Shizuko Nagao

Increased Water Intake Decreases Progression of Polycystic Kidney Disease in the PCK Rat

Renal enlargement in polycystic kidney disease (PKD) is caused by the proliferation of mural epithelial cells and transepithelial fluid secretion into the cavities of innumerable cysts. Arginine vasopressin (AVP) stimulates the proliferation of human PKD cells in vitro via cAMP-dependent activation of the B-Raf/MEK (MAPK/ERK kinase/extracellular signal-regulated kinase (ERK) pathway. ERK activity is elevated in cells that line the cysts in animals with PKD, and AVP receptor antagonists reduce ERK activity and halt disease progression. For suppression of the effect of AVP physiologically, water intake was increased in PCK rats, a model of PKD, and the effect on renal morphology, cellular mechanism, and function was determined. The addition of 5% glucose in the drinking water increased fluid intake approximately 3.5-fold compared with rats that received tap water. In PCK rats, increased water intake for 10 wk reduced urinary AVP excretion (68.3%), and urine osmolality fell below 290 mOsmol/kg. High water intake was associated with reduced renal expression of AVP V2 receptors (41.0%), B-Raf (15.4%), phosphorylated ERK (38.1%), and proliferating cell nuclear antigen-positive renal cells (61.7%). High water intake reduced the kidney/body weight ratio 28.0% and improved renal function. Taken together, these data demonstrate that water intake that is sufficient to cause persistent water diuresis suppresses B-Raf/MEK/ERK activity and decreases cyst and renal volumes in PCK rats. It is suggested that limiting serum AVP levels by increased water intake may be beneficial to some patients with PKD.

Methylprednisolone retards the progression of inherited polycystic kidney disease in rodents.

Polycystic kidney disease in adult laboratory animals and humans is associated with enlarged kidneys and a progressive decline of renal function, resulting in death from uremia. Interstitial inflammation and fibrosis typically are observed in association with the development of renal insufficiency. To determine whether amelioration of interstitial inflammation and fibrosis may diminish cyst expansion/kidney enlargement and stabilize renal function, we administered methylprednisolone, an anti-inflammatory drug with antifibrogenic effects, to mice and rats with hereditary polycystic kidney disease. The experiment was repeated once for each species. Mice were studied both in America and in Japan. Weanling male and female mice (DBA/FG pcy/pcy [cystic] and +/+ [normal], n = 87 and 20, respectively) and rats (Han:SPRD Cy/+ and +/+, n = 70 and 33, respectively) were administered methylprednisolone (1 to 2 mg/kg/d) in the drinking water for 100 days (mice) or 42 days (rats). Control animals drank distilled water. In normal DBA +/+ mice, methylprednisolone had no effect on serum urea nitrogen (SUN) levels, kidney weight, or kidney/body weight. Untreated male and female mice developed cystic kidneys and azotemia to an equal extent. Methylprednisolone administered in America to mice with renal cystic disease decreased kidney weight, kidney/body weight, SUN levels, volume density of cysts, and severity of interstitial fibrosis. In Japan, methylprednisolone decreased kidney weight and SUN levels of animals with cystic disease, but the effect on kidney/body weight did not reach statistical significance. In contrast to mice, male rats developed more severe renal cystic changes and were more azotemic than female rats. Methylprednisolone administered to male rats with cystic disease decreased SUN levels, kidney weight, kidney/body weight, volume density of cysts, and severity of interstitial fibrosis. Methylprednisolone had no effect on kidney/body weight or SUN levels in female rats with renal cystic disease. In normal Han:SPRD (+/+) rats of both sexes, kidney and body weight were decreased by methylprednisolone, but kidney/body weight and SUN levels were unchanged. On the basis of this study, we conclude that methylprednisolone decreased the extent of renal enlargement, reduced renal interstitial fibrosis, and preserved kidney function in mice and rats with relatively severe forms of inherited polycystic kidney disease.

PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft.

The programmed death‐1 (PD‐1)/B7‐H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD‐1/B7‐H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7‐H1 is highly expressed on the donor‐derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD‐1/B7‐H1 pathway via anti‐B7‐H1mAb or using B7‐H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody‐treated group in comparison to the controls. Taken together, these data revealed that the B7‐H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.

