Catherine M. Hoff

Transient Overexpression of TGF-β1 Induces Epithelial Mesenchymal Transition in the Rodent Peritoneum

Epithelial mesenchymal transition (EMT), a process involved in many growth and repair functions, has been identified in the peritoneal tissues of patients who undergo peritoneal dialysis. The sequence of changes in gene regulation and cellular events associated with EMT after TGF-beta1-induced peritoneal fibrosis is reported. Sprague-Dawley rats received an intraperitoneal injection of an adenovirus vector that transfers active TGF-beta1 (AdTGF-beta1) or control adenovirus, AdDL. Animals were killed 0 to 21 days after infection. Peritoneal effluent and tissue were analyzed for markers of EMT. In the animals that were treated with AdTGF-beta1, an increase in expression of genes associated with EMT and fibrosis, such as type I collagen A2, alpha-smooth muscle actin, and the zinc finger regulatory protein Snail, was identified. Transition of mesothelial cells 4 to 7 d after infection, with appearance of epithelial cells in the submesothelial zone 7 to 14 d after exposure to AdTGF-beta1, was demonstrated. This phase was associated with disruption of the basement membrane and increased expression of matrix metalloproteinase 2. By 14 to 21 d after infection, there was evidence of restoration of normal submesothelial architecture. These findings suggest that EMT occurs in vivo after TGF-beta1 overexpression in the peritoneum. Cellular changes and gene regulation associated with EMT are evident throughout the fibrogenic process and are not limited to early time points. This further supports the central role of TGF-beta1 in peritoneal fibrosis and provides an important model to study the sequence of events involved in TGF-beta1-induced EMT.

Inflammatory cytokines, angiogenesis, and fibrosis in the rat peritoneum

Peritonitis, a common complication of peritoneal dialysis, is followed by acute changes in the function of the peritoneum. The role of inflammatory cytokines in these processes is not clearly identified. We used adenoviral-mediated gene transfer to transiently overexpress interleukin (IL)-1 beta (AdIL-1 beta) or tumor necrosis factor (TNF)-alpha (AdTNF-alpha) in the rat peritoneum then used a modified equilibrium test to study the histological and functional changes. Overexpression of IL-1 beta or TNF-alpha led to an acute inflammatory response. Both inflammatory cytokines induced an early expression of the angiogenic cytokine, vascular endothelial growth factor, along with increased expression of the profibrotic cytokine, transforming growth factor-beta1, along with fibronectin expression and collagen deposition in peritoneal tissues. Both inflammatory cytokines induced angiogenesis, increased solute permeability, and ultrafiltration dysfunction at earlier time points. Changes in structure and function seen in AdTNF-alpha-treated animals returned to normal by 21 days after infection, whereas AdIL-1 beta-treated animals had persistently increased vasculature with submesothelial thickening and fibrosis. This was associated with up-regulation TIMP-1. TNF-alpha or IL-1 beta both induce acute changes in the peritoneum that mimic those seen in peritoneal dialysis patients who experience an episode of peritonitis. These functional changes were associated with early angiogenesis that resolved rapidly after exposure to TNF-alpha. IL-1 beta exposure, however, led to a different response with sustained vascularization and fibrosis. IL-1 beta inhibition may be a therapeutic goal in acute peritonitis to prevent peritoneal damage.

The role of mouse strain differences in the susceptibility to fibrosis: a systematic review.

