclear. The prominent inability to repair UV-induced DNA lesions in Cockayne cells from both man and mouse models has led to the hypothesis that hypersensitivity to DNA damage, presumably oxidative in nature, is primarily responsible for disease symptoms. Consistent with this, CSB mice are hypersensitive to the effects of whole-body ionizing irradiation, a potent oxidative stress (de Waard et al. 2003), and both CSA and CSB mice demonstrate an increase in photoreceptor cell loss following ionizing radiation (Gorgels et al. 2006). However, primary CSB cells (van de Ven et al. 2006) as well as immortalized CSA cells (de Waard et al. 2004) are not hypersensitive to oxidative stress.

A number of recent reports have described overlap between short-lived segmental progeroid NER models and long-lived hypopituitary dwarfs or dietary restricted wildtype mice on the level of physiology and gene expression (Wijnhoven et al. 2005; Niedernhofer et al. 2006; van de Ven et al. 2006; van der Pluijm et al. 2006; van de Ven et al. 2007; Schumacher et al. 2008). These phenotypes have been interpreted as an adaptive response to genotoxic stress overlapping with the starvation response. A previously untested prediction of this interpretation is increased resistance rather than hypersensitivity to acute oxidative stress in these models.

Consistent with this prediction, we showed here that mouse models of Cockayne syndrome deficient in either Csa or Csb are less susceptible to renal ischemia reperfusion injury than wildtype mice. Susceptibility was measured in terms of animal survival, kidney function and histological evidence of damage and organ regeneration. In all cases, CSB mice did significantly better than WT mice. On the molecular level, we observed less upregulation of the cell-cycle inhibitor p21 after injury in CSB mice, consistent with less damage and/or better proliferative potential. Upregulation of inflammatory markers ICAM-1 and P selectin were also significantly reduced following injury in CSB mice, consistent with either reduced damage and/or a dampened ability to mount an inflammatory response. Finally, improved glucose tolerance and insulin sensitivity at baseline were observed in overnight fasted CSB animals relative to WT controls. Taken together with previous studies demonstrating hypersensitivity to ionizing radiation in certain tissues, these data suggest that hypersensitivity and resistance to oxidative stress can coexist in the same animal, depending on the tissue or cell type involved.

Although the mechanism of protection against acute stress in the long-lived dwarf or dietary restricted models is not known, a number of plausible candidate mechanisms have been identified. These include increased antioxidant capacity, reduced inflammation and improved glucose homeostasis due to heightened insulin sensitivity (Bartke and Brown-Borg 2004). The induction of genes including hemeoxygenase-1 upon ischemic injury demonstrated that the capacity of CSB animals to mount a transcriptional response is normal despite a defect in the repair of RNA polymerase-stalling bulky DNA lesions. However, we did not observe any differences in antioxidant capacity on the level of gene expression between WT and CSB mice. We did find a difference in the upregulation of two markers of inflammation, P selectin and ICAM-1. Reduced inflammation is associated with improved outcome following renal ischemia (Friedewald and Rabb 2004), suggesting that these differences may in part underlie the stress resistance observed in the CSB model.

Glucose tolerance and insulin sensitivity tests were both consistent with improved response of CSB animals to the effects of insulin. Improved insulin sensitivity is associated with extended longevity in various models including genetic dwarfism and dietary restriction and is usually associated with reduced IGF-1 and improved glucose metabolism. How improved insulin sensitivity might underlie protection from acute stress observed here, or extended longevity observed in other models, remains unknown. As insulin and insulin-like growth factor signaling are pro-survival, and growth factor delivery can improve outcome after ischemic injury to other organs including the brain (Tang 2006), we speculate that increased sensitivity to these growth factors may result in both better cell survival as well as improved proliferation following ischemia reperfusion injury.

Which defective biochemical activity (or activities) of CSB is causative of disease phenotypes, possibly including protection from renal ischemic insult? We speculate that a defect in TC-NER is the likely culprit based on the following genetic argument. CSB and other proteins implicated in Cockayne syndrome are multifunctional and have roles in various cellular processes, defects in any of which could in principle give rise to disease symptoms. Nonetheless the symptoms of Cockayne syndrome are remarkably similar regardless of the underlying genetic lesion, particularly in mice including CSA, CSB, XPCS or TTD models in an XPA deficient background, as well as in XPG, XPF and ERCC1 single mutant animals (van de Ven et al. 2007). We speculate that this common phenotype likely stems from a defect in a shared process in which each of these proteins participates directly. To date, the only common pathway in which each of these multifunctional proteins has a direct role is TCNER. It should be noted, however, that the presumed unrepaired lesion or repair intermediate remains to be identified.

