Days Of Regeneration Research Articles

A simple "soaking method" for RNA interference in the planarian Dugesia japonica.

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.

Reorganization of monoaminergic systems in the earthworm, Eisenia fetida, following brain extirpation.

The present study describes the major aspects of how monoaminergic (serotonin, dopamine) systems change in the course of regeneration of the brain in the earthworm (Eisenia fetida), investigated by immunocytochemistry, HPLC assay, and ligand binding. Following brain extirpation, the total regeneration time is about 80 days at 10 degrees C. On the 3rd postoperative day serotonin, and on the 11th postoperative day tyrosine hydroxylase-immunoreactive neurons can be observed in the wound tissue. Thereafter the number of the immunoreactive cells increases gradually, and by the 76th-80th postoperative days all serotonin- and tyrosine hydroxylase-immunopositive neurons can be found in their final positions, similarly to those observed in the intact brain. Labeled neurons located in the dorsal part of the regenerated brain appear earlier than the cells in lateral and ventral positions. Both serotonin- and tyrosine hydroxylase-immunoreactive neurons of the newly formed brain seem to originate from undifferentiated neuroblasts situated within and around the ventral ganglia and the pleura. Dopaminergic (tyrosine hydroxylase-immunoreactive) elements may additionally derive from the proliferation of neurons localized in the subesophageal ganglion and the pharyngeal nerve plexus. Following brain extirpation, both serotonin and dopamine levels, assayed by HPLC, first increase in the subesophageal ganglion; by the 25th day of regeneration, the monoamine content decreases in it and increases in the brain. Hence it is suggested that monoamines are at least partly transported from this ganglion to the regenerating brain. At the same time, (3)H-LSD binding can be detected in the regenerating brain from the 3rd postoperative day, showing a continuous increase until the 80th postoperative day, suggesting a guiding role of postsynaptic elements in the monoaminergic reinnervation of the newly formed brain.

Plasma membrane Ca2+-ATPase mRNA expression in murine hepatocarcinoma and regenerating liver cells.

The plasma membrane calcium ATPase (PMCA) is an ubiquitous enzyme that extrudes calcium from the cytoplasm to the extracellular space. Four PMCA genes through alternative splicing produce a large diversity of isoforms of this enzyme. We reported previously that the PMCA contained in AS-30D hepatocarcinoma cells showed significant differences in activity in comparison to normal and regenerating liver. In the present study we investigate if the difference in PMCA activity could be related to differential expression of mRNAs encoding different isoforms of PMCA. Using RT-PCR we found that variants 1b, 1x, and 4b are expressed in all liver samples. The hepatoma AS-30 and liver at 2 days of regeneration express low amounts of isoforms 2w, 4b and 4x, and do not express isoforms 4a, 4d and 4z. Fetal and neonatal liver do not express variants 4a and 4d, but they do express variants 4x and 4z. Immunoblot analysis showed a higher ratio ATPase/total protein in the hepatoma AS-30D in comparison to normal liver. Our results suggest that the Ca2+-ATPase kinetic pattern previously observed by us in the AS-30D cells, could be at least partially explained by changes in the mRNA expression of several of the PMCA isoforms expressed in the liver.

Mitotic Activity in Tissues of the Regenerating Respiratory Tree of the Holothurian Apostichopus japonicus (Holothuroidea, Aspidochirota)

Mitotic activity in the regenerating respiratory tree was studied in the holothurian Apostichopus japonicus after evisceration. Significant proliferation was recorded in the regenerating tissues. The highest number of mitoses was revealed in the inner epithelium, the mitotic index (MI) of which reached 3.44 ± 0.01% on the 10th day of regeneration, which was comparable to MI values of blastema of a regenerating amphibian limb. In the coelomic epithelium the number of dividing cells varied from 0.49% in the upper part (20th day) up to 2.87% in the base (10th day). The proliferative activity gradually decreased in the further course of regeneration. In spite of high MI values, the distribution of mitoses in tissues was even at all stages of regeneration and blastema did not form.

Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.

Characterization of human muscle type cofilin (CFL2) in normal and regenerating muscle.