PPAR-γ agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-β-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Epithelial-to-mesenchymal transition in cyst lining epithelial cells in an orthologous PCK rat model of autosomal-recessive polycystic kidney disease

In polycystic kidney disease (PKD), cyst lining cells show polarity abnormalities. Recent studies have demonstrated loss of cell contact in cyst cells, suggesting induction of epithelial-to-mesenchymal transition (EMT). Recently, EMT has been implicated in the pathogenesis of PKD. To explore further evidence of EMT in PKD, we examined age- and segment-specific expression of adhesion molecules and mesenchymal markers in PCK rats, an orthologous model of human autosomal-recessive PKD. Kidneys from 5 male PCK and 5 control rats each at 0 days, 1, 3, 10, and 14 wk, and 4 mo of age were serially sectioned and stained with segment-specific markers and antibodies against E-cadherin, Snail1, β-catenin, and N-cadherin. mRNAs for E-cadherin and Snail1 were quantified by real-time PCR. Vimentin, fibronectin, and α-smooth muscle actin (α-SMA) expressions were assessed as mesenchymal markers. E-cadherin expression pattern was correlated with the disease pathology in that tubule segments showing the highest expression in control had much severer cyst formation in PCK rats. In PCK rats, E-cadherin and β-catenin in cystic tubules was attenuated and localized to lateral areas of cell-cell contact, whereas nuclear expression of Snail1 increased in parallel with cyst enlargement. Some epithelial cells in large cysts derived from these segments, especially in adjacent fibrotic areas, showed positive immunoreactivity for vimentin and fibronectin. In conclusion, these findings suggest that epithelial cells in cysts acquire mesenchymal features in response to cyst enlargement and participate in progressive renal fibrosis. Our study clarified the nephron segment-specific cyst profile related to EMT in PCK rats. EMT may play a key role in polycystic kidney disease.

Androgen Receptor Pathway in Rats with Autosomal Dominant Polycystic Kidney Disease

Androgens have been implicated in mediating disease escalation in autosomal dominant polycystic kidney disease (ADPKD). Dihydrotestosterone (DHT), an agonist, and flutamide (FLT), an antagonist, were administered to Han:SPRD rats with ADPKD, and the role of androgen receptor (AR) abundance and activation on the enlargement and function of cystic kidneys was evaluated. Renal AR abundance determined by immunoblots in 8- to 10-wk-old Cy/+ male rats was naturally increased four-fold above that of littermate +/+ controls. In male Cy/+, castration decreased AR abundance below control +/+ by -89.4%, and AR expression within cyst mural epithelial cells was strikingly decreased. Castration of Cy/+ male rats also reduced the usual increases in kidney weight by -49.7%, kidney cyst area by -34.0%, and serum urea nitrogen by -72.8%; these indices were restored to precastration levels by DHT. In Cy/+ male rats, FLT administration reduced the increase in kidney weight by -27.6% and serum urea nitrogen by -53.7% and decreased the increment in AR expression by -84.2% in comparison with untreated +/+ controls. There was no effect of FLT in female rats. Immunoblot expression of phospho-extracellular signal-regulated kinase 1/2 (P-ERK) and B-Raf, key intermediates in the mitogen-activated protein kinase pathway that are abnormally elevated in Cy/+, was unaffected by castration and/or administration of DHT or FLT. AR was not expressed in renal epithelial cell nuclei of androgen-deficient rats but was displayed in most tubule and mural cyst cell nuclei of androgen-replete rats. In androgen-deficient Cy/+, 80.6% of renal epithelial cells that had entered the cell cycle (proliferating cell nuclear antigen positive) also expressed P-ERK. In androgen-replete rats, proliferating cell nuclear antigen-positive cells co-expressed AR (12.7%), P-ERK (36.4%), and P-ERK + AR (45.0%); 5.9% were probably stimulated by other mitogenic mechanisms. It is concluded that androgens potentiate renal cell proliferation and cyst enlargement through ERK1/2-dependent and ERK1/2-independent signaling mechanisms in Han:SPRD. It is suggested that the basal rate of cell proliferation is determined by ERK1/2 signaling to a major extent and that androgens have additive effects.

