Biology subjects

Ramkumar, N.

Publications and source records attributed to Ramkumar, N..

10 recordsLinked to original sources

bioRxiv2026

Mitochondrial Bioenergetic Uncoupling and Strain-Dependent Adaptations in a High-Fat, High-Salt Model of Metabolic Dysfunction-Associated Kidney Disease

Metabolic dysfunction-associated kidney disease (MDAKD) is closely linked to dietary excess, but models that capture early kidney injury without obesity are limited. We fed male C57BL/6J (6J) and C57BL/6N (6N) mice a high-fat, high-sodium (HF/HNa) or control diet for 16 weeks. HF/HNa feeding did not alter body weight, adiposity, or total food intake; however, it increased dietary energy and sodium exposure, kidney mass, water intake, and urine volume. GFR declined modestly in 6J mice, whereas 6N mice maintained or slightly increased GFR. Both substrains showed increased urinary albumin, creatinine, KIM-1, and NGAL, while cystatin C rose predominantly in 6N mice, indicating strain-dependent tubular injury. Whole-kidney trichrome staining revealed increased fibrotic area with HF/HNa, particularly in 6N mice, without significant changes in glomerular morphology. In isolated renal mitochondria, oxygen consumption was preserved, but ATP production and ATP:O ratios were reduced, with unchanged citrate synthase activity and OXPHOS protein abundance, consistent with early mitochondrial bioenergetic uncoupling. Exploratory urinary proteomics in 6J mice identified HF/HNa-associated changes in proteins linked to tubular stress and extracellular matrix remodeling. These findings define an early MDAKD-like renal phenotype with strain-specific functional responses, tubular injury, fibrosis, and impaired mitochondrial ATP efficiency. Translational StatementMetabolic Dysfunction-Associated Kidney Disease (MDAKD) is a leading driver of chronic kidney disease (CKD) in the world. In addition to obesity and related comorbidities, renal mitochondrial dysfunction is thought to be a key contributor to the development of CKD in patients with MDAKD; however, few models recapitulate the progression of MDAKD. We couple well-established mouse models of obesity, namely the C57Bl/6J and C57Bl/6N mouse lines, with a high-fat, high-salt diet to induce renal mitochondrial dysfunction, leading to early stages of MDAKD as indicated by widespread fibrosis and mild reduction in glomerular filtration rate, though these effects were strain-dependent. We identify diet-induced mitochondrial dysfunction as a common feature in both mouse strains, suggesting impairments in mitochondrial respiration and oxidative ATP production are indeed a contributing factor to the development of MDAKD. This study highlights the role of energetic impairments in the pathogenesis of MDAKD and may guide future therapies for CKD.

physiology↗

bioRxiv2025

Loss of cell cycle gatekeeping by 1 CNOT3 impairs hematopoietic stem and progenitor cell division and repopulating activity

Adult mammalian hematopoietic stem cells (HSCs) constitute a heterogeneous population responsible for generating various cell types in the blood throughout adulthood. Gene expression programs underlying regulation of self-renewal and differentiation of HSCs are tightly regulated. However, how post-transcriptional regulation of gene expression influences HSCs and hematopoiesis remains largely unexplored. Here, we report the critical role of CNOT3, a subunit of the CCR4-NOT complex, in regulating hematopoietic stem cells (HSCs) function in adult hematopoiesis. We observed that Cnot3 mRNA is highly expressed in HSCs and CNOT3 ablation in the murine Cnot3 conditional knockout mouse model resulted in anemia, reduced bone marrow cellularity and enhanced extramedullary hematopoiesis in spleen. Deletion of Cnot3 resulted in the early expansion of immunophenotypic HSCs which were then progressively lost over time. Cnot3 knockout hematopoietic stem/progenitor cells (HSPCs) failed to reconstitute hematopoietic systems of recipient animals in transplantation assays. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs revealed disruptions in lineage development and loss of HSCs. Transcriptomic profiling and cell cycle analysis demonstrated that Cnot3 deletion led to increased cycling activity in HSCs. Our results indicate that CNOT3 is critical for maintenance of homeostasis in HSCs and the hematopoietic system.

developmental biology↗

bioRxiv2025

N-acetylcarnosine attenuates age-associated declines in multi-organ systems to improve survival

Histidine containing dipeptides (HCDs) such as N-acetylcarnosine are endogenous metabolites that are ergogenic and mitigate metabolic dysfunction. We previously demonstrated that short-term N-acetylcarnosine treatment is highly efficacious in protecting muscle atrophy induced by disuse. Here we demonstrate that a 6-months treatment of N-acetylcarnosine attenuates a broad spectrum of age-associated maladies and improved survival by [~]50% in female mice. A comprehensive survey of organ systems revealed that N-acetylcarnosine prevents decline in adiposity, diastolic function, vasodilation, muscle strength, and bone density. Together, N-acetylcarnosine substantially delays the onset of system-wide end-stage pathology to prolong lifespan. As an endogenously present metabolite, treatment with N-acetylcarnosine may be a safe and promising intervention to promote healthy aging in humans.