Cofilins are actin binding proteins and regulate actin assembly in vivo. Numerous cofilin homologues have been characterized in various organisms including mammals. In mice, a ubiquitously expressed cofilin (CFL1) and a skeletal muscle specific cofilin (CFL2) have been described. In the present study, we identified and characterized a human CFL2 gene localized on chromosome 14, with high homology to murine CFL2. Furthermore, we provide evidence for differentially spliced CFL2 transcripts (CFL2a and CFL2b). CFL2b is expressed predominantly in human skeletal muscle and heart, while CFL2a is expressed in various tissues. Genetic defects of CFL2 were excluded for one human muscle disorder, the chromosome 14 linked distal myopathy MPD1, and shown to be only possible to be a rare cause of another, nemaline myopathy. In a mouse model of mechanically induced muscle damage the changes of cofilin expression were monitored during the first 10 days of regeneration, with dephosphorylated CFL2 being the major isoform at later stages of muscle regeneration. A similar predominance of dephosphorylated CFL2 was observed in chronically regenerating dystrophin-deficient muscles of Duchenne muscular dystrophy patients. Therefore, the CFL2 isoform may play an important role in normal muscle function and muscle regeneration.

Myostatin levels in regenerating rat muscles and in myogenic cell cultures.

Myostatin is a newly described member of the TGF-beta superfamily acting as a secreted negative regulator of skeletal muscle mass in several species, but whose mode of action remains largely unknown. In the present work, we followed the myostatin mRNA and protein levels in rat soleus and extensor digitorum longus (EDL) muscles regenerating in vivo from notexin-induced necrosis, and the myostatin transcript levels in two different in vitro myogenic differentiation models: i.e. in mouse BC3H1 and C2Cl2 cultured cells. The in vivo regenerating rat skeletal muscles showed a characteristic time-dependent expression of myostatin mRNA. After notexin injection, the transcript levels dropped below the detection limit on day 1 in soleus and close to the detection limit on day 3 in EDL, then increased to a maximum on day 7 in soleus and after 28 days finally reached the control values in both types of muscles. In contrast, the myostatin protein levels increased dramatically on the first days of regeneration in both muscles, i.e. at the time when its transcript level was low. Later on the myostatin protein level gradually declined to normal in soleus while in EDL it stayed high some days longer and decreased to normal on days 21-28. In vitro proliferating myoblasts produced low level of myostatin mRNA, which increased upon induction of differentiation suggesting that functional innervation is no prerequisite for myostatin expression. Myostatin production in vitro seems not to be dependent on myocyte fusion either, since it is observed in differentiated BC3H1 cells, which are defective in myofiber formation.

Regeneration and Proliferation of Embryonic and Adult Rat Hippocampal Neurons in Culture

Adult mammalian CNS neurons appear to be terminally differentiated and postmitotic. However, this conclusion may be due to nonpermissive conditions in the brain or in culture media. If embryonic rat hippocampal neurons are cultured in Neurobasal/B27 with FGF2, nearly all neurons proliferated until a maximum density was reached. Similarly, adult neurons can be cultured that fire action potentials and display immunoreactivity for neurofilament, MAP2, tau, and glutamate. Seventy percent of the 3000 isolated adult cells per milligram of brain tissue began to proliferate after 3 days in culture and incorporated BrdU. By 4 days of regeneration in culture, virtually all neuron-like cells with asymmetric processes were glutamate positive and immunoreactive for neurofilament. Immunoreactivity of the intermediate filament stem cell marker nestin increased in adult cells to levels present in freshly isolated embryonic neurons. These are the first studies to demonstrate that over 50% of adult CNS cells with neuron-like characteristics retain regenerative and proliferative potential.

Effect of Triiodothyronine Administration on the Recovery of Liver Oxidative Capacity after Partial Hepatectomy

The aim of the present work was to investigate the role of triiodothyronine on liver regeneration after partial hepatectomy in the rat. During the first 3 days of liver regeneration, total protein, total DNA content and total cytochrome c oxidase activity of the residual tissue increased from 30% immediately after partial hepatectomy to 70% of the preoperative values in control rats. In triiodothyronine-treated rats, the increase in total protein was the same (70%), however, the increase in total liver DNA content and total cytochrome c oxidase acitivity was 100 and 135%, respectively during the first 3 days of regeneration. Triiodothyronine administration also increased significantly the activity of mitochondrial ATPase in regenerating liver.

