Medullary Atrophy Research Articles

Generation of an Inducible Avp Knockout Mouse Line Using CRISPR/Cas9 and Cre/LoxP

Background: Arginine vasopressin (AVP) is a peptide hormone coded by the Avp gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats are diffcult to breed owing largely to the fixed nature of the Avp mutation, resulting in increased neonatal death and behavioral effects in the adults. Consequently, commercial breeders have ceased production, despite a continued need. To fill this need, we have now created a mouse model with a tamoxifen-inducible deletion of AVP. Methods: We employed CRISPR/Cas9 to target the desired regions (first intron and 3' downstream) within the Avp gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes BamHI or EcoRI were used to confirm correct targeting. For additional confirmation, PCR products were cloned into the TA-cloning vector and sequenced. Mice harboring the floxed allele were mated to B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. Results: The resultant homozygous conditional knockout mice ( Cre+ Avpflx/ flx) are viable, fertile, and show no signs of polydipsia or polyuria prior to induction, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen (2-3 mg q.d., i.p. for 5 days) in 8-week-old mice resulted in a decrease in urine osmolality from 2076±138 to 122±6 mOsm/kgH2O on day 31 after induction (p<0.0001). Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. The kidney parenchyma was structurally normal with no evidence of medullary atrophy 6 weeks after induction. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in Cre+ Avpflx/ flx mice to 27±14 % of values in Cre− floxed control mice (p<0.05). Conclusion: We developed an inducible AVP knockout mouse line ( Cre+ Avpflx/ flx) that will be shared with the research community and are likely to be useful for further study of regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin. The work was funded by the Division of Intramural Research, National Heart, Lung and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vivo Detection of Age-Related Tortuous Cerebral Small Vessels using Ferumoxytol-enhanced 7T MRI.

Histopathological studies suggest that cerebral small vessel tortuosity is crucial in age-related blood flow reduction and cellular degeneration. However, in vivo evidence is lacking. Here, we used Ferumoxytol-enhanced 7T MRI to directly visualize cerebral small vessels (<300 µm), enabling the identification of vascular tortuosity and exploration of its links to age, tissue atrophy, and vascular risk factors. High-resolution 2D/3D gradient echo MRI at 7T enhanced with Ferumoxytol, an ultrasmall superparamagnetic iron oxide (USPIO), was obtained and analyzed for cerebral small medullary artery tortuosity from 37 healthy participants (21-70 years; mean/SD: 38±14 years; 19 females). Tortuous artery count and tortuosity indices were compared between young and old groups. Age effects on vascular tortuosity were examined through partial correlations and multiple linear regression, adjusting for sex, body mass index (BMI), blood pressure (BP), and other vascular risk factors. Associations between tortuous medullary arteries and tissue atrophy, perivascular spaces (PVS), and white matter (WM) hyperintensities were explored. Age and BMI, rather than BP, showed positive correlations with both tortuous artery count and tortuosity indices. A significant correlation existed between the number of tortuous arteries and WM atrophy. WM lesions were found in proximity to or at the distal ends of tortuous medullary arteries, especially within the deep WM. Moreover, the elderly population displayed a higher prevalence of PVS, including those containing enclosed tortuous arteries. Leveraging the blooming effect of Ferumoxytol, 7T MRI excels in directly detecting cerebral small arterial tortuosity in vivo, unveiling its associations with age, BMI, tissue atrophy, WMH and PVS.

Deep medullary veins score is associated with atrophy in patients with cerebral small vessel disease.

To explore the relationship between the deep medullary vein (DMV) score and atrophy in patients with cerebral small vessel disease (CSVD). Imaging and clinical data from 125 patients with CSVD from January to December 2022 were reviewed. Normalized gray matter volume (GM_N) was calculated by dividing the gray matter volume by the whole brain volume. DMV scoring is conducted using susceptibility-weighted magnetic resonance imaging, wherein the DMV area is partitioned into six distinct regions: bilateral frontal, parietal, and occipital regions. Each region undergoes assessment based on the clarity and consistency of DMV visibility. Subsequently, the scores from these six regions are summed, resulting in a score ranging from 0 to 18 points. DMV score was associated with GM_N (r = -0.376, p < 0.001). Comparisons among patients according to GM_N tertiles, differences in gender, age, current smoking, DMV score, and total CSVD magnetic resonance imaging score were demonstrated (p < 0.05). Adjusting for age, gender, vascular risk factors, and total CSVD MR score, the DMV score was independently associated with GM_N [β (95% CI): -0.347 (-0.525, -0.168), p < 0.001]. DMV scores are independently associated with GM_N, and DMV dysfunction may play a role in brain atrophy.

