Rodent Models Of Polycystic Kidney Disease Research Articles

#1460 Targeting cyst progression: evaluation of anthelmintic and proton pump inhibitor in a rodent PKD model

Abstract Background and Aims In polycystic kidney disease (PKD) cyst formation and growth leads to progressive kidney damage. Recognizing the need for effective cyst-inhibitory drugs, we previously performed high-content screening of approved, repurposable drugs in a zebrafish model and identified several compounds with cyst-inhibitory activity and low toxicity. Here, we further tested two of the identified compounds, an anthelmintic agent and proton pump inhibitor, in a rodent model of PKD (PCK rat) for their cyst inhibitory and GFR-preserving capacity in mammals. Method The identified compounds were administered to 3-week-old male and female PCK rats for a total of 10 weeks to evaluate early drug effects on kidney function. Untreated PCK rats fed a normal ROD16 diet served as controls. After 5 weeks of treatment, total kidney and cystic volume and number of cysts were assessed in vivo by ultrasound. Following 10 weeks of treatment, kidney function was assessed by transcutaneous measurement of FITC-Sinistrin. After perfusion fixation in the same week, the kidneys, liver, heart, and pancreas were harvested for subsequent histological and molecular biological analyses. Results In male rats, 5-week proton pump inhibitor treatment was associated with a 50% reduction in the number of cysts (p < 0.05) and a nominal 22% reduction of fractional cystic kidney volume (CKV). Females showed a 16% reduction in cyst count and a modest reduction in fractional CKV. Treatment with the anthelmintic tended to reduce fractional CKV and cyst count in female animals whereas fractional cyst volume in males increased. After 10 weeks of treatment, a trend towards better preservation of GFR was observed for both therapies. Conclusion In conclusion, our study demonstrates a potential therapeutic benefit of a proton pump inhibitor and an anthelmintic agent in attenuating the progression of polycystic kidney disease (PKD) in a PCK rat model. The observed reductions in fractional cystic kidney volume and cyst count, coupled with marginal improvements in renal function, document a potential therapeutic effect of these compounds. Our preliminary findings justify further exploration of novel treatment modalities targeting cyst progression and renal dysfunction in this challenging clinical condition.

Netrin-1 Overexpression Induces Polycystic Kidney Disease: A Novel Mechanism Contributing to Cystogenesis in Autosomal Dominant Polycystic Kidney Disease

Despite recent advances in understanding the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. This study describes a novel pathway involved in cyst formation. Transgenic mice overexpressing netrin-1 in proximal tubular cells showed increased production and urinary excretion of netrin-1. Although no cysts were detectable immediately after birth, numerous small cysts were evident by the age of 4 weeks, and disease was accelerated along with age. Surprisingly, cyst formation in the kidney was restricted to male mice, with 80% penetrance. However, ovariectomy induced kidney cyst growth in netrin-1-overexpressing female mice. Cyst development in males was associated with albuminuria and polyuria and increased cAMP excretion in netrin-1 transgenic mice. Netrin-1 overexpression significantly increased extracellular signal-regulated kinase and focal adhesion kinase phosphorylation and vimentin expression. Interestingly, p53 expression was increased but in an inactive form. Furthermore, netrin-1 expression was increased in cystic epithelia and urine of various rodent models of PKD. siRNA-mediated suppression of netrin-1 significantly reduced cyst growth and improved kidney function in netrin-1 transgenic mice and in two genetic animal models of PKD. Together, these data demonstrate that netrin-1 up-regulation induced cyst formation in autosomal dominant PKD.

