Ischemia causes kidney tubular cell damage and abnormal renal function. The kidney is capable of morphological restoration of tubules and recovery of function. Recently, it has been suggested that cells repopulating the ischemically injured tubule derive from bone marrow stem cells. We studied kidney repair in chimeric mice expressing GFP or bacterial beta-gal or harboring the male Y chromosome exclusively in bone marrow-derived cells. In GFP chimeras, some interstitial cells but not tubular cells expressed GFP after ischemic injury. More than 99% of those GFP interstitial cells were leukocytes. In female mice with male bone marrow, occasional tubular cells (0.06%) appeared to be positive for the Y chromosome, but deconvolution microscopy revealed these to be artifactual. In beta-gal chimeras, some tubular cells also appeared to express beta-gal as assessed by X-gal staining, but following suppression of endogenous (mammalian) beta-gal, no tubular cells could be found that stained with X-gal after ischemic injury. Whereas there was an absence of bone marrow-derived tubular cells, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. Upon i.v. injection of bone marrow mesenchymal stromal cells, postischemic functional renal impairment was reduced, but there was no evidence of differentiation of these cells into tubular cells of the kidney. Thus, our data indicate that bone marrow-derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult. It is likely that intrinsic tubular cell proliferation accounts for functionally significant replenishment of the tubular epithelium after ischemia.

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Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells

A wide variety of inherited lysosomal hydrolase deficiencies have been reported in animals and are characterized by accumulation of sphingolipids, glycolipids, oligosaccharides, or mucopolysaccharides within lysosomes Inhibitors of a lysosomal hydrolase, eg, swainsonine, may also induce storage disease Another group of lysosomal storage diseases, the ceroid-lipofuscinoses, involve the accumulation of hydrophobic proteins, but their pathogenesis is unclear Some of these diseases are of veterinary importance, and those caused by a hydrolase deficiency can be controlled by detection of heterozygotes through the gene dosage phenomenon or by molecular genetic techniques Other of these diseases are important to biomedical research either as models of the analogous human disease and/or through their ability to help elucidate specific aspects of cell biology Some of these models have been used to explore possible therapeutic strategies and to define their limitations and expectations

https://journals.sagepub.com/doi/pdf/10.1177/030098589703400601

Lysosomal Storage Diseases of Animals: An Essay in Comparative Pathology:

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.

/pdf/mouse-model-for-the-lysosomal-disorder-galactosialidosis-and-4zi4hru185.pdf

Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid beta-galactosidase, which hydrolyses the terminal beta-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted beta-galactosidase gene showed beta-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7-10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

Beta-galactosidase-deficient mouse as an animal model for GM1-gangliosidosis.

CHAPTER 3 – The Nervous System

Histopathologic, ultrastructural and Golgi impregnation studies disclosed lesions characteristic of a neuronal lysosomal storage disease in related sheep with onset of neurologic signs at 4-6 months. Biochemical and enzymatic evaluation disclosed storage of GM1 ganglioside, asialo-GM1, and neutral long chain oligosaccharides in brain, urinary excretion of neutral long chain oligosaccharides, and deficiencies of lysosomal beta-galactosidase and alpha-neuraminidase. Retrospective and limited prospective genetic studies suggested autosomal recessive inheritance. A gene-dosage effect on beta-galactosidase levels was documented in fibroblasts from putative heterozygous sheep. Fibroblasts from affected sheep did not have increased beta-galactosidase activity after incubation with the protease inhibitor, leupeptin. In some aspects this disease is similar to GM1 gangliosidosis, but is unique in that a genetic defect in lysosomal beta-galactosidase may cause the deficiency of lysosomal alpha-neuraminidase.

