Diane Felsen

Transforming Growth Factor-β, Basement Membrane, and Epithelial-Mesenchymal Transdifferentiation : Implications for Fibrosis in Kidney Disease

Interstitial fibrosis is characteristic of many clinical entities including diabetes, ureteral obstruction, transplant rejection, and glomerulonephritis. 1 The interstitial fibrosis that accompanies renal disease is a complex process involving derangements in both the synthesis and degradation of collagen and other extracellular matrix proteins. Cellular sources of the extracellular matrix laid down during the development of interstitial fibrosis include fibroblasts and infiltrating macrophages. 1 The fibroblasts may be resident renal fibroblasts, fibroblasts that migrate into the kidney from external sources, or a specialized population of fibroblasts known as myofibroblasts. 2 Furthermore, recent evidence suggests that during renal injury, renal epithelial cells may transform into fibroblasts in a process known as epithelial-mesenchymal transdifferentiation (EMT). 3,4 The synthesis and processing of extracellular matrix that malfunction in fibrosis are under the complex control of many cytokines and other factors. 1 One cytokine in particular that has been implicated in fibrotic disease, both in the kidney and in other tissues, is transforming growth factor-β (TGF-β). In the accompanying article “Renal Fibrosis: Collagen Composition and Assembly Regulates Epithelial-Mesenchymal Transdifferentiation,” by Zeisberg and colleagues 5 in this issue of The American Journal of Pathology, Zeisberg and co-workers use murine renal cell lines in culture to demonstrate that the integrity of basement membrane collagen has a significant effect on EMT in vitro. They further demonstrate that when basement membrane assembly is disrupted, the production of TGF-β is up-regulated. In this article we will discuss the emerging role of EMT in fibrosis and its regulation, both in vitro and in vivo, with emphasis on effects of TGF-β and collagen IV.

Toradol, an NSAID used for renal colic, decreases renal perfusion and ureteral pressure in a canine model of unilateral ureteral obstruction.

Toradol is a new parenteral, nonsteroidal anti-inflammatory drug which is efficacious in treating renal coli. In the present experiments, Toradol was administered to both control dogs and dogs with unilateral ureteral obstruction. In control dogs, Toradol had no effect on RBF or GFR, despite inhibition of renal prostaglandin synthesis (measured as urinary prostaglandin release). In contrast, RBF fell acutely by 35% (p < 0.001) within 15 minutes of Toradol administration in the setting of ureteral obstruction; contralateral RBF was unaffected. Ipsilateral ureteral pressure also fell. Changes in RBF and ureteral pressure, together with the known effects of NSAIDs on pain pathways, may contribute to the pain relief observed clinically with Toradol. However, the abrupt changes in renal hemodynamics brought on by Toradol to the obstructed kidney may compromise renal reserve, and Toradol should be used cautiously in treating renal colic.

Interstitial fibrosis of unilateral ureteral obstruction is exacerbated in kidneys of mice lacking the gene for inducible nitric oxide synthase

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function and increases in interstitial fibrosis. Previous studies have indicated that pharmacologic manipulations that increase nitric oxide (NO) are beneficial to the obstructed kidneys. NO is produced from arginine by nitric oxide synthase (NOS), an enzyme that exists in both constitutive and inducible (iNOS) forms. To determine the role of the inducible form of NOS in UUO, we used mice with a targeted deletion of iNOS (iNOS −/− mice) and compared them with wild-type (WT) mice. Kidneys were obstructed for 2 weeks in both WT and iNOS −/− mice, and were then removed and bisected. Half of the kidney was embedded in paraffin and tissue sections were examined for interstitial volume or the presence of macrophages. The remainder was flash-frozen and samples were used to measure tissue collagen (hydroxyproline) or transforming growth factor-β (TGF-β). This study demonstrates that both cortex and medulla of obstructed kidneys of iNOS −/− mice exhibit significantly increased interstitial volume and interstitial macrophages as compared with their WT counterparts. Furthermore tissue collagen was increased to 9.2 ± 1.3 μg/mg tissue in WT obstructed kidneys, whereas in iNOS −/− kidneys, collagen was increased to 13.2 ± 0.8 μg/mg tissue. The profibrotic cytokine TGF-β was also significantly increased in obstructed kidneys of iNOS −/− mice, as compared with WT mice. No differences were noted between the unobstructed kidneys of iNOS −/− mice compared with WT mice in any of the parameters examined. These results demonstrate that targeted deletion of the iNOS results in exacerbation of fibrotic events in the obstructed kidney. These results confirm previous pharmacologic studies, and suggest that NO produced via the inducible NOS normally serves a protective function in UUO.