Altered Expression and Function of Hepatic Transporters in a Rodent Model of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common form of inherited polycystic kidney disease (PKD) and is a leading cause of kidney failure. Fluid-filled cysts develop in the kidneys of patients with ADPKD, and cysts often form in their liver and other organs. Previous data have shown that bile acids are increased in the liver of polycystic kidney (PCK) rats, a rodent model of PKD; these changes may be associated with alterations in liver transporter expression and function. However, the impact of PKD on hepatic transporters has not been characterized. Therefore, this preclinical study was designed to investigate hepatic transporter expression and function in PCK compared with wild-type (WT) Sprague-Dawley rats. Transporter gene expression was measured by quantitative polymerase chain reaction, and protein levels were quantified by Western blot and liquid chromatography-tandem mass spectroscopy (LC-MS/MS)-based proteomic analysis in rat livers. Transporter function was assessed in isolated perfused livers (IPLs), and biliary and hepatic total glutathione content was measured. Protein expression of Mrp2 and Oatp1a4 was decreased 3.0-fold and 2.9-fold, respectively, in PCK rat livers based on Western blot analysis. Proteomic analysis confirmed a decrease in Mrp2 and a decrease in Oatp1a1 expression (PCK/WT ratios, 0.368 ± 0.098 and 0.563 ± 0.038, respectively; mean ± S.D.). The biliary excretion of 5(6)-carboxy-2',7'-dichlorofluorescein, a substrate of Oatp1a1, Mrp2, and Mrp3, was decreased 28-fold in PCK compared with WT rat IPLs. Total glutathione was significantly reduced in the bile of PCK rats. Differences in hepatic transporter expression and function may contribute to altered disposition of Mrp2 and Oatp substrates in PKD.

Increased salt intake does not worsen the progression of renal cystic disease in high water-loaded PCK rats.

The anti-diuretic hormone arginine vasopressin is thought to be a detrimental factor in polycystic kidney disease (PKD). We previously reported that high water intake (HWI) reduced urine osmolality and urinary arginine vasopressin, improved renal function, and reduced the kidney/body weight ratio in PCK rats, an orthologous model of human PKD. In PKD patients, however, it is reported that HWI increases total kidney volume, urine volume, and urine sodium excretion, which could be a consequence of high salt intake. In the current study, we loaded PCK rats with high salt concurrently with HWI to determine whether this human-imitated condition exacerbates disease progression. PCK rats were assigned into 4 groups: control group (CONT: distilled water), HWI group (HWI: 5% glucose in water), HWI with 0.2% NaCl group (HWI+0.2%NaCl), and HWI with 0.45% NaCl group (HWI+0.45%NaCl). Total water intake during the experimental period was increased by 1.86-, 2.02-, and 2.42-fold in HWI, HWI+0.2%NaCl, and HWI+0.45%NaCl, and sodium intake was increased by 2.55- and 5.83-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with CONT. Systolic blood pressure was higher in HWI+0.2%NaCl and HWI+0.45%NaCl than in both CONT and HWI. Serum urea nitrogen, kidney/body weight ratio, cAMP, cystic area, and fibrosis index were significantly lower in HWI compared with CONT, and these ameliorative effects were not abrogated in either HWI+0.2%NaCl or HWI+0.45%NaCl. The amount of sodium excreted into the urine was increased by 2.50- and 8.38-fold in HWI+0.2%NaCl and HWI+0.45%NaCl, respectively, compared with HWI. Serum sodium levels were not different between the groups. These findings indicate that the beneficial effect of HWI against the progression of cystic kidney disease was not affected even by high salt-overload in this rodent model of PKD.

Impact of prenatal and postnatal maternal environment on nephron endowment, renal function and blood pressure in the Lewis polycystic kidney rat.

Maternal insufficiency during fetal development can have long-lasting effects on the offspring, most notably on nephron endowment. In polycystic kidney disease (PKD), variability in severity of disease is observed and maternal environment may be a modifying factor. In this study, we first established that in a rodent model of PKD, the Lewis polycystic kidney (LPK) rat's nephron numbers are 25% lower compared with wildtype animals. We then investigated the effects of prenatal and postnatal maternal environment on phenotype and nephron number. LPK pups born from and raised by homozygous LPK dams (control) were compared with LPK pups cross-fostered onto heterozygous LPK dams to improve postnatal environment; with LPK pups born from and raised by heterozygous LPK dams to improve both prenatal and postnatal environment and with LPK pups born from and raised by Wistar Kyoto-LPK heterozygous dams to improve both prenatal and postnatal environment on a different genetic background. Improvement in both prenatal and postnatal environment improved postnatal growth, renal function and reduced blood pressure, most notably in animals with different genetic background. Animals with improved postnatal environment only showed improved growth and blood pressure, but to a lesser extent. All intervention groups showed increased nephron number compared with control LPK. In summary, prenatal and postnatal environment had significant effect in delaying progression and reducing severity of PKD, including nephron endowment.

Inhibition of cyst growth in PCK and Wpk rat models of polycystic kidney disease with low doses of peroxisome proliferator-activated receptor γ agonists.