We propose a model in which a congenital DNA repair deficiency results in activation of systemic protective mechanisms including a reduced inflammatory response and improved insulin sensitivity. These mechanisms would then lend resistance to certain forms of acute stress in some tissues (renal ischemia reperfusion injury to the kidney, as shown here) but not others (ionizing radiation in the photoreceptor layer of the eye (Gorgels et al. 2006)) . We can envision at least two distinct mechanisms by which this organ/tissue specificity could occur. Based on the neuronal phenotypes in Cockayne patients and mouse models, TC-NER deficiency could in principle result in neuronal deficiencies causing a state of real or perceived dietary restriction, for example through defective function of neurons involved in nutrient sensing, or neuronal control of the gut leading to malabsorption. Extended periods of dietary restriction in wildtype mice have been shown to protect against ischemic damage to organs including the heart (Chandrasekar et al. 2001) and the brain (Yu and Mattson 1999). Alternately, unrepaired TC-NER substrates in the genome may elicit an adaptive stress response similar to dietary restriction but in the absence of reduced food intake. Consistent with this latter interpretation is the adaptive response involving transient alterations in glucose homeostasis and serum IGF-1 levels observed in a related TC-NER deficient mouse model during the potentially stressful period of postnatal development (van de Ven et al. 2006).

Hormesis is a common biological phenomenon in which exposure to a low intensity stressor induces a general adaptive response that has net beneficial effects on the cellular and/or organismal level, including protection against subsequent, higher dose exposures as well as to different types of stress (Murray et al. 1986; Mattson 2008). Dietary restriction has been proposed to act as a mild stressor that extends longevity through hormetic mechanisms (Turturro et al. 2000; Sinclair 2005). Interestingly, ischemic preconditioning, a procedure used to protect against ischemic insult that entails brief period(s) of ischemia prior to a longer ischemia time, is also thought to function via hormesis (Arumugam et al. 2006). Our data suggest that specific types of unrepaired endogenous DNA lesions may also be hormetic in nature. The interplay between tissue-specific sensitivities to endogenous DNA damage and the resulting adaptive responses may underlie the complex phenotypes observed in segmental disorders such as Cockayne syndrome. Having identified such unexpected benefits associated with DNA repair deficiency, further elucidation of underlying mechanisms may allow exploitation of the benefits without suffering the severe complications associated with congenital DNA repair insufficiencies.

Materials and Methods

Animals

Animals were allowed free access to food and water throughout the experiments. All experiments were performed with the approval of the appropriate ethical board. Male Csb-/- (van der Horst et al. 1997) and Csa-/- (van der Horst et al. 2002) mice in a C57BL/6J background were bred at the animal facility of the Erasmus Medical Centre; C57BL/6J mice were purchased from Harlan, Horst, the Netherlands.

Ischemia model

Mice between 12 and 16 weeks of age were anaesthetized by isoflurane inhalation. Following a midline abdominal incision, an atraumatic microvascular clamp was used to occlude the left kidney for 25-37 minutes. After release of the clamp a contralateral nefrectomy was performed.

Kidney functional measurements

Blood samples were collected by retro-orbital puncture. Blood serum urea and creatinine levels were measured using QuantiChrom assay kits based on the improved Jung and Jaffe methods, respectively (DIUR-500 and DICT-500, Gentaur, Brussels, Belgium) according to the manufacturer’s instructions, or were determined using an ELAN analyzer (Eppendorf Merck, Hamburg, Germany) with Ecoline S+ reagents (Diagnostic Systems GmbH, Holzheim, Germany) according to manufacturer’s instructions.

Histology

Kidneys were harvested, bisected longitudinally, fixed for 24 hr in formalin and embedded in paraffin. 3 μm sections were stained with hematoxylin and eosin, modified Jones staining and periodic acid Schiff (PAS). Immunohistochemistry was performed on deparaffinzed sections following antigen retrieval in boiling 10 mM sodium citrate.

mRNA expression analysis

Total RNA was extracted from frozen kidney using TRIzol reagent (Invitrogen) and oligodT or hexamer-primed cDNA synthesized using SuperScript II (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using an Opticon2 DNA Engine (MJ Research) or a MyIQ (BioRad) with SYBR Green incorporation. Each sample was tested in duplo at least two times.