Identification of a novel biomarker gene set with sensitivity and specificity for distinguishing between allograft rejection and tolerance

Here we examined whether the expression of a novel immunoregulatory gene set could be used to predict outcomes in murine models of rapamycin‐induced cardiac tolerance, spontaneous hepatic tolerance, and cardiac rejection. The expression of the immunoregulatory gene set was assessed with the GeXP multiplex reverse‐transcription polymerase chain reaction (RT‐PCR) analysis system, and it was correlated to the pathological and biochemical parameters of the allografts. In rejecting cardiac grafts, the increased expression of an inflammatory set of genes, which included CD45, CD4, CD25, suppressor of cytokine signaling 2, cytotoxic T lymphocyte–associated protein 4 (CTLA4), selectin lymphocyte, interferon‐γ (IFN‐γ), programmed cell death 1 (Pdcd1), and granzyme B (Gzmb), was seen 8 days after transplantation along with histological evidence of severe allograft rejection. In tolerant cardiac allografts, the expression of fibrinogen‐like protein 2 (Fgl2), Pdcd1, killer cell lectin‐like receptor G1 (Klrg1), CTLA4, and lymphocyte‐activation gene 3 was associated with tolerance. In a model of liver allograft tolerance, the increased expression of lectin galactose‐binding soluble 1, Fgl2, CD39, phosphodiesterase 3B, Klrg1, forkhead box P3 (Foxp3), and transforming growth factor β as well as the inflammatory set of genes was observed 8 to 14 days after transplantation (ie, when there was severe inflammatory injury). At a later time when the liver allografts had been fully accepted and were histologically normal, the expression of the inflammatory set of genes returned to the baseline, but the expression of the tolerogenic set of genes was still increased. Genes that were expressed in tolerant cardiac and liver allografts included Fgl2, Klrg1, and Foxp3, whereas genes associated with rejection included CD25, Gzmb, and IFN‐γ. Our data indicate that monitoring the graft expression of a novel biomarker gene set with the GeXP multiplex RT‐PCR analysis system may allow differentiation between rejection and tolerance. Liver Transpl 18:444–454, 2012.

Cyst fluid from a murine model of polycystic kidney disease stimulates fluid secretion, cyclic adenosine monophosphate accumulation, and cell proliferation by Madin-Darby canine kidney cells in vitro

Cyst fluids from subjects with autosomal dominant polycystic kidney disease (ADPKD) cause polarized monolayers of MDCK cells to secrete fluid toward the apical compartment in vitro. To determine the extent to which secretagogue accumulation may be a general feature of polycystic diseases, cyst fluid from mice with a slowly progressive form of hereditary PKD (DBA/2FG-pcy/pcy) was added to polarized MDCK monolayers. Basolateral application of cyst fluids (diluted with culture medium to 15% final concentration) from 13 different animals 16 to 35 weeks old increased the fluid secretion rate from a baseline of 0.023 +/- 0.003 to 0.111 +/- 0.017 microL/cm2/h (P < 0.005). There was a direct relation between the concentration of cyst fluid and the rate of net fluid secretion. The secretory activity of cyst fluid was not altered by pronase treatment or boiling. Cyst fluid (10%) added to the basolateral surfaces of polarized MDCK monolayers for 24 hours increased cell cyclic adenosine monophosphate (AMP) levels from a baseline of 6.3 +/- 0.2 to 17.3 +/- 0.3 pmoles/monolayer (n = 3, P < 0.05). The capacity of cyst fluid to increase cyclic AMP levels was not changed by pronase treatment or boiling. There was a direct relation between the level of cellular cyclic AMP and the rate of transepithelial fluid secretion caused by cyst fluid. Cyst fluid increased thymidine incorporation by Madin-Darby canine kidney (MDCK) cells to an extent equal to that caused by epidermal growth factor and caused MDCK cells to form cysts in collagen matricies.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of probucol in a murine model of slowly progressive polycystic kidney disease.