The studies were designed to test the efficacy of two peroxisome proliferator-activated receptor γ (PPARγ) agonists in two rodent models of polycystic kidney disease (PKD). The PCK rat is a slowly progressing cystic model while the Wpk-/- rat is a rapidly progressing model. PCK rats were fed with a pharmacological (0.4 mg/kg body weight [BW]) and a sub-pharmacological (0.04 mg/kg BW) dose of rosiglitazone (week 4-28). Wpk-/- rats were fed with pharmacological (2.0 mg/kg BW) and sub-pharmacologic (0.2 mg/kg BW) doses of pioglitazone from day 5 to 18. At termination, kidney weights of treated versus untreated cystic animals were used to determine efficacy. The current studies were also compared with previous studies containing higher doses of PPARγ agonists. The concentrations used in the animals were calculated with reference to equivalent human doses for both drugs. The current studies demonstrate: 1) that low, pharmacologically relevant, doses of the PPARγ agonists effectively inhibit cyst growth; 2) there is a class action of the drugs with both commercially available PPARγ agonists, rosiglitazone, and pioglitazone, inhibiting cyst growth; 3) the drugs showed efficacy in two different preclinical cystic models. In the PCK rat, animals fed with a sub-pharmacological dose of rosiglitazone for 24 weeks had significantly lower kidney weights than untreated animals (3.68 ± 0.13 g vs. 4.17 ± 0. 11 g, respectively, P < 0.01) while treatment with a pharmacologic dose had no significant effect on kidney weight. The rapidly progressing Wpk-/- rats were fed with pharmacological and sub-pharmacologic doses of pioglitazone from day 5 to 18 and the kidneys were compared with non-treated, cystic animals. Kidney weights on the pharmacologic dose were not statistically lower than the untreated animals while rats fed a sub-pharmacologic dose showed a significant decrease compared with untreated animals (3.35 ± 0.15 g vs. 4.55 ± 0.46 g, respectively, P = 0.045). Concentrations of PPARγ agonists below the human equivalent diabetic doses are effective in slowing cyst growth in two rodent models of PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Missense Mutation in Sterile α Motif of Novel Protein SamCystin is Associated with Polycystic Kidney Disease in (cy/+) Rat

Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/+) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealed a C to T transition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence. It was determined that Pkdr1 is specifically expressed in renal proximal tubules and encodes a novel protein, SamCystin, that contains ankyrin repeats and a sterile alpha motif. The characterization of this protein, which does not share structural homologies with known polycystins, may give new insights into the pathophysiology of renal cyst development in patients.

C-myc antisense oligonucleotide treatment ameliorates murine ARPKD

Overexpression of c-myc is postulated to play a role in the pathogenesis of polycystic kidney disease (PKD). c-myc expression is increased in all rodent models of PKD that have been examined as well as in human ADPKD. To determine whether overexpression of renal c-myc contributes to renal cyst formation, C57BL/6J-cpk litters (an animal model of ARPKD) were treated with an antisense oligomer (ASO) to c-myc mRNA. Injections of 30 microg of a c-myc ASO were given to C57BL/6J-cpk litters on postnatal days 7-20. Control mice received either sham injections or injections of an equal amount of a scrambled ASO. At 20 days, kidney weight, body weight, serum urea nitrogen (SUN), hematocrit, and renal concentration of ASO were determined. In kidney, c-Myc and PCNA protein were assessed by immunoblotting and steady state levels of renal RNA for c-myc, EGF, SGP-2, and histone H4 were assessed by northern blot hybridization. c-Myc and PCNA proteins were localized by immunohistochemistry. Cystic mice treated with the c-myc ASO had a decreased relative kidney weight, improved renal function, and a reduced amount of cystic change compared with sham and scrambled ASO controls. The abnormal expression of several PKD related proteins and mRNAs were partially reversed by c-myc antisense treatment. c-myc staining appeared to be reduced in the noncystic tubules. Treatment with the c-myc ASO did not cause a reduction in hematocrit or total body weight indicating that the beneficial effects were not due to a generalized inhibition of cell proliferation in rapidly growing tissue. c-Myc appears to play a role in the cystogenesis of cpk-induced murine PKD and antisense targeting the overexpression of c-myc partially ameliorated the renal changes.