Glucose tolerance and insulin sensitivity tests

Mice were fasted overnight prior to testing. Following baseline blood glucose determination from tail blood of conscious, restrained mice, animals were injected with a bolus of glucose (1.5 mg glucose/gram body weight) or insulin (0.75 U/kg body weight) into the intraperitoneal cavity. Blood glucose determinations were performed as above at the indicated times following injection using a HemoCue glucose 201 RT blood glucose analyzer (HemoCue, Ängelholm, Sweden).

Statistics

Data are expressed as the mean ± SEM. Statistical analyses of data on urea, creatinine, histomorphology, immunohistochemistry and immunoblot was preformed using a Student’s T test. Survival was analyzed by Kaplan-Meier (SPSSv11).

Acknowledgements

This research was supported in part by the Netherlands Organization for Health Research and Development, Research Institute for Diseases of the Elderly (60-60400-98-004), the National Institutes of Health (1PO1 AG17242–02), the Association for International Cancer Research (05–280) and the European Commission RISC-RAD (FI6R-CT-2003-508842). JRM was a fellow of the Damon Runyon Cancer Research Fund (DRG 1677). MV was supported by a grant from the Dutch Kidney Foundation (C04.2105).

References

  1. Arumugam TV, Gleichmann M, Tang SC, Mattson MP. Hormesis/preconditioning mechanisms, the nervous system and aging. Ageing Res Rev. 2006;5:165–78. doi: 10.1016/j.arr.2006.03.003. [DOI] [PubMed] [Google Scholar]
  2. Bartke A, Brown-Borg H. Life extension in the dwarf mouse. Curr Top Dev Biol. 2004;63:189–225. doi: 10.1016/S0070-2153(04)63006-7. [DOI] [PubMed] [Google Scholar]
  3. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev. 1998;78:547–81. doi: 10.1152/physrev.1998.78.2.547. [DOI] [PubMed] [Google Scholar]
  4. Brooks PJ, Wise DS, Berry DA, Kosmoski JV, Smerdon MJ, Somers RL, Mackie H, Spoonde AY, Ackerman EJ, Coleman K, Tarone RE, Robbins JH. The oxidative DNA lesion 8,5′-(S)-cyclo-2′-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells. J Biol Chem. 2000;275:22355–62. doi: 10.1074/jbc.M002259200. [DOI] [PubMed] [Google Scholar]
  5. Chandrasekar B, Nelson JF, Colston JT, Freeman GL. Calorie restriction attenuates inflammatory responses to myocardial ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2001;280:H2094–102. doi: 10.1152/ajpheart.2001.280.5.H2094. [DOI] [PubMed] [Google Scholar]
  6. Citterio E, Van Den Boom V, Schnitzler G, Kanaar R, Bonte E, Kingston RE, Hoeijmakers JH, Vermeulen W. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcription-coupling factor. Mol Cell Biol. 2000;20:7643–53. doi: 10.1128/mcb.20.20.7643-7653.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. de Waard H, de Wit J, Andressoo JO, van Oostrom CT, Riis B, Weimann A, Poulsen HE, van Steeg H, Hoeijmakers JH, van der Horst GT. Different effects of CSA and CSB deficiency on sensitivity to oxidative DNA damage. Mol Cell Biol. 2004;24:7941–8. doi: 10.1128/MCB.24.18.7941-7948.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. de Waard H, de Wit J, Gorgels TG, van den Aardweg G, Andressoo JO, Vermeij M, van Steeg H, Hoeijmakers JH, van der Horst GT. Cell type-specific hypersensitivity to oxidative damage in CSB and XPA mice. DNA Repair (Amst) 2003;2:13–25. doi: 10.1016/s1568-7864(02)00188-x. [DOI] [PubMed] [Google Scholar]
  9. Devarajan P. Cellular and molecular derangements in acute tubular necrosis. Curr Opin Pediatr. 2005;17:193–9. doi: 10.1097/01.mop.0000152620.59425.eb. [DOI] [PubMed] [Google Scholar]
