Chronic Progressive Nephropathy Research Articles

Comparative Analysis of Hypertensive Tubulopathy in Animal Models of Hypertension and Its Relevance to Human Pathology.

Assessment of hypertensive tubulopathy for more than fifty animal models of hypertension in experimental pathology employs criteria that do not correspond to lesional descriptors for tubular lesions in clinical pathology. We provide a critical appraisal of experimental hypertension with the same approach used to estimate hypertensive renal tubulopathy in humans. Four models with different pathogenesis of hypertension were analyzed-chronic angiotensin (Ang) II-infused and renin-overexpressing (TTRhRen) mice, spontaneously hypertensive (SHR), and Goldblatt two-kidney one-clip (2K1C) rats. Mouse models, SHR, and the nonclipped kidney in 2K1C rats had no regular signs of hypertensive tubulopathy. Histopathology in animals was mild and limited to variations in the volume density of tubular lumen and epithelium, interstitial space, and interstitial collagen. Affected kidneys in animals demonstrated lesion values that are significantly different compared with healthy controls but correspond to mild damage if compared with hypertensive humans. The most substantial human-like hypertensive tubulopathy was detected in the clipped kidney of 2K1C rats. For the first time, our study demonstrated the regular presence of chronic progressive nephropathy (CPN) in relatively young mice and rats with induced hypertension. Because CPN may confound the assessment of rodent models of hypertension, proliferative markers should be used to verify nonhypertensive tubulopathy.

Causes of Mortality and Profile of Spontaneous Tumors in Young CD-1 Mice.

A retrospective study was performed to establish the causes of mortality and incidence patterns of tumors in young (<50 weeks) control CD-1® mice from Charles River Laboratories. Tumor incidences (fatal and nonfatal) and nonneoplastic causes of death observed during the first 50 weeks of the study were collected from 48 thirteen-week toxicity studies conducted between 2009 and 2018 and from 43 carcinogenicity studies conducted between 2005 and 2018. Thirteen-week studies had a mortality rate of 8/620 (1.3%) in males and 4/620 (0.65%) in females. The major factors contributing to death were integument lesions in males (3/8) and experimental procedure-related injuries in females (3/4). All tumors recorded were nonfatal. Bronchiolo-alveolar adenoma was the commonest tumor with the same incidence in both males and females (4/620, 0.65%); a single lymphoma (0.16%) and uterine leiomyosarcoma (1/620 0.16%) were reported in females. The mortality rates of males and females that died or were euthanized during the first 50 weeks in carcinogenicity studies were 192/2830 (6.8%) and 198/2830 (7%), respectively. The most common fatal tumor in this age group was lymphoma in both sexes, with an incidence of 18/192 (9.3%) and 41/198 (20.7%) in males and females, respectively. In males tumors were responsible for fewer deaths than in females (17% vs. 32.3%). The major nonneoplastic causes of death or moribundity were cutaneous lesions (44/192, 22.9%), and obstructive uropathy (39/192, 20.3%) in males, and chronic progressive nephropathy (40/198, 20.2%) in females. Only minor differences were evident compared to a similar study performed 15 years ago; these might reflect changes in terminology and diagnostic criteria, and stricter animal welfare endpoints.

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction.

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.

Confounders for kidney carcinogenesis in rodent cancer bioassays.

In the long-term safety testing of chemicals for carcinogenicity the toxicologist needs to be aware of a number of scenarios where renal tubule tumors, or their precursors, arise that are not due to a carcinogenic action of the test article. Situations producing false positive results in the kidney include exacerbation of chronic progressive nephropathy (CPN) in rats, confusion of atypical tubule hyperplasia (the obligate precursor of renal tubule tumor) with foci of benign CPN-related renal tubule cell proliferation, inclusion of spontaneous tumor entities, such as the amphophilic-vacuolar tumor, in the test article tumor count, the possibility of a link between spontaneous forms of tubule dilatation and renal tubule tumor formation in mice, and the supposed predictivity of chemically-induced karyomegaly for renal carcinogenicity in both rats and mice. Examples of these misleading situations are described and discussed.