physiology↗

bioRxiv2025

A Platform for Mitochondrial Profiling in Enriched Kidney Segments Under Thermodynamic Control

Mitochondrial function varies widely across kidney nephron segments, yet conventional approaches lack the resolution and control needed to assess cell-type-specific bioenergetics in situ. We present a methodological platform that enables segment-resolved profiling of mitochondrial respiration, conductance, and membrane potential in freshly isolated mouse and human nephron segments. Combining mechanical sieving and adhesion-based enrichment with permeabilized high-resolution respirometry, we adapted the creatine kinase clamp to quantify oxygen flux and mitochondrial membrane potential across defined free energies. Using this approach, we found that proximal tubules exhibit high respiratory conductance and dynamic mitochondrial polarization, while distal tubules and glomeruli maintain static membrane potential and low conductance. In a model of adenine-induced nephropathy, only proximal tubule mitochondria showed marked reductions in respiration and ATP production. This segment-specific dysfunction was not detectable in bulk mitochondrial isolates. We then demonstrate that these methods can be applied in human kidney samples. Our approach provides thermodynamically anchored, segment-resolved insight into mitochondrial adaptation under physiological and pathological conditions. It is broadly applicable to other tissues with metabolic heterogeneity and compatible with disease models, genetic tools, and pharmacological interventions. This platform bridges a critical gap between conventional respirometry and functional mitochondrial phenotyping in native tissue structures.

biochemistry↗

bioRxiv2024

Six months of physical inactivity is insufficient to cause chronic kidney disease in C57BL/6J mice

Chronic kidney disease (CKD) is a progressive disorder marked by a decline in kidney function. Obesity and sedentary behavior contribute to the development of CKD, though mechanisms by which this occurs are poorly understood. This knowledge gap is worsened by the lack of a reliable murine CKD model that does not rely on injury, toxin, or gene deletion to induce a reduction in kidney function. High-fat diet (HFD) feeding alone is insufficient to cause reduced kidney function until later in life. Here, we employed a small mouse cage (SMC), a recently developed mouse model of sedentariness, to study its effect on kidney function. Wildtype C57BL/6J male mice were housed in sham or SMC housing for six months with HFD in room (22{degrees}C) or thermoneutral (30{degrees}C) conditions. Despite hyperinsulinemia induced by the SMC+HFD intervention, kidneys from these mice displayed normal glomerular filtration rate (GFR). However, the kidneys showed early signs of kidney injury, including increases in Col1a1 and NGAL transcripts, as well as fibrosis by histology, primarily in the inner medullary/papilla region. High-resolution respirometry and fluorometry experiments showed no statistically significant changes in the capacities for respiration, ATP synthesis, or electron leak. These data confirm the technical challenge in modeling human CKD. They further support the notion that obesity and a sedentary lifestyle make the kidneys more vulnerable, but additional insults are likely required for the pathogenesis of CKD.

physiology↗

bioRxiv2024

Phased ERK-responsiveness and developmental robustness regulate teleost skin morphogenesis

Elongation of the vertebrate embryonic axis necessitates rapid expansion of the epidermis to accommodate the growth of underlying tissues. Here, we generated a toolkit to visualize and quantify signaling in entire cell populations of periderm, the outermost layer of the epidermis, in live developing zebrafish. We find that oriented cell divisions facilitate growth of the early periderm during axial elongation rather than cell addition from the basal layer. Activity levels of ERK, a downstream effector of MAPK pathway, gauged by a live biosensor, predicts cell cycle entry, and optogenetic ERK activation controls proliferation dynamics. As development proceeds, rates of peridermal cell proliferation decrease, ERK activity becomes more pulsatile and functionally transitions to promote hypertrophic cell growth. Targeted genetic blockade of cell division generates animals with oversized periderm cells, yet, unexpectedly, development to adulthood is not impaired. Our findings reveal stage-dependent differential responsiveness to ERK signaling and marked developmental robustness in growing teleost skin.

developmental biology↗

bioRxiv2021

DeepProjection: Rapid and structure-specific projections of tissue sheets embedded in 3D microscopy stacks using deep learning

The efficient extraction of local high-resolution content from massive amounts of imaging data remains a serious and unsolved problem in studies of complex biological tissues. Here we present DeepProjection, a trainable projection algorithm based on deep learning. This algorithm rapidly and robustly extracts image content contained in curved manifolds from time-lapse recorded 3D image stacks by binary masking of background content, stack by stack. The masks calculated for a given movie, when predicted, e.g., on fluorescent cell boundaries on one channel, can subsequently be applied to project other fluorescent channels from the same manifold. We apply DeepProjection to follow the dynamic movements of 2D-tissue sheets in embryonic development. We show that we can selectively project the amnioserosa cell sheet during dorsal closure in Drosophila melanogaster embryos and the periderm layer in the elongating zebrafish embryo while masking highly fluorescent out-of-plane artifacts.