  10. Dolle ME, Busuttil RA, Garcia AM, Wijnhoven S, van Drunen E, Niedernhofer LJ, van der Horst G, Hoeijmakers JH, van Steeg H, Vijg J. Increased genomic instability is not a prerequisite for shortened lifespan in DNA repair deficient mice. Mutat Res. 2006;596:22–35. doi: 10.1016/j.mrfmmm.2005.11.008. [DOI] [PubMed] [Google Scholar]
  11. Fousteri M, Vermeulen W, van Zeeland AA, Mullenders LH. Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol Cell. 2006;23:471–82. doi: 10.1016/j.molcel.2006.06.029. [DOI] [PubMed] [Google Scholar]
  12. Friedewald JJ, Rabb H. Inflammatory cells in ischemic acute renal failure. Kidney Int. 2004;66:486–91. doi: 10.1111/j.1523-1755.2004.761_3.x. [DOI] [PubMed] [Google Scholar]
  13. Gorgels TG, van der Pluijm I, Brandt RM, Garinis GA, van Steeg H, van den Aardweg G, Jansen GH, Ruijter JM, Bergen AA, van Norren D, Hoeijmakers JH, van der Horst GT. Retinal degeneration and ionizing radiation hypersensitivity in a mouse model for Cockayne syndrome. Mol Cell Biol. 2006 doi: 10.1128/MCB.01037-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R, Kisselev AF, Tanaka K, Nakatani Y. The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage. Cell. 2003;113:357–67. doi: 10.1016/s0092-8674(03)00316-7. [DOI] [PubMed] [Google Scholar]
  15. Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol. 1956;11:298–300. doi: 10.1093/geronj/11.3.298. [DOI] [PubMed] [Google Scholar]
  16. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature. 2001;411:366–74. doi: 10.1038/35077232. [DOI] [PubMed] [Google Scholar]
  17. Kelly KJ. Distant effects of experimental renal ischemia/reperfusion injury. J Am Soc Nephrol. 2003;14:1549–58. doi: 10.1097/01.asn.0000064946.94590.46. [DOI] [PubMed] [Google Scholar]
  18. Kher A, Meldrum KK, Wang M, Tsai BM, Pitcher JM, Meldrum DR. Cellular and molecular mechanisms of sex differences in renal ischemia-reperfusion injury. Cardiovasc Res. 2005;67:594–603. doi: 10.1016/j.cardiores.2005.05.005. [DOI] [PubMed] [Google Scholar]
  19. Martin GM, Oshima J. Lessons from human progeroid syndromes. Nature. 2000;408:263–6. doi: 10.1038/35041705. [DOI] [PubMed] [Google Scholar]
  20. Mattson MP. Hormesis defined. Ageing Res Rev. 2008;7:1–7. doi: 10.1016/j.arr.2007.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Megyesi J, Andrade L, Vieira JM, Jr., Safirstein RL, Price PM. Coordination of the cell cycle is an important determinant of the syndrome of acute renal failure. Am J Physiol Renal Physiol. 2002;283:F810–6. doi: 10.1152/ajprenal.00078.2002. [DOI] [PubMed] [Google Scholar]
  22. Murray CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124–1136. doi: 10.1161/01.cir.74.5.1124. [DOI] [PubMed] [Google Scholar]
  23. Nance MA, Berry SA. Cockayne syndrome: Review of 140 cases. Am. J. Med. Genet. 1992;42:68–84. doi: 10.1002/ajmg.1320420115. [DOI] [PubMed] [Google Scholar]
  24. Niedernhofer LJ, Garinis GA, Raams A, Lalai AS, Robinson AR, Appeldoorn E, Odijk H, Oostendorp R, Ahmad A, van Leeuwen W, Theil AF, Vermeulen W, van der Horst GT, Meinecke P, Kleijer WJ, Vijg J, Jaspers NG, Hoeijmakers JH. A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis. Nature. 2006;444:1038–43. doi: 10.1038/nature05456. [DOI] [PubMed] [Google Scholar]
  25. Padanilam BJ. Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol. 2003;284:F608–27. doi: 10.1152/ajprenal.00284.2002. [DOI] [PubMed] [Google Scholar]