Preclinical evaluation of [225Ac]Ac-DOTA-TATE for treatment of lung neuroendocrine neoplasms.

There is significant interest in the development of targeted alpha-particle therapies (TATs) for treatment of solid tumors. The metal chelator-peptide conjugate, DOTA-TATE, loaded with the β-particle emitting radionuclide 177Lu ([177Lu]Lu-DOTA-TATE) is now standard care for neuroendocrine tumors that express the somatostatin receptor 2 (SSTR2) target. A recent clinical study demonstrated efficacy of the corresponding [225Ac]Ac-DOTA-TATE in patients that were refractory to [177Lu]Lu-DOTA-TATE. Herein, we report the radiosynthesis, toxicity, biodistribution (BD), radiation dosimetry (RD), and efficacy of [225Ac]Ac-DOTA-TATE in small animal models of lung neuroendocrine neoplasms (NENs). [225Ac]Ac-DOTA-TATE was synthesized and characterized for radiochemical yield, purity and stability. Non-tumor-bearing BALB/c mice were tested for toxicity and BD. Efficacy was determined by single intravenous injection of [225Ac]Ac-DOTA-TATE into SCID mice-bearing human SSTR2 positive H727 and H69 lung NENs. RD was calculated using the BD data. [225Ac]Ac-DOTA-TATE was synthesized with 98% yield, 99.8% purity, and displayed 97% stability after 2days incubation in human serum at 37°C. All animals in the toxicity study appeared healthy 5months post injection with no indications of toxicity, except that animals that received ≥111kBq of [225Ac]Ac-DOTA-TATE had chronic progressive nephropathy. BD studies revealed that the primary route of elimination is by the renal route. RD calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. For both tumor models, a significant tumor growth delay and time to experimental endpoint were observed following a single administration of [225Ac]Ac-DOTA-TATE relative to controls. These results suggest significant potential for the clinical translation of [225Ac]Ac-DOTA-TATE for lung NENs.

Lipophilicity Determines Routes of Uptake and Clearance, and Toxicity of an Alpha-Particle-Emitting Peptide Receptor Radiotherapy.

Lipophilicity is explored in the biodistribution (BD), pharmacokinetics (PK), radiation dosimetry (RD), and toxicity of an internally administered targeted alpha-particle therapy (TAT) under development for the treatment of metastatic melanoma. The TAT conjugate is comprised of the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), conjugated to melanocortin receptor 1 specific peptidic ligand (MC1RL) using a linker moiety and chelation of the 225Ac radiometal. A set of conjugates were prepared with a range of lipophilicities (log D 7.4 values) by varying the chemical properties of the linker. Reported are the observations that higher log D 7.4 values are associated with decreased kidney uptake, decreased absorbed radiation dose, and decreased kidney toxicity of the TAT, and the inverse is observed for lower log D 7.4 values. Animals administered TATs with lower lipophilicities exhibited acute nephropathy and death, whereas animals administered the highest activity TATs with higher lipophilicities lived for the duration of the 7 month study and exhibited chronic progressive nephropathy. Changes in TAT lipophilicity were not associated with changes in liver uptake, dose, or toxicity. Significant observations include that lipophilicity correlates with kidney BD, the kidney-to-liver BD ratio, and weight loss and that blood urea nitrogen (BUN) levels correlated with kidney uptake. Furthermore, BUN was identified as having higher sensitivity and specificity of detection of kidney pathology, and the liver enzyme alkaline phosphatase (ALKP) had high sensitivity and specificity for detection of liver damage associated with the TAT. These findings suggest that tuning radiopharmaceutical lipophilicity can effectively modulate the level of kidney uptake to reduce morbidity and improve both safety and efficacy.

