Quantitative Renal Vascular Casting in Nephrology Research

The present paper describes the use of a quantitative renal vascular casting method to study the changes associated with kidney disease. Several animal models of hypertension (spontaneously hypertensive rat, SHR, with its normotensive rat the Wistar Kyoto, WKY; Dahl salt sensitive DS - hypertensive, and salt resistant DR -normotensive) were examined at time points when the systemic blood pressure was rising (6 and 12 weeks of age) and following renal denervation (in SHR-WKY rats). The SHR appears to have a smaller caliber afferent arteriole at both 6 and 12 weeks of age. This difference is probably not entirely due to sympathetic vasoconstriction since the strain related afferent arteriolar diameter difference was still present after renal denervation. In the Dahl rats, there is not much of an intrarenal vascular difference between the DS and DR rats with the only real finding of a smaller distal afferent arteriolar diameter found in outer cortical nephrons of the DR. The two models of acute renal failure (ARF) that were studied include, a) the glycerol model (known to initially cause an intense vasoconstriction) and b) gentamicin, a nephrotoxic antibiotic. Two time points were examined for each of these models. As expected in the glycerol model there was an intense vasoconstriction at three hours which essentially was gone at 3 days-a time when the renal failure was fulminant. The glomerulus appeared to be contracted at three hours as well. In the gentamicin model no renal vascular alteration was seen at 6 days, when renal failure was mild while at 10 days, when renal failure was pronounced, outer cortical afferent arterioles appeared to be moderately constricted. In the 5/6 nephrectomy model of chronic renal failure, the glomeruli were smaller in rats in renal failure than in the controls

Quantitative Vascular Casting of the Post-Ischemic Hydronephrotic Kidney

The renal microvasculature (afferent arteriole) and glomeruli were examined and quantitated by two methods in the post-ischemic hydronephrotic (PIH) kidney. The methods used were: 1) an in vivo examination and 2) controlled perfusion-fixation, quantitative vascular casting examined by scanning electron microscopy. The second method was also applied to the vasculature of the contralateral, functional kidney. The goals of the study were to: 1) validate the quantitative vascular casting method by comparing PIH renal microvascular data from the casting method with in vivo values and 2) determine the extent of microvascular dimensional difference of the PIH kidney from its contralateral functional counterpart. It was determined that the casting values were consistent with the data obtained from the in vivo examination of the afferent arteriole and glomeruli. This finding provides further support for the quantitative renal microvascular casting technique. Using that technique it was determined that the dimensions of the microvasculature and glomeruli of the PIH kidney were severely (and significantly, p\u3c0.05) reduced compared to its functional mate. Since these PIH vessels show a significant decrement in size, vascular reactivity and functional data based on the PIH vessels should be looked at cautiously. The vasculature and glomeruli of the PIH kidney might not be totally normal, however structurally, the glomeruli do not appear to be dramatically altered

Epidermal Growth Factor Ameliorates Autosomal Recessive Polycystic Kidney Disease in Mice

AbstractC57BL/6J mice homozygous for the cpk gene exhibit an autosomal recessive (AR) form of polycystic kidney disease (PKD), similar to human ARPKD, with massive collecting duct cysts. These cysts are lined by epithelia with an immature phenotype. Since renal expression of epidermal growth factor (EGF) is also significantly decreased in affected mice, we hypothesized that renal EGF is necessary for normal developmental maturation of the collecting duct. To determine if the lack of EGF may be a decisive factor in the initiation and/or growth of collecting duct cysts, we administered exogenous EGF (1 μg/g body wt subcutaneously) daily for Postnatal Days 3-9 (a critical period for collecting duct maturation) to C57BL/6J-cpk mice. EGF but not sham or albumin treatment retarded the development of PKD, reduced the degree of renal failure associated with the disease, and prolonged the survival of cystic mice. Sulfated glycoprotein-2 gene expression, a marker of immaturity in collecting duct cells, was reduced in cystic kidney by EGF treatment. This finding indicates that EGF treatment was associated with an increase in the maturation of the collecting duct epithelial cells. These findings support the view that decreased EGF may play a significant role in promoting the enlargement of collecting duct cysts in a hereditary model of ARPKD and that PKD involves defective and/or arrested collecting duct cell maturation

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

Background and Objectives 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). Materials and Methods 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. Results 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). Conclusion Concentrations of PPARγ agonists below the human equivalent diabetic doses are effective in slowing cyst growth in two rodent models of PKD

Parathyroid Hormone Enhances Mechanically Induced Bone Formation, Possibly Involving L-Type Voltage- Sensitive Calcium Channels