The levels of retinal mRNA for gefiltin, a neuronal intermediate filament protein, are regulated by the tectum during optic fiber regeneration in the goldfish

Reorganization of the intermediate filament (IF) network during axonal regeneration is accompanied by changes in the expression of various IF proteins. An increase in expression of the neuronal IF subunit gefiltin in goldfish retinal ganglion cells (RGCs) has been linked to the unique ability of the goldfish optic nerve to regenerate following injury. Evidence suggests that the optic tectum, the target of optic fibers, may regulate the expression of gefiltin during regeneration. To address this issue we examined gefiltin mRNA levels during optic fiber regeneration in the presence or absence of the tectum. We found that gefiltin mRNA levels in the RGCs of animals that received an optic nerve crush (ONC group) began increasing by 10 days, peaked from 20 to 38 days at 5.5-fold over normal, and declined to near normal values by 115 days. In animals that had the entire tectum removed as well as an optic nerve crush (ETR group), gefiltin mRNA levels increased by 10 days, peaked at 20 days at 5.5 to 6.5-fold over normal, and although they dropped slightly thereafter, they remained elevated at 5-fold over normal for at least 115 days. When axons regenerated to the ipsilateral tectal lobe as a result of a left tectal lobe removal and left eye removal surgery (LTR/LER group), the expression pattern of gefiltin mRNA paralleled that of the ONC group. We also found that the abundance of gefiltin subunits in the retina was elevated at 30 days of regeneration in ONC and ETR animals, and that levels in the nerve were reconstituted to 80% of normal by 30 days. These results demonstrate that increases in gefiltin mRNA and protein levels during optic nerve regeneration are independent of the tectum, whereas the downregulation of gefiltin mRNA levels in the late stages of regeneration is entirely dependent upon the tectum.

Developmentally regulated expression of cytochrome-c oxidase isoforms in regenerating rat skeletal muscle.

The developmental expression of tissue-specific isoforms of cytochrome-c oxidase (COX) subunit VIII [heart (COX VIII-H) and liver (COX VIII-L)] and the influence of innervation were examined in regenerating fast [extensor digitorum longus (EDL)] and slow (soleus) muscles. In adult muscles, COX VIII-H was the predominant isoform. The COX VIII-L mRNA was expressed 3 days after induction of regeneration, and it progressively decreased after 7, 10, 14, and 30 days of regeneration in both muscles. In contrast, the expression of COX VIII-H mRNA accumulated as myogenesis proceeded to the myotube stage between 7 and 10 days of regeneration and progressively increased to near control levels by 30 days. The influence of innervation on the expression of COX VIII and alpha-actin isoforms was examined in control, innervated, and denervated regenerating muscles at 3 and 10 days. The relative expression of COX VIII-L mRNA in denervated regenerating EDL muscles was significantly greater, while that of COX VIII-H was significantly less than in innervated regenerating EDL muscles after 10 days of regeneration. Similarly, cardiac alpha-actin mRNA levels were elevated in denervated regenerating EDL muscles after 10 days of regeneration. In conclusion, motor innervation influences the transition from the COX VIII-L to COX VIII-H isoform during myogenesis in regenerating muscles.

