Developmental regulation of hypomethylation of delta-crystallin genes in chicken embryo lens cells.

Sequences in the two delta-crystallin genes become hypomethylated when they are expressed in the chick lens. This system is particularly advantageous for studying temporal changes in hypomethylation, since lens tissue can be isolated at all developmental stages. In previous work we have shown that most HpaII sites become hypomethylated within the delta 1-crystallin gene long after delta-crystallin gene activation. One site is hypomethylated when crystallin mRNA begins to be synthesized at high levels at 50 h; we show here that this site maps to the 3' end (intron 15) of the delta 1-crystallin gene. In addition, we have examined the methylation status of HpaII and HhaI sites found near the 5' end of the delta 1-crystallin gene. Two HhaI sites adjacent to a viral core enhancer sequence in intron 2 are also first hypomethylated at 50 h. These findings point to regions of the delta 1 gene that should be investigated further for functional significance in regulating delta-crystallin transcription.

Autocrine, endocrine and paracrine regulation of growth abnormalities in autosomal dominant polycystic kidney disease.

Genetic polycystic kidney disease (ADPKD) is an autosomal dominant trait in man, the phenotypic expression of which is characterized by enormous cystic enlargement of renal tubules. Since this is, in part, a result of aberrant epithelial cell proliferation, the nature of this defect has been characterized by comparison of growth factor action on defined epithelial primary monolayer cultures derived from individually microdissected normal human renal proximal and distal tubules and ADPKD cyst-lining epithelia. Mitogenic assays showed an increased sensitivity of ADPKD epithelia to stimulation by the combination of the endocrine factors hydrocortisone (dexamethasone) and insulin, and Northern analysis suggested increased levels of insulin receptor steady state mRNA. The most potent, single mitogen was epidermal growth factor (EGF), and hypersensitivity to EGF in ADPKD epithelia was correlated with increased mRNA levels for EGF receptor in ADPKD kidneys. The localization in vivo of EGF immunoreactivity in ADPKD cyst-lining epithelia and in (apical) cyst fluids and the demonstration of EGF-receptor immunostaining and specific [125I]EGF binding to apical cell surfaces suggested an autocrine mechanism of growth stimulation by EGF in ADPKD epithelia. Transforming growth factor beta was an inhibitor of normal renal tubule proliferation but was unable to completely inhibit EGF stimulation in ADPKD cultures. Platelet-derived growth factor (PDGF) immunoreactivity which was also seen in ADPKD cyst epithelia, was not mitogenic to ADPKD epithelia but did stimulate division in ADPKD fibroblasts in vitro. This suggested a paracrine regulation of the observed fibrosis in ADPKD.

Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia.

The question of why chronic renal diseases progress is a topic only recently investigated. Putative causes such as proteinuria do not account for all aspects of progressive renal disease. An alternative mechanism, chronic hypoxia, is proposed that might better explain certain elements of progressive renal disease, but elements of the hypothesis remain subject to further study.

A rabbit model for nonunion of lumbar intertransverse process spine arthrodesis.

STUDY DESIGN: A rabbit model for lumbar intertransverse process spine arthrodesis was utilized, with blind evaluation of the control and experimental animals. OBJECTIVES: To establish an animal model for motion-induced nonunion of lumbar intertransverse process spine arthrodesis and to determine if there was an early time period during which excessive motion at the arthrodesis site was deleterious to fusion. SUMMARY OF BACKGROUND DATA: Most previously reported animal models for nonunion of lumbar spine arthrodesis do not use intertransverse process fusion and do not allow variable control of the nonunion-inducing factor. METHODS: Forty-two adult New Zealand white rabbits underwent posterolateral intertransverse process spine arthrodesis with autogenous iliac crest graft. Rabbits in the experimental group (n = 23) underwent a lifting protocol to produce motion at the arthrodesis site; those in the control group (n = 12) remained undisturbed in their cages. Arthrodeses were assessed radiographically and manually to determine if fusion had occurred. RESULTS: Lifted rabbits exhibited a 13% fusion rate compared to a 50% fusion rate in controls. Rabbits lifted during only the first 2 postoperative weeks exhibited a fusion rate that was significantly less (P = 0.03) than that of controls. CONCLUSIONS: A small animal model for motion-induced nonunion of lumbar intertransverse process spine arthrodesis has been established. With respect to excessive motion as a promoter of nonunion in spine arthrodeses, the early postoperative time period appears critical.