Background data of 2-year-old male and female F344 gpt delta rats.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

Long-term in vitro effects of exposing the human HK-2 proximal tubule cell line to 3-monochloropropane-1,2-diol.

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

A histopathological analysis of spontaneous neoplastic and non-neoplastic lesions in aged male RccHan:WIST rats.

Histopathological information about spontaneous lesions in aged Hannover Wistar rats is limited. In this study, we describe spontaneous lesions found in 39 male RccHan:WIST rats used as a control in a carcinogenicity study. Neoplastic lesions were frequently seen in the endocrine system, such as pituitary adenomas in the pars distalis. This strain exhibited a high incidence of thymoma (10.3%), compared to other strains. We encountered an oligodendroglioma, a pituitary adenoma of the pars intermedia, and a prostate adenocarcinoma, which are comparatively rare in rats. While the variety and incidence of non-neoplastic lesions were similar to those in other strains, several interesting lesions occurred with relatively high incidence, including “harderianization” of the extraorbital lacrimal gland, common bile duct ectasia, and hyperplasia of pulmonary endocrine cells in the lung. Furthermore, comparative analyses demonstrated that the severity of chronic progressive nephropathy and murine progressive cardiomyopathy in RccHan:WIST rats was less than that in F344 rats.

Methionine restriction delays aging-related urogenital diseases in male Fischer 344 rats.

Dietary methionine restriction (MR) has been found to enhance longevity across many species. We hypothesized that MR might enhance longevity in part by delaying or inhibiting age-related disease processes. To this end, male Fischer 344 rats were fed control (CF, 0.86% methionine) or MR (0.17% methionine) diets throughout their life until sacrifice at approximately 30 months of age, and histopathology was performed to identify the incidence and progression of two important aging-related pathologies, namely, chronic progressive nephropathy (CPN) and testicular tumorigenesis. Although kidney pathology was observed in 87% CF rats and CPN in 62% of CF animals, no evidence of kidney disease was observed in MR rats. Consistent with the absence of renal pathology, urinary albumin levels were lower in the MR group compared to controls throughout the study, with over a six-fold difference between the groups at 30 months of age. Biomarkers associated with renal disease, namely, clusterin, cystatin C, and β-2 microglobulin, were reduced following 18 months of MR. A reduction in testicular tumor incidence from 88% in CF to 22% in MR rats was also observed. These results suggest that MR may lead to metabolic and cellular changes providing protection against age-related diseases.

Proceedings of the 2019 National Toxicology Program Satellite Symposium.

The 2019 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Raleigh, North Carolina, at the Society of Toxicologic Pathology's 38th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included aging mouse lesions from various strains, as well as the following lesions from various rat strains: rete testis sperm granuloma/fibrosis, ovarian cystadenocarcinoma, retro-orbital schwannoma, periductal cholangiofibrosis of the liver and pancreas, pars distalis hypertrophy, chronic progressive nephropathy, and renal tubule regeneration. Other cases included polyovular follicles in young beagle dogs and a fungal blood smear contaminant. One series of cases challenged the audience to consider how immunohistochemistry may improve the diagnosis of some tumors. Interesting retinal lesions from a rhesus macaque emphasized the difficulty in determining the etiology of any particular retinal lesion due to the retina's similar response to vascular injury. Finally, a series of lesions from the International Harmonization of Nomenclature and Diagnostic Criteria Non-Rodent Fish Working Group were presented.