In humans, a number of genetic factors have been linked to the development of fibrosis in a variety of different organs. Seeking a wider understanding of this observation in man is ethically important. There is mounting evidence suggesting that inbred mouse strains with different genetic backgrounds demonstrate variable susceptibility to a fibrotic injury. We performed a systematic review of the literature describing strain and organ specific response to injury in order to determine whether genetic susceptibility plays a role in fibrogenesis. Data were collected from studies that were deemed eligible for analysis based on set inclusion criteria, and findings were assessed in relation to strain of mouse, type of injury and organ of investigation. A total of 44 studies were included covering 21 mouse strains and focusing on fibrosis in the lung, liver, kidney, intestine and heart. There is evidence that mouse strain differences influence susceptibility to fibrosis and this appears to be organ specific. For instance, C57BL/6J mice are resistant to hepatic, renal and cardiac fibrosis but susceptible to pulmonary and intestinal fibrosis. However, BALB/c mice are resistant to pulmonary fibrosis but susceptible to hepatic fibrosis. Few studies have assessed the effect of the same injury stimulus in different organ systems using the same strains of mouse. Such mouse strain studies may prove useful in elucidating the genetic as well as epigenetic factors in humans that could help determine why some people are more susceptible to the development of certain organ specific fibrosis than others.

Interleukin-1 gene cluster polymorphisms are associated with nutritional status and inflammation in patients with end-stage renal disease.

Background: Wasting and inflammation are two common risk factors for death in patients with end-stage renal disease (ESRD). Interleukin-1β (IL-1β) and its receptor antagonist (IL-1Ra) may play a pivotal role in the pathogenesis of wasting and inflammation. Methods: To investigate effects of the IL-1 gene cluster polymorphisms on wasting and inflammation, we studied 189 ESRD patients (52 ± 12 years, 62% males) close to the start of renal replacement therapy. 205 healthy volunteers served as controls. We analyzed the IL-1B –511C/T, –31C/T, and +3954C/T polymorphisms as well as a variable number of a tandem repeat (VNTR) in IL-1RN. Nutritional parameters included serum albumin level, subjective global nutritional assessment (SGA), and body composition evaluated by dual-energy X-ray absorptiometry (DXA). We used serum high-sensitivity C-reactive protein (hsCRP) as a marker of inflammation. Results: Wasting (SGA >1) was present in 31%, whereas inflammation (CRP ≧10 mg/l) was present in 36% of the patients. The male carriers of the –511T/T and –31C/C genotypes had a lower prevalence of wasting (p < 0.05), higher body mass index (BMI) (p < 0.05), and higher lean body mass (LBM) (p < 0.01). In a stepwise multiple regression model, age (p < 0.05), BMI (p < 0.01) and the IL-1B –511 genotype (p < 0.01) were independently associated with LBM. The carriers of the +3954T allele had a lower prevalence of inflammation (p < 0.05) and lower serum hsCRP (p < 0.05). The VNTR in IL-1RN was not associated with any markers. Conclusion: The investigated IL-1 gene cluster polymorphisms were associated with nutritional status and inflammation in ESRD patients, but marked differences were found between the genders. These polymorphisms could have prognostic utility for predicting wasting and inflammation in ESRD patients.

Transforming growth factor β-induced peritoneal fibrosis is mouse strain dependent*

BACKGROUND Encapsulating peritoneal sclerosis (EPS) is a rare but devastating complication of peritoneal dialysis. The etiology is unclear, but genetic predisposition may be a contributing factor. We used adenovirus-mediated gene transfer of transforming growth factor (TGF) β1 to the peritoneum in four genetically distinct laboratory mouse strains to assess differences in fibrogenic response. METHODS Mice from four genetic backgrounds (C57BL/6J, DBA/2J, C3H/HeJ and SJL/J) received an intraperitoneal injection of an adenovirus expressing TGFβ1 (AdTGFβ1) or control adenovirus (AdDL) and were assessed 4 and 10 days after infection. Submesothelial thickening, angiogenesis and gene expression were quantified from peritoneal tissue. Protein was extracted from omental tissue and assessed for collagen, E-cadherin and TGFβ signaling pathway proteins. RESULTS There was a graded response among the mouse strains to the peritoneal overexpression of TGFβ1. TGFβ1 induced a significant fibrogenic response in the C57BL/6J mice, whereas the SJL/J mice were resistant. The DBA/2J and the C3H/HeJ mice had intermediate responses. A similar graded response was seen in collagen protein levels in the omental tissue and in fibrosis-associated gene expression. TGFβ type 1 receptor and SMAD signaling pathways appeared to be intact in all the mouse strains. CONCLUSIONS There were significant differences in mouse strain susceptibility to peritoneal fibrosis, suggesting that genetic factors may play a role in the development of peritoneal fibrosis and possibly EPS. As early TGFβ1 signaling mechanisms appear to be intact, we hypothesize that fibrosis resistance in the SJL/J mice lies further down the wound-healing cascade or in an alternate, non-SMAD pathway.

