A case of hydronephrosis in a CD-1 outbred mouse with associated clinicopathological findings

Introduction

Laboratory animals are indispensable to biomedical and preclinical research, with mice and rats accounting for approximately 95% of laboratory animal use. The use of healthy animals is critical for the validity of experimental outcomes, as spontaneous diseases can confound results; reduce data reliability, and compromise reproducibility. Laboratory mice commonly develop naturally occurring lesions that may mimic, mask, or exacerbate experimentally induced phenotypes. Documented background pathologies include renal, respiratory, dermatological, cardiovascular, metabolic, reproductive, endocrine, and neoplastic disorders. Notably, renal diseases are the second leading cause of mortality in laboratory mice, emphasizing the importance of careful pathological assessment in experimental studies (Nemoto et al., 2024; Vigneshwar et al., 2021) [10,15] . Hydronephrosis is characterized by dilation of the renal pelvis and calyces due to impaired urine outflow, leading to urinary retention and progressive renal damage. In mice, spontaneous hydronephrosis occurs predominantly in certain inbred strains, with marked strain variability indicating a strong genetic component. Males are more frequently affected, often with greater involvement of the right kidney (Ninomiya et al., 1999; Yoshioka et al., 2016) [11,16] . Although generally uncommon, spontaneous hydronephrosis has been reported more frequently in inbred strains such as C57BL/6 and DDD mice, while occurrences in outbred strains are rare, with only a single documented case in CD-1 mouse (Chandra and Frith, 1994) [2] . The etiology of hydronephrosis in mice is multifactorial, encompassing genetic predisposition, infection, urolithiasis, neoplasia, and congenital or acquired urinary tract abnormalities, particularly in aging animals (Springer et al., 2014) [14] . In humans, hydronephrosis most commonly results from urinary tract obstruction caused by conditions such as calculi, neoplasms, strictures, or prostatic enlargement, while pediatric cases are often congenital, paralleling spontaneous disease in certain mouse strains. Across species, hydronephrosis is defined by progressive dilation of the renal pelvis with compression and atrophy of the renal parenchyma (Davisson et al., 2015) [3] .

Case History

At the Laboratory Animal House of Christian Medical College (CMC), Vellore, mice colonies were maintained in groups on sterilized bedding within Individually Ventilated Cages (IVCs). The animals were housed in separate rooms designated for breeding, holding, and experimental purposes. All husbandry and animal care procedures were carried out in strict compliance with the guidelines prescribed by the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Govt. of India. During routine health monitoring, a six-month-old male CD-1 mouse from the founder colony exhibited clinical signs suggestive of distress, including back arching, abdominal pressing, reduced activity, right-sided abdominal distension, decreased urination, and reduced feed and water intake. Physical examination revealed a large, soft, fluid-filled swelling in the neck region (Fig. 1). The animal was anesthetized and subjected to fine-needle aspiration biopsy; clear fluid was aspirated using a tuberculin syringe (Fig. 2). The right kidney was markedly enlarged and softer in consistency compared to the left (Fig. 3), with dilation of the renal pelvis and thinning with atrophy of the renal parenchyma. The left kidney was also enlarged and congested. In addition, seminal vesiculitis was observed. Based on the gross pathological findings, a diagnosis of bilateral hydronephrosis was confirmed.

Materials and Methods

Smears were prepared from the aspirated fluid, air-dried, fixed in methanol, and stained with Giemsa. Urine samples were aseptically collected directly from the urinary bladder following euthanasia and initially examined under lowpower magnification (10×) for sediment evaluation. Further, urinalysis was performed using Medi-Test Combi 10 VET urine dipsticks as per the manufacturer's instructions. Blood samples were collected immediately after euthanasia via cardiac puncture. Hematological parameters were assessed using an automated blood analyzer. Serum was separated by centrifugation, and biochemical analyses, including blood urea nitrogen (BUN), creatinine, total protein, and electrolytes, were performed using an automated clinical chemistry analyzer in accordance with standard operating procedures. All values obtained were compared with established reference ranges for CD-1 mice (Serfilippi et al., 2003) [13] .

Semen samples were collected immediately after euthanasia from the cauda epididymis and examined microscopically at 40× magnification to assess sperm motility. Thin smears were prepared on clean glass slides and stained with Eosin-Nigrosin for evaluation of sperm morphology and viability. The stained smears were examined under oil immersion at 100× magnification. Tissue samples from the kidneys, urinary bladder, and seminal vesicles were collected and fixed in 10% neutral buffered formalin. The tissues were subsequently dehydrated through graded concentrations of isopropyl alcohol, cleared in xylene, embedded in paraffin wax, sectioned at 5 µm thickness, and stained with Hematoxylin and Eosin (H&E). Histopathological examination was performed using a light microscope.