INTERACTION OF NITRIC OXIDE AND TRANSFORMING GROWTH FACTOR-β1 INDUCED BY ANGIOTENSIN II AND MECHANICAL STRETCH IN RAT RENAL TUBULAR EPITHELIAL CELLS

PURPOSE Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch. MATERIALS AND METHODS NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. RESULTS Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch. CONCLUSIONS These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.

MECHANISM OF HEALING FOLLOWING THE SNODGRASS REPAIR

PURPOSE It has been suggested that healing after tubularized incised plate urethroplasty occurs through re-epithelialization with normal tissue ingrowth or by secondary intent through scarring. We investigated healing in tubularized incised plate urethroplasty. MATERIALS AND METHODS Hypospadias was created in 5 dogs by incising the ventral urethra, allowed to heal for 21 days and subsequently repaired. During hypospadias creation a tattoo was made longitudinally in the midline dorsal urethral plate. The tattoo was bisected during repair, thus creating 2 distinct lines marking the edges of the incision. A neourethra was tubularized and closed in 2 layers. At 21 days the phallus was harvested, inspected and embedded for histology. RESULTS The dorsal urethral plate incision contained 2 distinct lines in all samples representing the area of separation between the native and ingrowing urethras. The distance between these lines was 0.9 +/- 0.1 mm. Proximal urethral lumen diameter (3. 3 +/- 0.1 mm.) was not significantly different from that of the neourethral lumen (3.1 +/- 0.1 mm.). Histologically all repairs had intact squamous epithelium. There was normal appearing subepithelial architecture with scant perivascular lymphocytic infiltrates between the tattoos. In contrast, the area around the sutures showed a desmoplastic (fibroblastic) reaction with an inflammatory, primarily neutrophilic response. CONCLUSIONS Healing of the incision in the dorsal urethral plate during tubularized incised plate urethroplasty occurs by re-epithelialization with normal tissue ingrowth. In contrast, the sutured closure heals with a desmoplastic and inflammatory response.

A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function, increased interstitial fibrosis, tubular apoptosis, and cellular infiltration. It has been suggested that inhibition of tubular apoptosis may protect against renal damage in obstruction. We have recently developed a series of peptides which are concentrated in the inner mitochondrial membrane and prevent cell death. These peptides are also active in vivo, in myocardial infraction, ischemic brain injury, and amyotrophic lateral sclerosis models. We therefore used SS-31, a prototype of these peptides, and assessed its effects on renal damage and oxidative stress in a 14-day obstruction model. SS-31 (1 or 3 mg/kg) or saline was given 1 day before and throughout the 14 days of obstruction. Kidneys were harvested and assessed for apoptosis (terminal transferase-dUTP-nick-end labeling, caspase 3 expression), fibrosis (trichrome staining), macrophage infiltration, fibroblast expression (immunoperoxidase), and oxidative damage (8-OH deoxyguanosine and heme oxygenase-1 expression), cytokines, and signaling pathways (transforming growth factor-beta, CCR-1, p38-MAPK, NF-kappaB). SS-31 significantly attenuated the effects of obstruction on all aspects of renal damage which were examined, with both the 1 and 3 mg/kg doses showing efficacy. We noted increased oxidative stress in obstruction, which was also attenuated by SS-31 treatment. Signaling via NF-kappaB and p38 MAPK pathways were both affected by SS-31 treatment. This study provides a proof of concept that peptides which protect mitochondria in vitro can provide protection from renal damage in a UUO model. The mechanism by which protection is afforded requires further studies both in vitro and in vivo.

In Vivo Cystometric Evaluation of Progressive Bladder Outlet Obstruction in Rats

PURPOSE Bladder outlet obstruction in man is a common medical disorder that may result from benign prostatic hyperplasia, urethral stricture disease, or congenital anomaly. The functional changes that develop in response to obstruction include detrusor instability, elevated voiding pressures, and the presence of a residual urine. The aim of this study was to document the development of progressive bladder outlet obstruction over time in a rat model using conscious, in vivo urodynamics. MATERIALS AND METHODS Infravesical bladder outlet obstruction was created in female rats by placing a jewelers jump ring loosely around the proximal urethra. Gradual development of outlet obstruction was followed urodynamically in awake animals at 3, 7, 14, 21, and 28 days post obstruction using a subcutaneously implanted mediport. For each group n = 5-8 animals. RESULTS Animals developed large capacity bladders with increased compliance, a high residual urine volume, and spontaneous activity. Bladder capacity increased from 0.20 + 0.02 ml. to 6.30 + 1.59 ml. at 28 days post obstruction (p < 0.05). Residual volume increased from 0.06 + 0.01 ml. to 5.95 + 1.54 ml. (p < 0.05). Percent void decreases from 72 + 3.7% in sham controls to 6.7 + 2.5% at 28 d (p < 0.05). Voiding pressure increased from 12 + 1.6 mm. Hg in sham animals to a maximum of 42 + 6.1 mm. Hg at 21 d (p < 0.05). Compliance was significantly higher at 28 d when compared to all other time points. 89% of obstructed animals developed bladder instability. CONCLUSIONS This study provides clear evidence of the progressive change in bladder function which occurs following outlet obstruction. Implantation of a subcutaneous mediport allows in vivo recording of both the filling and voiding phases of micturition in awake animals that have intact neural responses. This is a precise and easily reproducible method for producing obstruction in a small animal which can provide a continuum of tissue and urodynamic data that may be used to further study the pathophysiologic changes underlying bladder outlet obstruction or other models of bladder dysfunction.