Gremlin Promotes Peritoneal Membrane Injury in an Experimental Mouse Model and Is Associated with Increased Solute Transport in Peritoneal Dialysis Patients

The peritoneal membrane becomes damaged in patients on peritoneal dialysis (PD). Gremlin 1 (GREM1) inhibits bone morphogenic proteins (BMPs) and plays a role in kidney development and fibrosis. We evaluated the role of gremlin in peritoneal fibrosis and angiogenesis. In a cohort of 32 stable PD patients, GREM1 concentration in the peritoneal effluent correlated with measures of peritoneal membrane damage. AdGrem1, an adenovirus to overexpress gremlin in the mouse peritoneum, induced submesothelial thickening, fibrosis, and angiogenesis in C57BL/6 mice, which was associated with decreased expression of BMP4 and BMP7. There was evidence of mesothelial cell transition to a mesenchymal phenotype with increased α smooth muscle actin expression and suppression of E-cadherin. Some of the GREM1 effects may be reversed with recombinant BMP7 or a pan-specific transforming growth factor β (TGF-β) antibody. Neovascularization was not inhibited with a TGF-β antibody, suggesting a TGF-β-independent angiogenic mechanism. Swiss/Jackson Laboratory (SJL) mice, which are resistant to TGF-β-induced peritoneal fibrosis, responded in a similar fashion to AdGrem1 as did C57BL/6 mice with fibrosis, angiogenesis, and mesothelial-to-mesenchymal transition. GREM1 was associated with up-regulated TGF-β expression in both SJL and C57BL/6 mice, but SJL mice demonstrated a defective TGF-β-induced GREM1 expression. In summary, GREM1 induces fibrosis and angiogenesis in mouse peritoneum and is associated with increased solute transport in these PD patients.

ULTRAFILTRATION CHARACTERISTICS OF GLUCOSE POLYMERS WITH LOW POLYDISPERSITY

♦ Background: Icodextrin, a glucose polymer with a polydispersity [ratio of weight-average molecular weight (Mw) to number-average molecular weight] of approximately 2.6, has been shown, compared with glucose, to provide superior ultrafiltration (UF) efficiency [ratio of UF to carbohydrate (CHO) absorbed] when used as an osmotic agent during a long-dwell peritoneal dialysis exchange. In an experimental rabbit model, we evaluated the effect of Mw on the UF and UF efficiency of glucose polymers with low polydispersity. ♦ Methods: A crossover trial in female New Zealand White rabbits (2.20 - 2.65 kg) with surgically implanted peritoneal catheters evaluated two glucose polymers at nominal concentrations of 7.5 g/dL: a 6K polymer (Mw: 6.4 kDa; polydispersity: 2.3) and a 19K polymer (Mw: 18.8 kDa; polydispersity: 2.0). Rabbits were randomized to receive either the 6K (n = 11) or the 19K (n = 12) solution during the first exchange (40 mL/kg body weight). The alternative solution was evaluated in a second exchange 3 days later. During each 4-hour dwell, the UF and total glucose polymer CHO absorbed were determined. ♦ Results: The UF was higher for the 6K (p < 0.0001) than for the 19K polymer (mean ± standard deviation: 73.6 ± 30.8 mL vs. 43.0 ± 20.2 mL), as was the amount of CHO absorbed (42.5% ± 9.8% vs. 35.7% ± 11.0%, p = 0.021). In spite of higher CHO absorption, an approximately 50% higher (p = 0.029) UF efficiency was achieved with the 6K polymer (28.3 ± 18.8 mL/g) than with the 19K polymer (19.0 ± 11.3 mL/g). The results were independent of the order of the experimental exchanges. ♦ Conclusions: Glucose polymers with low polydispersity are effective osmotic agents in a rabbit model. The low-Mw polymer was more effective at generating UF and had a higher UF efficiency, but those results came at the expense of the polymer being more readily absorbed from the peritoneal cavity.