PTH and mechanical loading might act synergistically on bone formation. We tested the in vivo effect of the L-type voltage-sensitive calcium channel (VSCC) blocker, verapamil, on bone formation induced by human PTH-(1–34) (PTH) injection with or without mechanical loading. Adult rats were divided into eight groups: vehicle, verapamil, PTH, or verapamil plus PTH with or without mechanical loading. Verapamil (100 mg/kg) was given orally 90 min before loading. PTH (80 μg/kg) was injected sc 30 min before loading. Loading applied to tibia and ulna for 3 min significantly increased the bone formation rate on both the endocortical surface of tibia and the periosteal surface of ulna (P < 0.0001). Treatment with PTH enhanced load-induced bone formation by 53% and 76% (P < 0.001) on the endocortical and periosteal surfaces, respectively. Treatment with verapamil suppressed load-induced bone formation rate by 77% and 59% (P < 0.01). Furthermore, verapamil suppressed bone formation in rats subjected to PTH plus loading by 74% and 68% (P < 0.0001) at the tibia and ulna, respectively. In the groups without loading, neither verapamil nor PTH treatment significantly changed any bone formation parameter. This study indicates that L-type VSCCs mediate load-induced bone formation in vivo. Furthermore, PTH enhances load-induced bone adaptation through involvement of L-type VSCCs

Structural and Functional Analyses of Liver Cysts from the BALB/c-cpk Mouse Model of Polycystic Kidney Disease

Liver cysts arising from hepatic bile ducts are a common extra-renal pathology associated with both autosomal dominant and recessive polycystic kidney disease in humans. To elucidate the functional and structural changes inherent in cyst formation and growth, hepatic bile duct epithelia were isolated from the BALB/ c-cpk mouse model of polycystic kidney disease. Light and transmission electron microscopy revealed substantial fibrosis in the basal lamina surrounding hepatic bile duct cysts isolated from heterozygous (BALB/c-cpk/+) and homozygous (BALB/c-cpk/cpk) animals. Scanning electron microscopy and length analysis of normal, precystic and cystic bile ducts provided the unique observation that primary cilia in cholangiocytes isolated from bile ducts and cysts of animals expressing the mutated cpk gene had lengths outside the minimal and maximal ranges of those in cells lining bile ducts of wild-type animals. Based on the hypothesis that PKD is one of several diseases characterized as ciliopathies, this abnormal variability in the length of the primary cilia may have functional implications. Electrophysiological analyses of freshly isolated cysts indicate that the amiloride-sensitive epithelial Na(+) channel (ENaC) is inactive/absent and cAMP-mediated anion secretion is the electrogenic transport process contributing to cyst fluid accumulation. Anion secretion can be stimulated by the luminal stimulation of adenylyl cyclase

Renal Cyst Fluid From Human Polycystic Kidney Disease Patients Stimulates Cl- Transport: Active Factor and Cl- Channels

poster abstractAutosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of fluid-filled cysts predominately in the kidney and in liver bile ducts. The factors involved in modifying the rate of cyst growth through epithelial proliferation or secretion are critical to understanding the progression of the disease. In addition, elucidation of mechanisms that potentiate the normal progression to renal failure will provide the basis for therapeutic intervention. Of note are the observations that the decline in renal function in middle age is precipitous and that renal injury results in an exacerbation of cyst growth. Using electrophysiological and biochemical techniques, we identified LPA (lysophosphatic acid) as a component of cyst fluid that stimulates secretory Cl- transport via two anion channels, CFTR and TMEM16a, in the mpkCCDcl4 model of renal principal cells. The LPA effect is manifested through receptors located on the basolateral membrane of polarized renal cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage into the interstitial space and/or leakage of vascular LPA are capable of stimulating epithelial cell secretion resulting in cyst enlargement. Research Support: IUPUI Membrane Biosciences Signature Center Gran

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse. The C57BL/6J-cpk mouse has a form of autosomal-recessive polycystic kidney disease characterized by the rapid growth of large collecting duct cysts and the development of severe renal failure usually by three to four weeks of age. Previous studies had shown higher steady-state levels of proto-oncogene mRNA in these cystic kidneys. It is now shown using nuclear run-on transcription that the c-fos and c-myc proto-oncogenes are transcribed at higher rates in cystic kidneys, and thus that increased transcription, in part, may account for the increased mRNA levels. c-myc mRNA was detected by in situ hybridization in nephron anlagen and elongating tubules of normal and cystic kidneys during late fetal and early neonatal kidney development. Localization of c-myc expression in the normal kidney decreased with age over the three-week postnatal period. By contrast, c-myc mRNA was found in cysts as early as three days of age, with increased levels at two and three weeks, c-myc expression was also elevated in apparently normal, non-dividing proximal tubules in three-week-old cystic animals. On the basis of these findings, we suggest that c-myc expression is linked to the proliferation of cells engaged in the primary cystogenic process, and that expression of this gene in proximal tubule cells of severely azotemic animals reflects the compensatory response of residual tubular epithelial cells to progressive renal dysfunction