Tetrahydrofuran-induced tumors in rodents are not relevant to humans: Quantitative weight of evidence analysis of mode of action information does not support classification of tetrahydrofuran as a possible human carcinogen

Inhalation of tetrahydrofuran (THF) causes a marginal increase in the incidence of renal tumors in male rats and an increase in the incidence of liver tumors in female mice. Quantitative weight of evidence (QWoE) was applied to assess experimental support for biologically plausible modes of action (MoA) of tumor formation by THF and their human relevance. QWoE did not obtain support for a MoA to induce kidney tumors in male rats from THF exposure via α2u -globulin nephropathy, exacerbation of chronic progressive nephropathy (CPN), DNA-damage, or recurrent cytotoxicity but obtained moderate to good support for a constitutive androgen receptor (CAR)-mediated MoA for the induction of liver tumors in female mice. Tumors as a consequence of CAR-activation are not considered relevant to humans. Considering the previous conclusion that the increases in kidney tumors in male rats are unlikely related to THF-exposure and the support for a CAR-mediated MoA in mice obtained here, these tumors should not be used as a basis for THF cancer classification.

Intrarenal Renin-Angiotensin System Involvement in the Pathogenesis of Chronic Progressive Nephropathy-Bridging the Informational Gap Between Disciplines.

Chronic progressive nephropathy (CPN) is the most commonly encountered spontaneous background finding in laboratory rodents. Various theories on its pathogenesis have been proposed, but there is a paucity of data regarding specific mechanisms or physiologic pathways involved in early CPN development. The current CPN mechanism of action for tumorigenesis is largely based on its associated increase in tubular cell proliferation without regard to preceding subcellular degenerative changes. Combing through the published literature from multiple biology disciplines provided insight into the preceding cellular events. Mechanistic pathways involved in the progressive age-related decline in rodent kidney function and several key inflexion points have been identified. These critical pathway factors were then connected using data from renal models from multiple rodent strains, other species, and mechanistic work in humans to form a cohesive picture of pathways and protein interactions. Abundant data linked similar renal pathologies to local events involving hypoxia (hypoxia-inducible factor 1α), altered intrarenal renin-angiotensin system (RAS), oxidative stress (nitric oxide), and pro-inflammatory pathways (transforming growth factor β), with positive feedback loops and downstream effectors amplifying the injury and promoting scarring. Intrarenal RAS alterations seem to be central to all these events and may be critical to CPN development and progression.

Investigations on the Relationship Between Ovarian, Endocrine, and Renal Findings in Nonclinical Safety Studies of the γ-Secretase Inhibitor Avagacestat.

Avagacestat, a gamma (γ)-secretase inhibitor that was in development for treatment of Alzheimer's disease, produced ovarian granulosa-thecal cell tumors in rats and dogs and a glomerulopathy with profound proteinuria in female rats. This report describes the results of follow-up investigative studies, including the use of ovariectomized (OVX) rats, to further characterize these findings and determine their mechanism(s). Ovarian proliferative changes in rats likely resulted from: (1) inhibition of Notch signaling pathways regulating ovarian follicular differentiation/development, characterized microscopically as altered ovarian cyclicity and/or ovarian follicular degeneration; (2) subsequent disruption of the hypothalamic-pituitary-ovarian axis due to ovarian atrophy with decreases in serum estrogen and progesterone (as low as 0.45× and 0.21× controls, respectively); and (3) chronic gonadotropin stimulation and pituitary hypertrophy/hyperplasia in response to the absence of negative feedback. Gonadotropin stimulation in rats was confirmed by increases in serum follicle-stimulating hormone (up to 7.75× controls) and luteinizing hormone (up to 5.84×). A similar nongenotoxic mechanism was likely responsible for the ovarian findings in dogs although changes in serum hormone levels were not detected. The dose- and time-dependent glomerulopathy with progression to chronic progressive nephropathy in female rats appears to be a direct effect of avagacestat and was not ameliorated with coadministration of 17β-estradiol or an antihypertensive (enalapril) and was not present in control OVX rats. In contrast, adrenocortical hypertrophy in female rats was considered secondary to ovarian changes based on the absence of this finding in avagacestat-treated OVX rats and no increase in adrenocorticotropic hormone staining in the pituitary.