Cell population changes during atrophy and regeneration of rat parotid gland

Limited data exist regarding the changes in number and location of myoepithelial cells during salivary gland atrophy and regeneration. Through the use of double immunohistochemical labeling for muscle-specific actin and amylase coupled with morphometric analysis, this study investigated the changes in distribution and proportion of cell types during salivary gland atrophy/regeneration phases in a model previously used to study proliferation in rat parotid gland. The double immunohistochemical labeling clearly showed the changes in proportion of cell types in the atrophying and regenerating glands. The morphometric analysis showed that the relative myoepithelial area increased (as did the intercalated duct and striated duct areas) as the gland atrophied. Myoepithelial cells occupied 19.0% of the total epithelial area by day 7 of atrophy, up from 2.7% in the resting gland. Regeneration of acinar cells was obvious 1 day after duct release. The myoepithelial cell area decreased to 4.3% of the total epithelial area by day 14 of regeneration; this value was higher than the percentage of area in the resting gland ( p = 0.02). The relative areas of acinar, striated duct, and intercalated duct cells returned to resting levels after 14 days of regeneration. The morphometric and histologic results of this study show that the parotid gland is capable of regenerating to essentially normal anatomic condition after 7 days of gland atrophy and then 14 days of regeneration. Each type of cell, however, responded to the atrophy and regeneration differently. Atrophy of salivary glands from radiation therapy, Sjögren's syndrome, or sialadenitis is an important clinical problem. Study of the salivary gland response to atrophy and regeneration may provide a framework for designing strategies for the radioprotection of salivary glands or methods by which to treat or reverse the effects of gland atrophy.

Activation of the skeletal alpha-actin promoter during muscle regeneration.

Little is known concerning promoter regulation of genes in regenerating skeletal muscles. In young rats, recovery of muscle mass and protein content is complete within 21 days. During the initial 5-10 days of regeneration, mRNA abundance for IGF-I, myogenin and MyoD have been shown to be dramatically increased. The skeletal alpha-actin promoter contains E box and serum response element (SRE) regulatory regions which are directly or indirectly activated by myogenin (or MyoD) and IGF-I proteins, respectively. We hypothesized that the skeletal alpha-actin promoter activity would increase during muscle regeneration, and that this induction would occur before muscle protein content returned to normal. Total protein content and the percentage content of skeletal alpha-actin protein was diminished at 4 and 8 days and re-accumulation had largely occurred by 16 days post-bupivacaine injection. Skeletal alpha-actin mRNA per whole muscle was decreased at day 8, and thereafter returned to control values. During regeneration at day 8, luciferase activity (a reporter of promoter activity) directed by -424 skeletal alpha-actin and -99 skeletal alpha-actin promoter constructs was increased by 700% and 250% respectively; however, at day 16, skeletal alpha-actin promoter activities were similar to control values. Thus, initial activation of the skeletal alpha-actin promoter is associated with regeneration of skeletal muscle, despite not being sustained during the later stages of regrowth. The proximal SRE of the skeletal alpha-actin promoter was not sufficient to confer a regeneration-induced promoter activation, despite increased serum response factor protein binding to this regulatory element in electrophoretic mobility shift assays. Skeletal alpha-actin promoter induction during regeneration is due to a combination of regulatory elements, at least including the SRE and E box.

Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tigrina.

Freshwater planarians (Platyhelminthes, Turbellaria) show a great degree of morphological plasticity, making them a useful model for studying cell differentiation and pattern restoration processes during regeneration. Using confocal microscopy and a monoclonal antibody specific for muscle cells (TMUS-13), we have monitored the restoration of the body wall musculature during head regeneration in whole-mount organisms. Our results show that until the 4th day of regeneration the blastema is occupied by very disorganized muscle fibers, that from this moment become progressively organized restoring the original muscle pattern. In addition to recognizing mature muscle cells, TMUS-13 also recognizes differentiating myocytes, allowing us to trace the origin of newly formed muscle cells. We report that myocytes are detected in the postblastema region as early as day 1 of regeneration. This is the first demonstration that, in addition to serving as a proliferative zone as previously described, overt differentiation begins in the postblastema, at least for muscle cells. We also show that the TMUS13 antigen is the myosin heavy-chain gene from planarians.