The effect of different surgical releases on thoracic spinal motion. A cadaveric study.

STUDY DESIGN: Two separate experiments (A and B), each involving six human cadaveric torsos with intact rib cages and sternums, were done to determine the effect of two different sequences of surgical releases (at T8-T9) on thoracic spinal motion. OBJECTIVES: Experiment A was designed to test the effects of three releases in sequence from anterior to posterior, analogous to a two-stage operative treatment with anterior and posterior releases. Experiment B, which involved three releases, was designed to determine 1) if unilateral posterior total facetectomy alone allowed a significant increase in motion and 2) if rib head resection without discectomy allowed a significant increase in motion. SUMMARY OF BACKGROUND DATA: In the surgical treatment of thoracic spinal deformity, surgical release is often done to impart additional flexibility to the spine. Available releases include discectomy, rib head resection, and facetectomy. There is little work to date on the relative importance of the disc, rib head, and facet joint in the stability of the thoracic spine. METHODS: In experiment A and experiment B, the cadaveric torsos were mounted on a custom-made loading frame. Mechanical testing (using weights, pulleys, and digital goniometers) was done after each surgical release to measure the extent of angular rotation in the coronal plane (right lateral bending and left lateral bending) and in the sagittal plane (flexion and extension). RESULTS: The combination of rib head resection and radical discectomy provided the greatest increase in thoracic spinal motion. Standard discectomy alone did not allow a significant increase in motion. Rib head resection without discectomy did not allow a significant increase in motion. Unilateral posterior total facet excision did not allow a significant increase in motion. CONCLUSIONS: These experiments indicate that the combination of rib head resection and radical discectomy may be the optimal thoracic spinal release.

Strength of fixation of anterior vertebral body screws.

STUDY DESIGN: The technique of hole preparation and the placement or orientation of anterior transvertebral screws in relation to the endplates were the subject of two experiments. OBJECTIVES: Experiment 1--to determine if anterior transvertebral body screws have higher pull-out strengths after unicortical predrilling compared with a bicortical predrilling; Experiment 2--to determine the effect of the screw's orientation in the vertebral body on its resistance to loosening. SUMMARY AND BACKGROUND DATA: There are a variety of surgical techniques for using anterior transvertebral screws in the vertebral body without any clear guidelines as to which techniques optimize the strength of fixation. METHODS: In experiment 1, 31 cadaveric vertebral bodies were tested for pull-out strength of transvertebral body screws placed after unicortical predrilling compared with bicortical predrilling. In experiment 2, 48 cadaveric vertebral bodies were tested with transvertebral screws inserted using four different screw orientations. RESULTS: There is no statistically significant difference in pull-out strength of anterior transvertebral body screws inserted after unicortical compared with bicortical predrilling (Experiment 1). The resistance to loosening is greatest when transvertebral screws are oriented in the "superior oblique" position (Experiment 2). CONCLUSIONS: Experiment 1--preparation of the far cortex by predrilling is not necessary. Experiment 2--transvertebral screws should be obliquely oriented so that the forces applied to the screws are resisted by both of the endplates.

The role of angiotensin II in renal growth.

Angiotensin II (AII) has many of the features of the archetypical growth factors and appears to be a growth regulator in the kidney. AII binds to specific cell surface receptors present on a number of different renal cell types including mesangial, vascular smooth muscle, tubular and interstitial cells, and activates many of the intracellular signalling pathways associated with cell growth. In vitro AII can potentiate the mitogenic effect of other growth factors such as EGF. AII induces hypertrophy of vascular smooth muscle cells but the role of AII in the growth of other renal cell types has not been systematically studied.

Regulation of gene expression in renal compensatory growth.