Histopathology re-examination of the NTP toxicity/carcinogenicity studies of tert-butyl alcohol to identify renal tumor and toxicity modes of action

Tert-butyl alcohol (TBA) targets the rat kidney following repeated exposures, including renal tubule tumors. The mode of action (MOA) of these tumors, concluded by a pathology working group, involves both alpha2u-globulin nephropathy (α2u-gN) and exacerbated chronic progressive nephropathy (CPN), but has been disputed and an undefined MOA proposed. This study further reviews the histology slides of male and female rat kidneys from the NTP drinking water 13-week toxicity and 2-year carcinogenicity studies, including the 15-month interim sacrifice group. The papillary epithelial lining alteration formerly referred to as “transitional cell hyperplasia” develops as part of advanced CPN and does not represent a separate toxicity. No changes were observed in the kidney pelvis urothelium. The only alterations in subchronic male rats involved α2u-gN and CPN, without test article-related alterations in females. Focused examination of areas of parenchyma unaffected by CPN in TBA-treated male and female rats of the chronic studies revealed no renal tubule abnormalities other than from the effects of α2u-gN and CPN. Unrelated to toxicity were spontaneous amphophilic or vacuolar tubule proliferative lesions. All observed TBA-associated non-neoplastic and neoplastic histopathological changes in the kidney can be explained by α2u-gN or enhanced CPN, neither of which are relevant to humans.

Mechanisms of Rodent Renal Carcinogenesis Revisited.

The important renal tumors that can be induced by exposure of rats to chemical carcinogens are renal tubule tumors (RTTs) derived from tubule epithelium; renal pelvic carcinoma derived from the urothelial lining of the pelvis; renal mesenchymal tumors (RMTs) derived from the interstitial connective tissue; and nephroblastoma derived from the metanephric primordia. However, almost all of our knowledge concerning mechanisms of renal carcinogenesis in the rodent pertains to the adenomas and carcinomas originating from renal tubule epithelium. Currently, nine mechanistic pathways can be identified in either the rat or mouse following chemical exposure. These include direct DNA reactivity, indirect DNA reactivity through free radical formation, multiphase bioactivation involving glutathione conjugation, mitotic disruption, sustained cell proliferation from direct cytotoxicity, sustained cell proliferation by disruption of a physiologic process (alpha 2u-globulin nephropathy), exaggerated pharmacologic response, species-dominant metabolic pathway, and chemical exacerbation of chronic progressive nephropathy. Spontaneous occurrence of RTTs in the rat will be included since one example is a confounder for interpreting kidney tumor results in chemical carcinogenicity studies in rats.

Dipeptiven® improves kidney pathology in a rat model of chronic kidney disease

Summary Background & aims Administration of glutamine in patients with renal dysfunction is considered to be potentially adverse. In a rat model of moderate kidney dysfunction dose-dependent effects of intravenous alanyl-glutamine infusion on possible biochemical and histological signs of toxicity were investigated. Methods Rats with renal dysfunction resulting from 5/6 nephrectomy received a 9 days continuous intravenous infusion of either saline or 0.5 g/kg/day or 3.0 g/kg/day alanyl-glutamine (Dipeptiven®) or 3.0 g/kg/day alanine. Dose-dependent effects on kidney and other organs were assessed by analyzing blood levels of creatinine, ammonia, urea, ALT, AST, ALP, pH, pO2, pCO2, glutamine, and histopathology. Results Continuous intravenous infusion of 3.0 g/kg/day alanyl-glutamine increased plasma glutamine concentrations up to 60% without aggravating the underlying kidney injury. In contrast, the morphology of the kidneys was improved due to reduced glomerulosclerosis and tubular proteinaceous casts. An increase in plasma urea concentrations observed in the 3.0 g/kg/day alanyl-glutamine group only was not associated with worsening of the phenotype. Conclusions Continuous intravenous infusion of alanyl-glutamine at 0.5 and 3.0 g/kg/day up to 9 consecutive days is safe in a rat model of chronic moderate kidney dysfunction and improved the renal morphology by reducing glomerulosclerosis and tubular proteinaceous casts. In these animals a decreased incidence and severity of chronic progressive nephropathy was observed compared to the saline and alanine treated animals.