Results

Giemsa-stained FNAB smears revealed the presence of numerous leukocytes, indicative of an inflammatory response. Hematological analysis demonstrated elevated total white blood cell (WBC) and platelet counts, further supporting the presence of inflammation. Reduced lymphocyte levels and decreased packed cell volume (hematocrit) were observed, suggestive of edema (Table 1). Biochemical evaluation showed increased serum concentrations of blood urea nitrogen (BUN) and creatinine, measuring 36 mg/dL and 0.6 mg/dL, respectively. These values exceeded the established reference ranges for CD-1 male mice (BUN: 16-22 mg/dL; creatinine: 0.1-0.3 mg/dL), confirming renal dysfunction. In contrast, serum total protein and albumin levels were reduced, with values of 4.7 g/dL and 2.2 g/dL, respectively, compared with normal reference ranges (total protein: 5.4-6.4 g/dL; albumin: 3.2-3.9 g/dL), indicating hypoproteinemia. Electrolyte imbalances was also observed (Table 2). Microscopic examination of urine revealed the presence of a few red blood cells, and dipstick analysis was positive for protein, indicating proteinuria. Semen analysis demonstrated poor sperm motility (30%), a high proportion of morphologically abnormal spermatozoa (50%), and a substantial percentage of non-viable sperm (60%), confirming infertility. Histopathological analysis of kidney sections revealed loss of the tubular brush border accompanied by severe degenerative alterations in the renal tubules. Both proximal and distal tubules were markedly dilated and lined by flattened epithelial cells. Eosinophilic casts were present within the lumina of some tubules, along with mild congestion of the interstitial capillaries (Fig. 4A-C). Microscopic examination of the seminal vesicle showed numerous empty tubules which contained eosinophilic secretory material and a few exfoliated cells (Fig 4 -D).

Discussion

Experimental hydronephrosis is commonly induced in animals by ureteral ligation; however, naturally occurring models are considered more desirable due to their simplicity and translational relevance. Spontaneous hydronephrosis has been reported in several inbred mouse strains, including C57BL/6 and C57BL/KsJ, as well as in B6-based congenic strains such as MRL/MpJ and B6/DBA F2, which serve as models for obstructive uropathy and developmental hydronephrosis by inflammatory and hyperplastic narrowing of the ureteropelvic 6 ujunction. In contrast, CD-1 is an outbred mouse strain, and only a single report of spontaneous hydronephrosis has been documented to date (Chandra and Frith, 1994) [2] , highlighting its potential value for investigating genetic and pathological associations of the condition. Nakajima et al., (1983) [9] classified hydronephrosis in laboratory rodents into two types: obstructive hydronephrosis secondary to identifiable urinary tract obstruction and non-obstructive hydronephrosis of unknown etiology. The present case corresponds to the latter category, as no definitive obstructive mechanism was identified, indicating the need for further investigation into its pathogenesis. Lozzio et al., (1967) [7] described three types of macroscopic renal lesions-solitary macrocysts, hydronephrosis, and cicatricial scarring of the kidney surface-in the hereditary renal disease of Gunn rats. Similarly, Nakajima et al., (1983) [9] reported medullary atrophy with loss of the medulla and cortical atrophy in cases of spontaneous hydronephrosis in DOD mice. Additionally, lymphocytopenia alone or in conjunction with C-reactive protein (CRP) could be a surrogate marker for infected and obstructed renal conditions (Ali et al., 2015) [1] , these findings align with the present observations. Previous studies have reported marked kidney damage in KK-A y male mouse with hydronephrosis, including an enlarged renal pelvis, thinning of the cortex and medulla, shrunken glomeruli, dilated tubules, tubular epithelial degeneration, and interstitial fibrosis (Ninomiya et al., 1999) [11] . Cortical atrophy and dilation of renal tubules and the pelvis were demonstrated as key histological features of hydronephrosis by Frazier et al., (2012) [4] . Formation of eosinophilic crystals in the cytoplasm of hyperplastic urothelial cells has also been observed in affected mice (Ichii et al., 2017; Ozaki et al., 2014) [6,12] . However, the timing and mechanisms underlying the initiation of inflammation and epithelial proliferation before the onset of hydronephrosis remain unclear. The histopathological findings of the present study are consistent with these earlier reports.

Renal failure is known to impair male fertility through various spermatotoxic mechanisms including hormonal dysregulation, hypogonadism, disrupted spermatogenesis and erectile dysfunction leading to oligospermia, oligoasthenozoospermia, azoospermia or sertoli cell atrophy (Holley, 2004; Lundy and Vij, 2019) [5,8] . The reproductive abnormalities observed in this study are consistent with these established mechanisms.

Conclusion

Spontaneous hydronephrosis is uncommon in mice and has been predominantly reported in genetically predisposed inbred strains such as C57BL/6 and DDD. In contrast, CD-1 is an outbred strain, suggesting that the condition in the present case was acquired rather than hereditary; however, the exact pathogenesis could not be determined. Although infectious diseases are effectively controlled in SPF facilities, systemic non-infectious conditions cannot be entirely excluded. Therefore, careful maintenance and review of pedigree records during breeder selection are recommended to reduce breeding-related complications.

Acknowledgement

AcknowledgmentsThe authors thank the technical staff of the Laboratory Animal Facility for their assistance in animal housing, sample collection and slides preparation for histopathological analysis.

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