Clonidine displacing substance is biologically active on smooth muscle

A substance has been isolated from brain which potently inhibits the binding of clonidine to brain membranes (clonidine displacing substance, CDS). We sought to determine if CDS is biologically active on smooth muscle. CDS had no effect on vascular smooth muscle. In contrast, CDS potently contracted rat gastric fundus strips in a dose dependent manner. The contractile effect of CDS was not blocked by antagonists selective for biologically active substances known to contract the fundus strip. These results demonstrate that CDS has a unique and potent ability to selectively contract smooth muscle.

Renal hemodynamic and ureteral pressure changes in response to ureteral obstruction: the role of nitric oxide.

PURPOSE Triphasic changes in renal blood flow and ureteral pressure after unilateral ureteral obstruction have long been known. The contribution of nitric oxide to the decline in renal blood flow and ureteral pressure in unilateral ureteral obstruction was studied in this model using arginine infusion and by studying the effect of 2 inhibitors of nitric oxide synthase (NOS). MATERIALS AND METHODS Left ureteral obstruction was created in dogs. Renal blood flow and ureteral pressure were monitored. Groups 1 to 4 underwent unilateral ureteral obstruction and group 5 dogs underwent sham operation. Groups 2 to 5 received an infusion of arginine at hour 18 of obstruction that was sustained for 1 hour. In addition, NOS inhibitors were administered to dogs in groups 3 (N-monomethyl-L-arginine) and 4 (triamcinolone diacetate). RESULTS Arginine administration at 18 hours of obstruction caused a significant increase in renal blood flow and ureteral pressure compared to sham operated animals. Triamcinolone diacetate eliminated the increase in renal blood flow and ureteral pressure, whereas N-monomethyl-L-arginine did not, reflecting the competitive nature of its inhibition of NOS. CONCLUSIONS Arginine infusion 18 hours after unilateral ureteral obstruction led to increases in renal blood flow and ureteral pressure that were not seen in control animals. These results suggest that the nitric oxide system of the kidney is activated in unilateral ureteral obstruction. Since the addition of arginine is accompanied by an increase in renal blood flow and ureteral pressure, it further suggests that a lack of availability of substrate for NOS may explain the decrease in renal blood flow and ureteral pressure in obstruction. Providing substrate may be a way of maintaining renal blood flow in unilateral ureteral obstruction.

Effects of Transforming Growth Factor-β1 on Human Vocal Fold Fibroblasts

Objectives: We studied the effect of transforming growth factor (TGF)–β on immortalized human vocal fold fibroblasts. Methods: Normal human vocal fold fibroblasts were subjected to sequential lentiviral transduction with genes for human telomerase (hTERT) and SV40 large T antigen in order to produce an “immortalized” cell line of normal phenotype. After confirmation of vocal fold fibroblast transfection, these cells, referred to as HVOX, were treated with various concentrations of exogenous TGF-β1 and assayed for collagen secretion, migration, and proliferation. In addition, components of the TGF-β signaling pathway were examined in this cell line. Results: TGF-β stimulated collagen secretion and migration without altering proliferation of HVOX. HVOX constitutively expressed type I and II TGF-β receptors, as well as messenger RNA for the Smad signaling proteins and for all TGF-β isoforms. Exogenous TGF-β1 induced temporally dependent alterations in Smad2 and Smad3 gene expression. TGF-β increased Smad7 expression at both 4 and 24 hours. Prolonged exposure to TGF-β decreased TGF-β1 gene expression. Conclusions: Insight into the underlying pathophysiology of vocal fold fibrosis is likely to yield improved therapeutic strategies to mitigate vocal fold scarring. Our data suggest that TGF-β signaling may be both paracrine and autocrine in this vocal fold fibroblast cell line, and we therefore propose that TGF-β may be a reasonable target for therapies to prevent and/or treat vocal fold fibrosis, given its putative role in both acute and chronic vocal fold injury, as well as its effects on vocal fold fibroblasts.