Epithelial proliferation, extracellular matrix remodeling, and interstitial inflammation are central elements in the pathogenesis of slowly progressive polycystic kidney disorders. Probucol, an antioxidant that lowers plasma cholesterol, has been shown to decrease smooth muscle cell proliferation and macrophage accumulation in blood vessels and to prevent restenosis after coronary angioplasty. We determined in 30-day-old male BDF1-pcy hybrid mice (derived from mating DBA/2FG-pcy and C57BL/6FG-pcy) the effect of probucol administered in the diet (1%) for 200 days on kidney weight relative to body weight (KW/BW), cyst expansion, renal interstitial fibrosis, and serum urea nitrogen (SUN) concentration. Animals were fed a moderately high-protein diet (HPD, 36%) to accentuate the development of renal cysts and to promote interstitial fibrosis. Probucol decreased serum cholesterol from 68 to 16 mg/dL but had no effect on food intake or body weight. Probucol decreased relative kidney size from 4.16% +/- 0.55% to 2.64% +/- 0.12% KW/BW (P < 0.01), SUN from 30.5 +/- 1.8 to 25.9 +/- 1.0 mg/dL (P < 0.05), cystic index from 2.45 +/- 0.11 to 1.36 +/- 0.10 (P < 0.01), and fibrosis index from 2.40 +/- 0.11 to 1.82 +/- 0.08 (P < 0.01). We conclude that probucol ameliorates the progressive deterioration in renal function and structure in pcy mice ingesting a relatively high level of dietary protein.

The effect of paclitaxel on the progression of polycystic kidney disease in rodents

Woo et al (Nature 368:750-753) reported that parenteral administration of paclitaxel arrested the striking renal enlargement and prolonged life in C57BL/6J-cpk/cpk mice with a rapidly progressive form of polycystic kidney disease (PKD). In the current study, we sought to determine whether paclitaxel could alter the progression of other forms of hereditary PKD in rodents. Paclitaxel was administered by intraperitoneal injection to C57BL/6J-cpk/cpk mice and Han:SPRD-Cy/Cy rats with rapidly progressive PKD and to DBA/2FG-pcy/pcy mice and Han:SPRD-Cy/+ rats with slowly progressive PKD. Paclitaxel (150 micrograms/wk) prolonged the survival of cpk/cpk mice from 24.5 days to more than 65 days and decreased kidney weight relative to body weight from 16.5% at 21 days of age to 8.2% at more than 65 days of age. Mortality attributable to paclitaxel was 12%. By contrast, the administration of paclitaxel (0.1 to 15 mg/kg/wk) to 7- to 10-day-old Han:SPRD-Cy/Cy rats with rapidly progressive PKD had no effect on the course of the disease; moreover, paclitaxel caused severe side effects and premature death in all the Cy/Cy animals. Heterozygous male Cy/+ rats develop slowly progressive renal enlargement and azotemia. Paclitaxel, administered at 7, 15, or 27 mg/kg/wk to male Cy/+ rats from 4 until 10 weeks of age, reduced body weight gain, had an inconsistent effect on kidney weight relative to body weight, and had no effect on the serum urea nitrogen concentration. Mortality associated with the 7, 15, and 27 mg/kg/wk doses of paclitaxel was 0%, 15.4%, and 28.5%, respectively. DBA/2FG-pcy/pcy mice of either sex developed slowly progressive renal enlargement and azotemia. The administration of paclitaxel (100 to 150 micrograms/wk) from 2 to 10 weeks of age to DBA/2FG-pcy/pcy mice with cystic disease had no effect on the increase in kidney weight or on the level of serum urea nitrogen in comparison to untreated cystic animals. Mortality associated with 100- and 150-micrograms/wk doses of paclitaxel was 0% and 20%, respectively. We conclude that paclitaxel diminished the rate of renal enlargement and increased the life span of cpk/cpk mice but not Cy/Cy rats with rapidly progressive forms of PKD. Paclitaxel had no apparent benefit in Cy/+ rats nor pcy/pcy mice with slowly progressive PKD. On the basis of these studies in rodents, it appears that paclitaxel has limited potential usefulness as a therapeutic agent in the treatment of PKD.