developmental biology↗

bioRxiv2021

Invivo and systematic analysis of random multigenic deletions associated with human diseases during epithelial morphogenesis using Drosophila

Random loss of multigenic loci on chromosomes, a crucial drive for evolution, occurs frequently in all living organisms. Analysis of such chromosomal disruption and understanding the consequences of their impact on the growth and development of multicellular organisms is challenging. In this report, we have addressed this issue using invivo mosaic analysis of deficiency lines in Drosophila. Genes on fly deficiency lines were compared with human orthologs for their implications in disease development during cytoskeletal processes and epithelial morphogenesis. The cytoskeletal phenotypes from the fly has been utilized to predict the function of human orthologs. In addition, as these Drosophila deficiency lines are equivalent to human microdeletions, based on the clonal behaviour and phenotypes generated, a systematic analysis has been carried out to establish the critical loci that correspond to Microdeletion Syndromes and Mendelian Disorders in humans. Further we have drawn the synteny that exists between these chromosomes and have identified critical region corresponding to defects. A few potential candidates that might have an implication in epithelial morphogenesis are also identified.

genetics↗

bioRxiv2020

Targeted Next-Generation Sequencing Identifies Pathogenic Variants in Kidney Disease-Related Genes in Patients with Diabetic Kidney Disease

Diabetes is the most common cause of chronic kidney disease (CKD). For patients with diabetes and CKD, the underlying cause of their kidney disease is often assumed to be a consequence of their diabetes. Without histopathological confirmation, however, the underlying cause of their kidney disease is unclear. Recent studies have shown that next-generation sequencing (NGS) provides a promising avenue toward uncovering and establishing precise genetic diagnoses in various forms of kidney disease. Here, we set out to investigate the genetic basis of disease in non-diabetic kidney disease (NDKD) and diabetic kidney disease (DKD) patients by performing targeted NGS using a custom panel comprised of 345 kidney disease-related genes. Our analysis identified rare diagnostic variants that were consistent with the clinical diagnosis of 19% of the NDKD patients included in this study. Similarly, 22% of DKD patients were found to carry rare pathogenic/likely pathogenic variants in kidney disease-related genes included on our panel. Genetic variants suggestive of NDKD were detected in 3% of the diabetic patients included in this study. Our findings suggest that rare variants in kidney disease-related genes in the context of diabetic pathophysiology may play a role in the pathogenesis of kidney disease in patients with diabetes. Key MessagesO_LIWhat is already known about this subject? O_LIFor patients with diabetes and chronic kidney disease, the underlying cause of their kidney disease is often assumed to be a consequence of their diabetes; without histopathological confirmation, however, the underlying cause of their kidney disease is unclear. C_LIO_LINext-generation sequencing (NGS) provides a promising avenue toward uncovering and establishing precise genetic diagnoses in various forms of kidney disease. C_LI C_LIO_LIWhat are the new findings? O_LIUsing targeted NGS and a custom panel comprised of 345 kidney disease-related genes, we found that 22% of diabetic kidney disease patients were found to carry rare pathogenic/likely pathogenic variants in kidney disease-related genes included on our panel. C_LIO_LIGenetic variants suggestive of non-diabetic kidney disease were detected in 3% of the diabetic patients included in this study. C_LI C_LIO_LIHow might these results change the focus of research or clinical practice? O_LIOur findings suggest that rare variants in kidney disease-related genes in the context of diabetic pathophysiology may play a role in the pathogenesis of kidney disease in patients with diabetes. C_LIO_LIImportantly, improved understanding of the underlying disease process in diabetic kidney disease could have major implications in terms of patient care and monitoring as well as for research studies in this field. C_LI C_LI

genetics↗

bioRxiv2018

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle

The ability of epithelial cells to divide along their long cell axis, known as \"Hertwigs rule\", has been proposed to play an important and wide-ranging role in homogenising epithelial cell packing during tissue development and homeostasis. Since the position of the anaphase spindle defines the division plane, how divisions are oriented requires an understanding of the mechanisms that position the mitotic spindle. While many of the molecules required to orient the mitotic spindle have been identified in genetic screens, the mechanisms by which spindles read and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly orient their divisions depends both on cortical cues and on local tissue tension. Thus, spindles align with the long cell axis in tissues in which isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low. Importantly, these region-specific differences in spindle behaviour can be reversed by decreasing or increasing the activity of non-muscle Myosin II. In addition, spindles in a tissue experiencing isotropic stress fail to align with the long cell axis if cells are mechanically isolated from their neighbours. These data lead us to propose that isotropic tension is required within an epithelium to provide cells with a mechanically stable substrate upon which localised cortical Dynein can pull on astral microtubules to orient the spindle.

cell biology↗