Cep120 is essential for kidney stromal progenitor cell growth and differentiation

Mutations in genes that disrupt centrosome structure or function can cause congenital kidney developmental defects and lead to fibrocystic pathologies. Yet, it is unclear how defective centrosome biogenesis impacts renal progenitor cell physiology. Here, we examined the consequences of impaired centrosome duplication on kidney stromal progenitor cell growth, differentiation, and fate. Conditional deletion of the ciliopathy gene Cep120, which is essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of interstitial lineages including pericytes, fibroblasts and mesangial cells. These phenotypes were caused by a combination of delayed mitosis, activation of the mitotic surveillance pathway leading to apoptosis, and changes in both Wnt and Hedgehog signaling that are key for differentiation of stromal cells. Cep120 ablation resulted in small hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, Cep120 and centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis after renal injury via enhanced TGF-β/Smad3-Gli2 signaling. Our study defines the cellular and developmental defects caused by loss of Cep120 and aberrant centrosome biogenesis in the embryonic kidney stroma.

Adult-onset Alexander disease among patients of Jewish Syrian descent.

Alexander disease (AxD) is a rare autosomal dominant leukodystrophy caused by heterozygous mutations in the glial fibrillary acid protein (GFAP) gene. The age of symptoms onset ranges from infancy to adulthood, with variable clinical and radiological manifestations. Adult-onset AxD manifests as a chronic and progressive condition, characterized by bulbar, motor, cerebellar, and other clinical signs and symptoms. Neuroradiological findings typically involve the brainstem and cervical spinal cord. Adult-onset AxD has been described in diverse populations but is rare in Israel. We present a series of patients diagnosed with adult-onset AxD from three families, all of Jewish Syrian descent. Five patients (4 females) were diagnosed with adult-onset AxD due to the heterozygous mutationc.219G > A, p.Met73Ile in GFAP. Age at symptoms onset ranged from 48 to 61 years. Clinical characteristics were typical and involved progressive bulbar and gait disturbance, followed by pyramidal and cerebellar impairment, dysautonomia, and cognitive decline. Imaging findings included medullary and cervical spinal atrophy and mostly infratentorial white matter hyperintensities. A newly recognized cluster of adult-onset AxD in Jews of Syrian origin is presented. This disorder should be considered in differential diagnosis in appropriate circumstances. Genetic counselling for family members is required in order to discuss options for future family planning.

Teaching Neuro Images : Prominent spinal cord atrophy and white matter changes in adult polyglucosan body disease

A 48-year-old man of Ashkenazi Jewish descent developed neurogenic bladder and mild cognitive dysfunction. Neurologic examination and CSF analysis were unremarkable. Brain MRI demonstrated white matter changes in the internal capsule, optic radiations, mesencephalon, superior cerebellar peduncles, pons, and medulla (figure 1) and prominent medullary and spinal cord atrophy (figure 2). These findings are classically found in adult polyglucosan body disease (APBD).1 Glycogen branching enzyme GBE1 gene sequencing revealed that the patient was compound heterozygous (c.691+2T>C and c.986 A>C). APBD should be considered in patients with adult-onset leukodystrophy and spinal atrophy; genetic testing is crucial to prevent misdiagnosis with multiple sclerosis and other leukodystrophies.2

Cytokine signatures of end organ injury in COVID-19

Increasing evidence has shown that Coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunologic response. We aimed to assess the differences in inflammatory cytokines in COVID-19 patients compared to contemporaneously hospitalized controls and then analyze the relationship between these cytokines and the development of Acute Respiratory Distress Syndrome (ARDS), Acute Kidney Injury (AKI) and mortality. In this cohort study of hospitalized patients, done between March third, 2020 and April first, 2020 at a quaternary referral center in New York City we included adult hospitalized patients with COVID-19 and negative controls. Serum specimens were obtained on the first, second, and third hospital day and cytokines were measured by Luminex. Autopsies of nine cohort patients were examined. We identified 90 COVID-19 patients and 51 controls. Analysis of 48 inflammatory cytokines revealed upregulation of macrophage induced chemokines, T-cell related interleukines and stromal cell producing cytokines in COVID-19 patients compared to the controls. Moreover, distinctive cytokine signatures predicted the development of ARDS, AKI and mortality in COVID-19 patients. Specifically, macrophage-associated cytokines predicted ARDS, T cell immunity related cytokines predicted AKI and mortality was associated with cytokines of activated immune pathways, of which IL-13 was universally correlated with ARDS, AKI and mortality. Histopathological examination of the autopsies showed diffuse alveolar damage with significant mononuclear inflammatory cell infiltration. Additionally, the kidneys demonstrated glomerular sclerosis, tubulointerstitial lymphocyte infiltration and cortical and medullary atrophy. These patterns of cytokine expression offer insight into the pathogenesis of COVID-19 disease, its severity, and subsequent lung and kidney injury suggesting more targeted treatment strategies.