Compensatory renal hypertrophy is characterized by an increase in cell protein content. There are a number of different cellular mechanisms by which such an increase could be mediated. The relative contributions of transcriptional and posttranscriptional regulation of gene expression to the increase in constitutively expressed cellular proteins were examined in mouse kidneys undergoing compensatory growth following unilateral nephrectomy (UNI-NX). Over the 14-day study period, total protein per kidney increased by up to 37%. Northern blot analysis of the expression of the mRNAs for a number of genes associated with cell growth indicated that there was no significant increase in steady-state levels of these selected genes in nephrectomized compared with sham-operated animals. In vitro assays to measure transcriptional regulation of the same set of genes in compensatory growth showed no increase in the rate of transcription. However, transcription of the 18S ribosomal RNA gene was increased 24 hours after nephrectomy. Immunoblots of two representative proteins showed that the concentration of these proteins in total kidney protein remained constant in the different experimental groups. Since total protein increased, the concentration of these proteins in the hypertrophied kidney must have increased. Taken together, these data suggest that posttranscriptional mechanisms may account for the increase in at least some constitutively expressed proteins in cells undergoing compensatory growth.

Post-transcriptional regulation of gene expression in compensatory renal hypertrophy.

An increase in cell protein content is a characteristic of renal cells undergoing compensatory hypertrophy. The contribution of transcriptional and post-transcriptional regulation of mRNAs to the increase in constitutively expressed proteins in hypertrophy was examined in mouse kidneys after unilateral nephrectomy (UNI-NX). No significant increases in steady state levels or transcription of mRNAs for eight growth-related genes (Na+K+ATPase, ADP/ATP translocase, beta-actin, ornithine decarboxylase, poly A(+)-binding protein and two genes expressed in androgen-induced hypertrophy) were observed from 30 min to 10 days after UNI-NX. In contrast, transcription of the 18S ribosomal gene increased by 24% within 24 h after surgery. By Western blotting the proportion of two specific proteins relative to total cell protein was constant over 10 days. Since total protein/kidney increased by 28% over this period the concentration of these specific proteins/kidney must also have increased. Although it is possible that one or more 'hypertrophy specific' genes are transcriptionally regulated to initiate the growth process, the present data suggest that protein accumulation in compensatory hypertrophy is regulated, predominantly, by post-transcriptional mechanisms.

Renal growth responses to acute and chronic injury: routes to therapeutic intervention.

Knowledge of the control of cell growth and extracellular matrix deposition has assumed center stage in the understanding of how the diseased kidney responds to injury. After acute tubular injury, there may be reversible, partial depolarization of renal cells or cell necrosis. The latter requires a regenerative response, which could be under the control of growth factors such as epidermal growth factor (EGF). Up-regulation of EGF receptors on viable cells provides the cell with an enhanced growth response despite a reduction in EGF production by the kidney. Acute glomerular injury involves a highly complex network of cytokines and growth inhibitors, the most important of which appear to be platelet-derived growth factor as a mitogen and transforming growth factor beta as an activator of extracellular matrix deposition. The long-term growth responses of the kidney to injury, reflected by chronic renal diseases, include tubular hypertrophy in those nephrons which are less affected by the primary disease. Tubular cell enlargement appears to proceed along a pathway that is different from the growth in cell size which precedes cell division, at least as indicated by a fundamentally different pattern of early gene expression. This pattern is not suggestive of a classical growth factor-initiated process. Other chronic changes that seem to correlate well with the progression of human disease are tubular atrophy and interstitial fibrosis. Growth factors produced by tubular cells may cause proliferation and matrix deposition by adjacent interstitial fibroblasts. A scheme is proposed in which low-grade ischemic injury to tubular cells, secondary to microvascular injury, leads to tubular atrophy, the release of growth factors, interstitial fibrosis, and the obliteration of peritubular capillaries. This would aggravate primary glomerular injury by compromising the vascular outflow from the glomerulus and would account for the long-recognized association between tubulo-interstitial injury and the progression of a variety of renal diseases. The use of growth factors to stimulate specific growth responses, antibodies, or inhibitory molecules to inhibit scarring generated by cytokines and the potential for genetic manipulation of the kidney provide future avenues for manipulating the growth response of the diseased kidney.

Progressive renal disease: fibroblasts, extracellular matrix, and integrins.