NaOH Regeneration of Pb and Phenol-Laden Activated Carbon. I. Batch Study Results

Granular activated carbon (GAC) has been used to remove organics and metals simultaneously from wastewaters (4). For a regeneration procedure to be effective on GAC containing both metals and organics, it must remove both the metals and organic compounds. In this study, GAC saturated with Pb and phenol was regenerated using an NaOH rinse in batch mode. Four variables were investigated: 1) regenerant concentration (0, 0.1, and 1.0 N), 2) regenerant to carbon mass ratio (5:1 and 10:1), 3) regenerant temperature (22 and 80°C), and 4) regeneration time (0.33, 1, 3, 7, and 21 days). Maximum regenerations of 47% for Pb and 48% for phenol were observed. The 1.0 N NaOH regenerated a higher percentage of both Pb and phenol when compared to the other regenerants. The 10:1 regenerant to carbon ratio resulted in increased regeneration over the 5:1 regenerations. Pb regeneration was slightly higher at 80°C than at 22°C whereas phenol regeneration was slightly lower at 80°C than at 22°C. Maximum regenerations for both Pb and phenol were observed within the first day of regeneration, in most cases within the first 8 hours.

Activity of mu- and m-calpain in regenerating fast and slow twitch skeletal muscles.

Calpains--non-lysosomal intracellular calcium-activated neutral proteinases, form a family consisting of several distinct members. Two of the isoenzymes: mu (calpain I) and m (calpain II) responded differently to the injury during complete regeneration of Extensor digitorum longus (EDL) muscle and partial regeneration of Soleus muscle. In the crushed EDL the level of m-calpain on the 3rd and 7th day of regeneration was higher than in non-operated muscles, whereas the activity of this calpain in injured Soleus decreased. The level of mu-calpain in EDL oscillated irregularly during regeneration whereas in Soleus of both injured and contralateral muscles its level rapidly rose. Our results support the hypothesis that m-calpain is involved in the process of fusion of myogenic cells whereas mu-calpain plays a significant but indirect role in muscle regeneration.

Changes in mRNA levels of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms in the rat soleus muscle regenerating from notexin-induced necrosis.

The relative mRNA levels corresponding to the different sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase isoforms (SERCA1a, SERCA1b, SERCA2a, SERCA2b and SERCA3) were measured by reverse transcriptase-PCR in rat soleus muscles regenerating after notexin-induced necrosis. The succession of appearance of the different types of SERCA mRNA species in regenerating muscle largely recapitulates those observed during normal ontogenesis. The mRNA levels of the muscle-specific isoforms SERCA1a and SERCA2a became very low on the first and third days after injection of the snake venom. It was only on the fifth day of regeneration that the mRNA of the neonatal variant of the fast-twitch skeletal SERCA1b isoform began to rise, well before the other SERCA transcripts. At 7 and 10 days, i.e. at a time when the new myofibres normally become reinnervated, the mRNA level of SERCA1a and SERCA2a increased markedly, but the fast-twitch skeletal SERCA1a isoform was still the most prominent. On day 21, in the advanced stage of regeneration, a switch in the relative expression levels of SERCA1a and SERCA2a mRNA was observed and the ratio of both isoforms became similar to that found in the normal soleus muscles. This was followed by a decline in the level of all SERCA mRNA species, so that on day 28 the levels of the sarcoplasmic/endoplasmatic-reticulum Ca(2+)-pump RNAs was again lower but their ratio remained similar to that of the untreated control soleus.

Multiple neurotrophic factors including NGF-like activity in nerve regeneration chamber fluids

Silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve of adult rats. Neurotrophic activities in cell-free fluids collected from the chambers were determined using bioassays for survival of embryonic chick ciliary and sympathetic neurons in culture. Separation by molecular exclusion HPLC of the components of fluids collected l, 2 or 3 days after implantation revealed the presence of a multitude of neurotrophic factors differing in their molecular weights, specificity towards the two types of neurons, and time course. Antiserum to nerve growth factor partially blocked sympathetic activity of fluids collected at 1 day. Affinity purified antibody was also effective and completely eliminated bioactivity of HPLC fractions corresponding to the molecular weight of nerve growth factor. The presence in the fluids of 13–18 and 20–32 kD components active towards ciliary neurons is consistent with the release of fibroblast growth factor and ciliary neurotrophic factor respectively. The stimulation of sympathetic neurons by the 13–18 kD material, and also by 4–6 and 7–11 kD components cannot be entirely accounted for by known factors. This study demonstrates that a number of neurotrophic factors, which differ in their specificity towards sympathetic and parasympathetic neurons, are made available to the region of axonal regrowth over the first few days of regeneration. Contrary to earlier reports, nerve growth factor-like activity was shown to be present in nerve regeneration chambers.