Linear brain atrophy measures in multiple sclerosis and clinically isolated syndromes: a 30-year follow-up

ObjectiveTo determine 30-year brain atrophy rates following clinically isolated syndromes and the relationship of atrophy in the first 5 years and clinical outcomes 25 years later.MethodsA cohort of 132 people...

A single combination gene therapy treats multiple age-related diseases

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-β receptor 2 [sTGFβR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

Polycystic Kidney-like Disease in a Red-ear Slider Turtle (Trachemys scripta elegans)

A 14-year-old red-ear slider turtle (Trachemys scripta elegans) with no history of pre-existing clinical disease died and was referred for necropsy examination. Grossly, oesophageal prolapse, bilateral renal cysts and a paraduodenal cystic mass were detected. Tissues were processed routinely for histology and immunohistochemistry (IHC) with primary antibodies specific for Wilm's tumour suppressor gene-1 (WT-1), insulin, glucagon and pancytokeratins. Microscopically, renal medullary cysts and medullary atrophy resembled the changes associated with polycystic kidney disease (PKD). The cysts of the paraduodenal mass were lined by ciliated epithelial cells resembling embryonal cells and were intensely positive for glucagon and insulin by IHC. There was no cytokeratin expression in either lesion. WT-1 expression in the paraduodenal mass was cytoplasmic and appeared non-specific. Lesions were consistent with renal PKD-like disease and a pancreatic cystic hamartoma.

Autoimmune-Mediated Thymic Atrophy Is Accelerated but Reversible in RelB-Deficient Mice.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB−/− mice, which have spontaneous multiorgan autoimmune disease. RelB−/− thymi were organized, with medullary structures containing AIRE− mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB−/− thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Respiratory difficulty with palatal, laryngeal and respiratory muscle tremor in adult-onset Alexander’s disease

Sleep apnoea and respiratory difficulties are reported in adult-onset Alexander’s disease (AOAD), an autosomal-dominant leukodystrophy that presents mainly with progressive ataxia. We demonstrate for the first time that the respiratory...

Dejerine: The concept of peripheral neuropathy

Dejerine: The concept of peripheral neuropathy

ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice.

ABSTRACTCollagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.

Longitudinal assessment of mouse renal injury using high-resolution anatomic and magnetization transfer MR imaging

The purpose of this study is to evaluate the utility of high-resolution non-invasive endogenous high-field MRI methods for the longitudinal structural and quantitative assessments of mouse kidney disease using the model of unilateral ureter obstruction (UUO). T1-weighted, T2-weighted and magnetization transfer (MT) imaging protocols were optimized to improve the regional contrast in mouse kidney. Conventional T1 and T2 weighted images were collected in UUO mice on day 0 (~3h), day 1, day 3 and day 6 after injury, on a 7T small animal MRI system. Cortical and medullary thickness, corticomedullary contrast and Magnetization Transfer Ratio (MTR) were assessed longitudinally. Masson trichrome staining was used to histologically assess changes in tissue microstructure. Over the course of UUO progression there were significant (p<0.05) changes in thickness of cortex and outer medulla, and regional changes in T2 signal intensity and MTR values. Histological changes included tubular cell death, tubular dilation, urine retention, and interstitial fibrosis, assessed by histology. The MRI measures of renal cortical and medullary atrophy, cortical–medullary differentiation and MTR changes provide an endogenous, non-invasive and quantitative evaluation of renal morphology and tissue composition during UUO progression.

UT-B-deficient mice develop renal dysfunction and structural damage.

BackgroundUrea transporter UT-B is the major urea transporter in erythrocytes and the descending vasa recta in the kidney. In this study, we investigated the effects of long-term UT-B deficiency on functional and structural defect in the kidney of 16-and 52-week-old UT-B-null mice.MethodsUT-B-knockout mice were generated by targeted gene disruption and lacked UT-B protein expression in all organs. The urinary concentrating ability of mice was studied in terms of daily urine output, urine osmolality, and urine and plasma chemistries. Changes in renal morphology were evaluated by hematoxylin and eosin staining.ResultsThe UT-B-null mice showed defective urine concentrating ability. The daily urine output in UT-B-null mice (2.5 ± 0.1 ml) was 60% higher and urine osmolality (985 ± 151 mosm) was significantly lower than that in wild-type mice (1463 ± 227 mosm). The 52-week-old UT-B-null mice exhibited polyuria after water deprivation, although urine osmolality was increased. At 52 weeks of age, over 31% of UT-B-null mice exhibited renal medullary atrophy because of severe polyuria and hydronephrosis.ConclusionsLong-term UT-B deficiency causes severe renal dysfunction and structural damage. These results demonstrate the important role of UT-B in countercurrent exchange and urine concentration.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Autoreactive T Cells Escaping Thymic Deletion in REL-B Deficient Mice Depend on Dendritic Cell-encoded REL-B for Control of Autoimmunity