Genetic Modification of the Peritoneal Membrane: Potential for Improving Peritoneal Dialysis Through Gene Therapy

In peritoneal dialysis (PD), the peritoneal membrane functions as a semipermeable membrane that regulates the selective transport of water and solutes between the systemic circulation and the peritoneal cavity. The efficacy of PD and its success as a long-term treatment depend on the maintenance of the dialyzing capacity and the integrity of the peritoneal membrane. There is increasing evidence, however, that peritoneal membrane structure and function may become adversely affected with increasing time on PD (1-8). These changes may compromise dialyzing efficiency and eventually lead to membrane failure, precluding continuation of this mode of therapy. New and innovative strategies to alleviate or prevent peritoneal membrane injury during PD and increase the longevity of the therapy must be investigated. One such strategy utilizes a molecular genetic approach to change properties of the membrane through genetic modification. This approach, based on the use of genetically altered tissue to treat a disease or pathophysiological condition, is called gene therapy.

LOW-POLYDISPERSITY GLUCOSE POLYMERS AS OSMOTIC AGENTS FOR PERITONEAL DIALYSIS

♦ Background: Peritoneal dialysis (PD) solutions containing icodextrin as the osmotic agent have advantages during long dwells. The glucose polymers that constitute icodextrin are a heterogeneous mix of molecules with a polydispersity [ratio of weight-average to number-average molecular weight (Mw/Mn)] of approximately 2.6. The present study evaluates whether modifications in the polydispersity and concentration of glucose polymers can improve ultrafiltration (UF) without an associated increase in carbohydrate absorption (CA). ♦ Methods: Computer simulations using a three-pore model of peritoneal transport during a long dwell in PD patients predict that, in general, compared with 7.5% icodextrin, glucose polymers with a Mw greater than or equal to 7.5 kDa, a polydispersity less than 2.6, and concentrations greater than 7% could achieve higher UF without higher CA. Based on the simulations, we hypothesized that, compared with 7.5% icodextrin, glucose polymers with a Mw of 18 – 19 kDa and a polydispersity of 2.0 at 11% concentration could achieve higher UF without a higher CA. We tested this hypothesis in experimental studies using 8-hour dwells in New Zealand White rabbits. In those studies, UF was measured by complete fluid collection, and CA was measured by subtracting the total carbohydrate in the collected fluid from the carbohydrate initially infused. ♦ Results: The UF was higher with 11% 19 kDa glucose polymer than with 7.5% icodextrin (mean ± standard deviation: 89 ± 31 mL vs 49 ± 15 mL; p = 0.004) without higher CA (5.2 ± 0.9 g vs 5.0 ± 0.9 g, p = 0.7). Similar results were seen with the 11% 18 kDa glucose polymer, which, compared with 7.5% icodextrin, resulted in higher UF (mean ± standard deviation: 96 ± 18 mL vs 66 ± 17 mL; p < 0.001) without higher CA (4.8 ± 0.7 g vs 5.2 ± 0.6 g, p = 0.2). ♦ Conclusions: The findings demonstrate that, compared with 7.5% icodextrin solution, long-dwell PD solutions containing 11% glucose polymers with a Mw of 18–19 kDa and a polydispersity of 2.0 can provide higher UF without higher CA.