  26. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–22. doi: 10.1002/(SICI)1097-4652(200003)182:3<311::AID-JCP1>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  27. Schumacher B, van der Pluijm I, Moorhouse MJ, Kosteas T, Robinson AR, Suh Y, Breit TM, van Steeg H, Niedernhofer LJ, van Ijcken W, Bartke A, Spindler SR, Hoeijmakers JH, van der Horst GT, Garinis GA. Delayed and accelerated aging share common longevity assurance mechanisms. PLoS Genet. 2008;4:e1000161. doi: 10.1371/journal.pgen.1000161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sinclair DA. Toward a unified theory of caloric restriction and longevity regulation. Mech Ageing Dev. 2005;126:987–1002. doi: 10.1016/j.mad.2005.03.019. [DOI] [PubMed] [Google Scholar]
  29. Takada M, Nadeau KC, Shaw GD, Marquette KA, Tilney NL. The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand. J Clin Invest. 1997;99:2682–90. doi: 10.1172/JCI119457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tang BL. SIRT1, neuronal cell survival and the insulin/IGF-1 aging paradox. Neurobiol Aging. 2006;27:501–5. doi: 10.1016/j.neurobiolaging.2005.02.001. [DOI] [PubMed] [Google Scholar]
  31. Turturro A, Hass BS, Hart RW. Does caloric restriction induce hormesis? Hum Exp Toxicol. 2000;19:320–9. doi: 10.1191/096032700678815981. [DOI] [PubMed] [Google Scholar]
  32. van de Ven M, Andressoo JO, Holcomb VB, Hasty P, Suh Y, van Steeg H, Garinis GA, Hoeijmakers JH, Mitchell JR. Extended longevity mechanisms in short-lived progeroid mice: identification of a preservative stress response associated with successful aging. Mech Ageing Dev. 2007;128:58–63. doi: 10.1016/j.mad.2006.11.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van de Ven M, Andressoo JO, Holcomb VB, von Lindern M, Jong WM, Zeeuw CI, Suh Y, Hasty P, Hoeijmakers JH, van der Horst GT, Mitchell JR. Adaptive Stress Response in Segmental Progeria Resembles Long-Lived Dwarfism and Calorie Restriction in Mice. PLoS Genet. 2006;2:e192. doi: 10.1371/journal.pgen.0020192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van der Horst GT, Meira L, Gorgels TG, de Wit J, Velasco-Miguel S, Richardson JA, Kamp Y, Vreeswijk MP, Smit B, Bootsma D, Hoeijmakers JH, Friedberg EC. UVB radiation-induced cancer predisposition in Cockayne syndrome group A (Csa) mutant mice. DNA Repair (Amst) 2002;1:143–57. doi: 10.1016/s1568-7864(01)00010-6. [DOI] [PubMed] [Google Scholar]
  35. van der Horst GTJ, van Steeg H, Berg RJW, van Gool A, de Wit J, Weeda G, Morreau H, Beems RB, van Kreijl CF, de Gruijl FR, Bootsma D, Hoeijmakers JHJ. Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition. Cell. 1997;89:425–35. doi: 10.1016/s0092-8674(00)80223-8. [DOI] [PubMed] [Google Scholar]
  36. van der Pluijm I, Garinis GA, Brandt RM, Gorgels TG, Wijnhoven SW, Diderich KE, de Wit J, Mitchell JR, van Oostrom C, Beems R, Niedernhofer LJ, Velasco S, Friedberg EC, Tanaka K, van Steeg H, Hoeijmakers JH, van der Horst GT. Impaired genome maintenance suppresses the growth hormone--insulin-like growth factor 1 axis in mice with Cockayne syndrome. PLoS Biol. 2006;5:e2. doi: 10.1371/journal.pbio.0050002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wijnhoven SW, Beems RB, Roodbergen M, van den Berg J, Lohman PH, Diderich K, van der Horst GT, Vijg J, Hoeijmakers JH, van Steeg H. Accelerated aging pathology in ad libitum fed Xpd(TTD) mice is accompanied by features suggestive of caloric restriction. DNA Repair (Amst) 2005 doi: 10.1016/j.dnarep.2005.07.002. [DOI] [PubMed] [Google Scholar]
  38. Yu ZF, Mattson MP. Dietary restriction and 2-deoxyglucose administration reduce focal ischemic brain damage and improve behavioral outcome: evidence for a preconditioning mechanism. J Neurosci Res. 1999;57:830–9. [PubMed] [Google Scholar]

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