Progressive renal disease is characterized by expansion of the tubulo-interstitium and accumulation of extracellular matrix within this tissue compartment. Interstitial fibroblasts are the primary producers of the interstitial matrix, and in the evolution of tubulo-interstitial fibrosis these cells undergo changes, namely increased proliferation, differentiation to myofibroblasts, and altered extracellular matrix metabolism, all of which, in other cell types, have been shown to be regulated by the major family of extracellular matrix receptors, the integrins. In the normal kidney, interstitial fibroblasts express alpha1, alpha4, alpha5, and beta1 integrins, and fibrosis is associated with increased expression of alpha1, alpha2, alpha5, alphav, and beta1 integrins. In particular, alpha5, beta1, and alphav are suggested to be linked with the fibrotic process. In vitro, renal fibroblasts express a similar range of integrins, and ligation of selected receptors is associated with specific functions. Ligation of alpha6 stimulates proliferation, while alpha5 promotes expression of myofibroblastic phenotype, and beta1 integrin has been implicated in cell contraction. Recent studies suggest that renal fibroblasts also express the non-integrin matrix receptors, discoidin domain receptors, and that changes in activation of these receptors may be associated with fibrogenic events. Thus the current, albeit limited, data suggest an important role for receptors for extracellular matrix molecules in the pathogenesis of progressive renal fibrosis.

Differential response of activated versus non-activated renal fibroblasts to tubular epithelial cells: a model of initiation and progression of fibrosis?

BACKGROUND/AIMS: The mechanisms of initiation and maintenance of renal fibrosis remain obscure, but one hypothesis highlights the importance of tubular epithelial cell-interstitial fibroblast interactions and suggests that tubular injury may be a precipitating factor. The aims of the study were to examine the effects of factors of proximal tubular origin on renal fibroblasts expressing different levels of smooth muscle actin (SMA; a putative marker of fibroblast activation) and to examine the modulation of SMA by extracellular matrix (ECM) proteins and transforming growth factor beta 1 (TGF-beta 1), a major profibrotic cytokine. METHODS: Primary cultures of rat cortical fibroblasts (CF) and the rat kidney fibroblast cell line NRK-49F were (1) cultured on different ECM proteins; (2) treated with medium conditioned by rat proximal tubular epithelial cells (PTE), and (3) treated with TGF-beta 1. SMA protein expression was examined by immunocytochemistry, while expression of MMP-2, MMP-3, TIMP-1, and collagen I mRNA was determined by Northern blot analysis or reverse-transcriptase polymerase chain reaction. RESULTS: In cells with low basal levels of SMA (CF), serum was the most potent inducer of increased SMA expression, although ECM proteins also modulated expression. With high basal levels of SMA expression (NRK), ECM proteins alone had little or no effect, but acted synergistically with serum to stimulate expression. In CF, PTE-conditioned medium (CM) had no effect on SMA and TIMP mRNA levels, but suppressed expression of MMP mRNAs. In NRK-49F, PTE-CM stimulated SMA and TIMP-1 mRNA levels, but had no effect on MMP mRNA levels. Although TGF-beta 1 modulated some cellular responses in NRK-49F, neutralizing antibody studies showed it was not the main mediator of the PTE-CM-induced effects. CONCLUSIONS: These data suggest (1) that in renal fibroblasts SMA expression can be modulated by both serum and ECM proteins and (2) that PTE induce a fibrogenic phenotype in both non-activated (low SMA) and activated (high SMA) fibroblasts via different mechanisms.

Mechanisms of tubulo-interstitial injury in progressive renal diseases.

A vast amount of evidence, based upon human renal biopsy material, indicates that the presence of tubular atrophy and interstitial fibrosis is a better indicator of outcome of renal function than is the extent of glomerular sclerosis. The pathophysiological basis for this surprising fact has not been adequately addressed. In this review we point out that the systemic hypertension which accompanies most forms of chronic renal disease could impact adversely upon the vasodilated interstitial vascular compartment which, together with a component of primary capillary injury related to the disease process, could cause progressive obliteration of particular capillaries. This would initiate a process of chronic tubular ischaemia ultimately leading to tubular atrophy. Since tubular cells have been shown to produce an array of cytokines and growth factors which modulate fibroblast proliferation, extracellular matrix production and chemo-attracts for infiltrating cells, it is further proposed that it is the tubular injury which initiates the deleterious cascade of events. Tubular injury may be aggravated by the filtration of potentially 'noxious' molecules through the diseased glomerulus and by infiltrating cells. As the vascular bed into which glomerular blood flow empties is progressively obliterated, glomerular function declines and renal failure advances in relation to the degree of tubulo-interstitial fibrosis.

The induction of new ductal growth in adult prostatic epithelium in response to an embryonic prostatic inductor.