Inherited lysosomal storage disease associated with deficiencies of β‐galactosidase and α‐neuraminidase in sheep

Lectin histochemistry is a useful technique to identify and to localize in cells and tissues the terminal carbohydrate moieties of glycoproteins and glycolipids. The specific diagnosis of some glycoprotein storage diseases was accomplished using lectin staining patterns, and such methods of diagnosis have been attempted for some glycolipid storage diseases. This technique was applied to formalin-fixed paraffin-embedded and frozen neural, hepatic, and renal tissues of sheep with an inherited lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase. The cytoplasm of central nervous system neurons of affected sheep in paraffin-embedded sections stained with peanut agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I), Dolichos biflorus agglutinin (DBA), and soybean agglutinin (SBA). The cytoplasm of neurons in frozen sections of these tissues stained with PNA, RCA-I, wheat germ agglutinin (WGA), and Ulex europaeus agglutinin-I (UEA-I). The cytoplasm of frozen and paraffin-embedded sections of liver and kidney of affected sheep stained with PNA, whereas paraffin-embedded sections also stained with RCA-I. These results suggest the stored material in this disease has terminal saccharide moieties consisting of beta-galactose, N-acetylneuraminic acid, and N-acetylgalactosamine. Paraffin processing altered lectin staining patterns. Although the staining pattern in this glycolipid storage disease was complex, lectin histochemistry may prove to be a useful technique for the characterization of storage products and for the diagnosis of lysosomal storage diseases.

Lectin histochemistry of an ovine lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase.

An inherited disease associated with deficiencies of beta-galactosidase and alpha-neuraminidase has been identified recently in sheep. The clinical signs, the deficiency of lysosomal enzymes, and the familial nature of the disorder suggested that the condition was a lysosomal storage disease. Four affected sheep were necropsied and their tissues were examined by histopathologic and histochemical methods to determine if the lesions were consistent with a lysosomal storage disease. Central nervous system neurons were enlarged with finely to coarsely granular cytoplasmic material, or less often, neurons were distended with multiple, variably-sized vacuoles. Loss of neurons without gliosis was evident and the Nissl substance was either dispersed and fragmented or condensed around the nuclei of remaining neurons. Neurons of intestinal and other peripheral ganglia, retinal ganglion cells, and heart Purkinje fibers were enlarged similarly. White matter of the cerebrum and spinal cord had numerous spheroid to ellipsoid axonal enlargements. Periportal hepatocytes and renal epithelial cells were enlarged with marked vacuolation. The neuronal storage material stained intensely with periodic acid-Schiff/alcian blue, with Luxol fast blue, for acid phosphatase, and moderately with oil red O stains. Renal and hepatocyte storage material stained intensely with oil red O and moderately with periodic acid-Schiff/alcian blue and Sudan black B stains. The lesions in these sheep were consistent with those of a lysosomal storage disease. Both neuronal and visceral storage occurred, but the neuronal storage was more severe.

The lesions of an ovine lysosomal storage disease. Initial characterization.

Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of β-galactosidase and α-neuraminidase was homologous with any of four β-galactosidase-deficient human diseases. Fibroblasts from β-glactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for β-galactosidase, with 5-bromo-4-chloro-3-indolyl β-d-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from β-galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from β-galactosidase-deficient sheep and fibroblasts from human or feline GM1, gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM1, gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the β-galactosidase structural gene.

Interspecific genetic complementation analysis of human and sheep fibroblasts with beta-galactosidase deficiency.

Tissues and fibroblasts of sheep affected with an inherited, neuronal lysosomal storage disease expressed a deficiency of β-galactosidase activity. Cerebrum, kidney, lung, spinal cord, and spleen from affected sheep had less than 8% of the β-galactosidase activity present in the respective tissues of normal sheep. No evidence for the presence of an endogenous inhibitor in affected sheep was detected by mixing studies. Liver of affected sheep expressed a deficiency of β-galactosidase activity only in the presence of the β-d-glycosidase inhibitors, glucono-δ-lactone and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. In these studies, we demonstrated the existence of tissue-specific β-galactosidases in sheep and showed that the affected sheep have a deficiency of the lysosomal β-galactosidase. Our results suggest that the high residual β-galactosidase activity in liver of affected sheep can be attributed to a nonlysosomal β-galactosidase that has a neutralpH optimum and may be under temporal regulation.

Robert D. Murnane

Papers

Inherited lysosomal storage disease associated with deficiencies of β‐galactosidase and α‐neuraminidase in sheep

Lectin histochemistry of an ovine lysosomal storage disease with deficiencies of beta-galactosidase and alpha-neuraminidase.

The lesions of an ovine lysosomal storage disease. Initial characterization.

Interspecific genetic complementation analysis of human and sheep fibroblasts with beta-galactosidase deficiency.

β-galactosidase activity in fibroblasts and tissues from sheep with a lysosomal storage disease