Adverse mandibular bone effects associated with kidney disease are only partially corrected with bisphosphonate and/or calcium treatment

Bone Biology Laboratory http://www.iupui.edu/~bonelab/ Department of Anatomy and Cell Biology Indiana University School of Medicine Department of Biomedical Engineering IUPUIBackground/Aims: Patients with chronic kidney disease (CKD) have high prevalence of periodontal disease that may predispose to tooth loss and inflammation. The goal of this study was to test the hypotheses that a genetic rat model of progressive CKD would exhibit altered oral bone properties and that treatment with either bisphosphonates or calcium could attenuate these adverse changes. Methods: At 25 weeks of age, rats were treated with zoledronate, calcium gluconate, or their combination for 5 or 10 weeks. Mandible bone properties were assessed using micro-computed tomography to determine bone volume (BV/TV) and cementenamel junction to alveolar crest distance (CEJ-AC). Results: Untreated CKD animals had significantly lower BV/TV at both 30 (-5%) and 35 (-14%) weeks of age and higher CEJ-AC (+27 and 29%) compared to normal animals. CKD animals had significantly higher PTH compared to normal animals yet similar levels of C-reactive protein. Zoledronate-treatment normalized BV/TV over the first 5 weeks but this benefit was lost by 10 weeks. Calcium treatment, alone or in combination with zoledronate, was effective in normalizing BV/TV at both time points. Neither zoledronate nor calcium was able to correct the higher CEJ-AC caused by CKD. Calcium, but not zoledronate, significantly reduced serum parathyroid hormone (PTH) while neither treatment affected C-reactive protein. Conclusions: 1) this progressive animal model of chronic kidney disease shows a clear mandibular skeletal phenotype consistent with periodontitis, 2) the periodontitis is not associated with systemic inflammation as measured by C-reactive protein, and 3) reducing PTH has positive effects on the mandible phenotype.This work was supported by NIH grant (AR058005). We would like to thank Dr. Xianming Chen, Mr. Alex Carr and Mr. Drew Brown for their assistance with the biochemical assays, breeding colony and micro CT scanning/analysis, respectively

A comparison of calcium to zoledronic acid for improvement of cortical bone in an animal model of CKD

Bone Biology Laboratory http://www.iupui.edu/~bonelab/ Department of Anatomy and Cell Biology Indiana University School of MedicinePatients with chronic kidney disease (CKD) have increased risk of fractures, yet the optimal treatment is unknown. In secondary analyses of large randomized trials, bisphosphonates have been shown to improve bone mineral density and reduce fractures. However, bisphosphonates are currently not recommended in patients with advanced kidney disease due to concern about over-suppressing bone remodeling, which may increase the risk of developing arterial calcification. In the present study we used a naturally occurring rat model of CKD with secondary hyperparathyroidism, the Cy/+ rat, and compared the efficacy of treatment with zoledronic acid, calcium given in water to simulate a phosphate binder, and the combination of calcium and zoledronic acid. Animals were treated beginning at 25 weeks of age (approximately 30% of normal renal function) and followed for ten weeks. The results demonstrate that both zoledronic acid and calcium improved bone volume by microCT and both equally suppressed mineral apposition rate, bone formation rate, and mineralizing surface of trabecular bone. In contrast, only calcium treatment with or without zoledronic acid improved cortical porosity and cortical biomechanical properties (ultimate load and stiffness) and lowered parathyroid hormone (PTH). However, only calcium treatment led to the adverse effects of increased arterial calcification and fibroblast growth factor 23 (FGF23). These results suggest zoledronic acid may improve trabecular bone volume in CKD in the presence of secondary hyperparathyroidism, but does not benefit extraskeletal calcification or cortical biomechanical properties. Calcium effectively reduces PTH and benefits both cortical and trabecular bone yet increases the degree of extra skeletal calcification.This work was supported by the NIH NIAMS R01 5R01AR058005 (SMM) and S10-RR023710 (microCT equipment grant). We thank Drew Brown for tissue dissections, CT scanning and analysis