Tissue recombinants were prepared with a single epithelial ductal tip from adult prostate and mesenchyme from either the embryonic urogenital sinus or adult urinary bladder. Recombinants were grown in vivo beneath the renal capsule of male hosts. After 4 weeks of in vivo growth, extensive growth of arborizing ducts was apparent in recombinants composed of urogenital sinus mesenchyme and a single adult prostatic ductal tip. One-dimensional polyacrylamide gel electrophoresis indicated that these recombinants contained many of the proteins of the mature prostate. Heterospecific recombinants (rat urogenital sinus mesenchyme and mouse prostatic epithelium) showed the ductal tissue to be derived solely from the prostatic epithelium. In recombinants of a prostatic ductal tip with mesenchyme from the urinary bladder, ductal growth was absent, the ductal tip was maintained as a single, discrete, epithelial structure, and the protein composition of these recombinants more closely resembled that of the bladder. The results demonstrate that the epithelial cells of the adult prostate can participate in new ductal growth in response to an embryonic prostatic inductor. These data provide experimental evidence to support the hypothesis that human benign prostatic hyperplasia may result from the anomalous reactivation of embryonic growth potential in the adult prostate.

Hypoxia promotes fibrogenesis in human renal fibroblasts.

BACKGROUND: The mechanisms underlying progressive renal fibrosis are unknown, but the common association of fibrosis and microvascular loss suggests that hypoxia per se may be a fibrogenic stimulus. METHODS: To determine whether human renal fibroblasts (HRFs), the primary matrix-producing cells in the tubulointerstitium, possess oxygen-sensitive responses relevant to fibrogenesis, cells were exposed to 1% O2 in vitro. RESULTS: Hypoxia simultaneously stimulated extracellular matrix synthesis and suppressed turnover with increased production of collagen alpha1(I) (Coll-I), decreased expression of collagenase, and increased tissue inhibitor of metalloproteinase (TIMP)-1. These effects are time dependent, require new RNA and protein synthesis, and are specific to hypoxia. The changes in Coll-I and TIMP-1 gene expression involve a heme-protein O2 sensor and protein kinase- and tyrosine kinase-mediated signaling. Although hypoxia induced transforming growth factor-beta1 (TGF-beta1), neutralizing anti-TGF-beta1-antibody did not block hypoxia-induced Coll-I and TIMP-1 mRNA expression. Furthermore, hypoxic-cell conditioned-medium had no effect on the expression of these mRNAs in naive fibroblasts, suggesting direct effects on gene transcription. Transient transfections identified a hypoxia response element (HRE) in the TIMP-1 promoter and demonstrated HIF-1-dependent promoter activation by decreased ambient pO2. CONCLUSIONS: These data suggest that hypoxia co-ordinately up-regulates matrix production and decreases turnover in renal fibroblasts. The results support a role for hypoxia in the pathogenesis of fibrosis and provide evidence for novel, direct hypoxic effects on the expression of genes involved in fibrogenesis.

Acidic FGF regulation of hyperproliferation of fibroblasts in human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cystic tubule enlargement and expansion of the interstitium associated with fibrosis. Our previous studies have analyzed the increased proliferation of cystic epithelial cells and this study examines the basis of increased proliferation of interstitial fibroblasts associated with ADPKD disease progression. ADPKD fibroblasts show phenotypic alterations in vitro, have acquired the capacity to grow in soft agar, and show an increased mitogenic response to a variety of growth factors particularly acidic FGF (aFGF). ELISA, Western immunoblot analysis, and immunocytochemistry showed increased aFGF content in ADPKD tissues and fibroblasts in culture, and aFGF was secreted into the extracellular matrix and conditioned medium, respectively. No alterations in aFGF receptor number were found, but Scatchard analysis of 125I-aFGF binding suggested an increased affinity of binding to the low affinity receptor, and covalent cross-linking analysis suggested the presence of novel putative receptors (120 kDa) in ADPKD fibroblasts. Signaling abnormalities were found, since aFGF incubation resulted in the tyrosine phosphorylation of additional substrates, more rapidly and for a more sustained duration in ADPKD fibroblasts than in normal fibroblasts. These findings suggest an important role for acidic FGF in the hyperproliferation of interstitial fibroblasts associated with disease progression in human ADPKD.