Hypothyroidism prolongs and increases mdx muscle precursor proliferation and delays myotube formation in normal and dystrophic limb muscle.

Hypothyroidism (induced by 8 weeks of oral 0.05% propylthiouracil) heightened the phenotype of mdx mouse dystrophin-deficient myopathy to more closely resemble human Duchenne muscular dystrophy. Muscle repair after crush injury to the tibialis anterior muscle (TA) in hypothyroid mdx mice showed decreased myotube formation and delayed debris removal. To investigate whether reduced muscle precursor cell proliferation can account for the effects of hypothyroidism on repair from injury, immunocytochemistry for neural cell adhesion molecule (NCAM) on muscle precursor cells and autoradiography to detect DNA synthesis were performed in control and mdx TA. The proportions of labelled polymorphonuclear leukocyte nuclei (PMN), myotube nuclei (MN), and total mononuclear cell nuclei (TLN, the majority being muscle precursors) were counted in defined areas of regenerating TA after 2 and 4 days recovery. MN and the numbers of activated satellite cell nuclei on intact fibers were counted in surviving areas. In the same muscle, earlier phases of regeneration were observed in areas distal than proximal to the injury. At 2 days of regeneration, labelled PMN were increased in treated compared with untreated mdx TA. In distal areas at 4 days, fewer muscle precursors had recently fused to myotubes in treated than in untreated mdx. In proximal areas 4 days (relatively late in repair), TLN data suggested that muscle precursor proliferation was greater in hypothyroid compared with untreated mdx TA. NCAM immunostaining was consistent with proliferation data and confirmed that there were more muscle precursors in mdx than in control regenerating muscle. These results suggest that hypothyroidism prolongs and increases the phase of replication by mdx muscle precursors and delays precursor fusion into myotubes in regeneration.

Clusterin expression and apoptosis in tissue remodeling associated with renal regeneration

To analyze the role of clusterin in renal diseases involving a regenerative process, we have used a novel rodent model to compare temporal and spatial expression of clusterin mRNA. Thus, renal artery stenosis was used to induce unilateral non-infarctive renal atrophy. After several weeks, when cellular pathology of atrophic kidneys involved minimal apoptosis or inflammatory response and mitosis was at normal levels, regeneration of atrophic kidneys was stimulated by removal of the contralateral healthy kidneys. The regrowth response was very rapid and involved renal hyperplasia rather than hypertrophy. Regenerating kidneys were studied 0, 4, 8, 24 hours and 2, 3, 5, 7, and 14 days after contralateral nephrectomy. Several parameters were compared: level and localization of clusterin mRNA; cell proliferation; cell dedifferentiation and redifferentiation and apoptosis. During the acute regenerative phase (first 24 hr) clusterin expression was markedly increased, decreasing to untraceable levels by five days of regeneration. Clusterin mRNA was localized in dilated or collapsed atrophic tubules that had lost identifying surface structures of normal tubular epithelium (termed dedifferentiated). Clusterin was also localized in the periphery of some blood vessel walls. Cell proliferation peaked at three to five days of regeneration, and was also localized in dedifferentiated tubules. Despite the regenerative stimulus, an unexpected result was a transient but marked increase in apoptotic cell death in atrophic tubules in the first 24 hours of regeneration. Our results provide evidence of a temporal association between increased clusterin expression and apoptosis, but in situ localization showed clusterin mRNA over apparently viable, as well as apoptotic, cells in the epithelium of tubules showing clusterin expression. Clusterin mRNA was rarely identified over epithelial cells in foci of non-atrophic (non-dedifferentiated) nephrons that responded to the regenerative stimulus by cellular hypertrophy. The dramatic response after initiation of regeneration, especially the initiation of apoptosis in the tubular epithelium, may have applications for the study of genetic changes leading to renal oncogenesis.