In humans and mice, autoimmunity is associated with mutations that attenuate T cell receptor (TCR) signaling capacity, which alter thymic selection, and signaling of the peripheral T cell repertoire. The RelB transcription factor controls maturation of antigen presenting cell function by dendritic cells (DCs). Similar to TCR signaling mutants, RelB-/- mice have severe T cell-dependent autoimmune disease and relative lymphopenia, but normal numbers of FoxP3+ regulatory T cells. By analysis of effector and regulatory T cell phenotype and function, distribution and function of antigen presenting cells (APCs) and DC transfer studies in RelB knockout and RelB-/- bone marrow chimeras, we addressed whether reduced capacity of thymic and peripheral APCs to signal T cells drives autoimmunity in RelB-/-mice. Th2-type RelB-/- CD44hi effector T cells, which escape thymic negative selection due to medullary atrophy and APC deficiency, are susceptible to RelB-/- Treg suppression only when signalled by wild type DCs, with enhanced costimulatory activity. Transfer of RelB-sufficient DCs, to RelB-/- mice reversed peripheral organ inflammatory disease, Th2 cytokine over-production, thymic atrophy and lymphopenia, and rendered effector T cells susceptible to suppression by Treg cells. Thus, similar to defects in TCR signaling, autoimmunity may also result from defects in APC function. In the absence of DC-encoded RelB, progressive T cell and myeloid cell-mediated inflammation, uncontrolled by Treg cells, destroys thymus and other organs. Genetic and environmental factors control the level of RelB expression, and reduced expression is predicted to exacerbate the effect of mutations, such as PTPN22R620W that attenuate TCR signaling capacity. Copyright © 2010 Published by Elsevier Inc.

Resonancia magnética en flexoextensión en el diagnóstico de la mielopatía cervical en la enfermedad de Hirayama

We report the case of a young man who presented with atrophy of the musculature of the right forearm and hand. The clinician suspected cervical myelopathy in Hirayama's disease on the basis of the findings at electromyography and recommended an MRI examination in both flexed and neutral position. The typical imaging findings for cervical myelopathy in Hirayama's disease were observed and the diagnosis was confirmed. The key findings for Hirayama's disease consist of asymmetrical medullary atrophy with involvement of the anterior horns, dorsal detachment of the dura mater, and dilatation of the epidural venous plexus.

Immunosuppressive activity of FK-506 in rats: flow cytometric analysis of lymphocyte populations in blood, spleen and thymus during treatment and following drug withdrawal

Rats were immunized systemically with sheep red blood cells (SRBC) and given either FK-506 (1 mg/kg) or drug vehicle by i.m. injection for 7 days. In animals receiving FK-506, there was suppression (87%) of the splenic plaque-forming cell response on day 4 and marked reductions in the serum antibody titre throughout the 3-week period following immunization. Sequential flow cytometric analyses of blood lymphocytes revealed statistically significant attenuation, by FK-506, of the increase in relative numbers of OX-12+ (B) cells between days 4 and 7. Following drug withdrawal, OX-12 values remained elevated, whereas in control animals a decline was observed. These changes were reflected in concomitant increases in the relative numbers of OX-19+ (CD3+), W3/25+ (CD4+) and OX-8+ (CD8+) T cells; however, due to an overall reduction in lymphocytes by day 7, absolute values were not significantly affected compared with controls. The pattern of changes in OX-6+ (MHC class II+) cells in blood was similar to that observed for B cells. FK-506 also suppressed increases in the small proportion of blood-borne OX-40+ (activated CD4+) cells and OX-39+ (interleukin-2 receptor+) cells in the 7 day period following immunization; thereafter values for activation marker expression between treatment and control groups were similar. In the spleen, there were fewer significant differences between FK-506 and control groups in the incidences of cells expressing the above markers. OX-8+ cells, however, were significantly higher in drug-treated animals on day 7, and there were also reductions in the small proportions of OX-39+ and OX-40+ cells when compared with controls. In the thymus, reversible medullary atrophy induced by FK-506 was accompanied on day 7 by increases in the incidence of CD4+ and CD8+ cells and by a concomitant reduction in OX-44+ mature, medullary thymocytes. Two weeks after drug withdrawal, the phenotypic marker expression profile had been restored to normal in blood, spleen and thymus. These data provide new information on the apparent capacity of FK-506 to interfere with T cell maturation and its influence on lymphocyte activation in vivo.