Long-Term Safety and Tolerability of Dasiglucagon, a Stable-in-Solution Glucagon Analog

Several groups are developing fully integrated dual-hormone artificial pancreas systems, which deliver insulin or glucagon in response to changes in glucose to maintain euglycemia. These systems hold the potential to transform management of type 1 diabetes, but in order to be realized these systems require a stable-in-solution glucagon analog suitable for chronic use. Dasiglucagon is a novel glucagon analog stable in liquid formulation. The present studies evaluated safety and tolerability of chronically administered dasiglucagon in rats and dogs. Rats and dogs were dosed s.c. daily for 26 and 39 weeks, respectively, in 4 groups of 20 rats with 0 (vehicle), 0.5, 2, or 8 mg/kg/day; 4 groups of 4 beagle dogs with 0, 0.02, 0.1, or 0.3 mg/kg/day. Glucose and insulin levels increased in a dose-dependent manner in all treated animals. In dogs, heart rate increased at the 0.1 and 0.3 mg/kg/day dose level. Liver and kidney weights increased at all dose levels for in both species, and microscopic pathology demonstrated increase in hepatocyte glycogen vacuolation and increase in chronic progressive nephropathy (a normal finding in aging rats). Heart weights increased with no histopathological correlate. All findings showed full/partial recovery after 4-week treatment free periods. Some animals developed anti-drug-antibodies but with no apparent effect on pharmacodynamics. The NOAEL (no observed adverse effect level) dose of 2 mg/kg/day in rats and 0.1 mg/kg/day in dogs represents exposure multiples (AUC) of 22 and 2 compared to anticipated human doses of up to 1 mg/day. Chronic administration of dasiglucagon multiple times above relevant human doses was well tolerated. All findings were consistent with the known pharmacological effects of glucagon. The risk of glycogen accumulation is not considered relevant for humans treated with dasiglucagon to maintain euglycemia. These findings support long-term human testing of dasiglucagon in dual-hormonal artificial pancreas systems. Disclosure J.R. Castle: Consultant; Self; Zealand Pharma A/S. Advisory Panel; Self; Novo Nordisk Inc. M. Elander: Employee; Self; Zealand Pharma A/S. S.A. O'Halloran: None.

Epithelium Lining Rat Renal Papilla: Nomenclature and Association with Chronic Progressive Nephropathy (CPN).

Chronic progressive nephropathy (CPN) occurs commonly in rats, more frequently and severely in males than females. High-grade CPN is characterized by increased layers of the renal papilla lining, designated as urothelial hyperplasia in the International Harmonization of Nomenclature and Diagnostic Criteria classification. However, urothelium lining the pelvis is not equivalent to the epithelium lining the papilla. To evaluate whether the epithelium lining the renal papilla is actually urothelial in nature and whether CPN-associated multicellularity represents proliferation, kidney tissues from aged rats with CPN, from rats with multicellularity of the renal papilla epithelium of either low-grade or marked severity, and from young rats with normal kidneys were analyzed and compared. Immunohistochemical staining for uroplakins (urothelial specific proteins) was negative in the papilla epithelium in all rats with multicellularity or not, indicating these cells are not urothelial. Mitotic figures were rarely observed in this epithelium, even with multicellularity. Immunohistochemical staining for Ki-67 was negative. Papilla lining cells and true urothelium differed by scanning electron microscopy. Based on these findings, we recommend that the epithelium lining the papilla not be classified as urothelial, and the CPN-associated lesion be designated as vesicular alteration of renal papilla instead of hyperplasia and distinguished in diagnostic systems from kidney pelvis urothelial hyperplasia.

Short-term Low-Dose mTORC1 Inhibition in Aged Rats Counter-Regulates Age-Related Gene Changes and Blocks Age-Related Kidney Pathology.

Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes.