Luise Anne Cullen-McEwen

Key Points: Maternal low protein diet reduced kidney size and altered branching without changing ureteric tip number, with fewer nephrons at birth and adulthood. Nephron progenitor proliferation and commitment were impaired, pretubular aggregate formation decreased, consistent with a reduced nephron number. Background: Low nephron number has a direct effect on the development of hypertension and CKD later in life. Although intrauterine growth restriction caused by maternal low-protein diet (LPD) is believed to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes that drive nephron formation are poorly understood. Methods: We conducted a comprehensive characterization of the effect of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphologic features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during development of the kidneys to affect nephron number. Results: Single-cell analysis revealed differential expression across metabolic, cell cycle, epigenetic, and reciprocal inductive signaling pathways in most cell types, shifting cellular energy production and developmental trajectories. In nephron progenitor cells, LPD impeded commitment and differentiation toward pretubular aggregates and renal vesicles, accompanied by downregulated Wnt signaling. Confocal microscopy showed fewer pretubular aggregates and reduced progenitor proliferation, consistent with impaired commitment. Critically, nephron progenitor cell proliferation remained reduced through P0, whereas ureteric tip proliferation, although reduced earlier, had normalized by P0. Branching morphology also changed, with optical projection tomography showing shorter tip and tip-parent lengths, consistent with subtle patterning defects. Conclusions: This study demonstrates that gestational LPD reduced nephron endowment by impairing nephron progenitor cell commitment, with concurrent alterations in branching morphogenesis.

Low birthweight is a risk factor for hypertension and chronic kidney disease. Kidneys of low birthweight babies typically have a low nephron endowment, which is permanent. Therefore, strategies to boost or rescue nephron endowment in low birthweight offspring might be expected to decrease the prevalence of these chronic conditions. We previously reported that a high-fat diet (17% protein, 43% carbohydrate, 40% fat) fed to mice before mating and until weaning boosted nephron endowment in mice by 20%. Here, we show that offspring from dams fed a normal diet during pregnancy and switched to a high-fat diet at birth had a 14% augmented nephron endowment. Additionally, transition to a high-fat diet at birth completely rescued a 20% nephron deficit induced by feeding dams a low-protein diet (8% protein, 76% carbohydrate, 16% fat) during gestation. The augmentation and rescue of nephron endowment were associated with increased maternal caloric intake on day 1, as well as increased maternal fat and reduced carbohydrate intake during the postnatal period of rapid nephrogenesis. These findings indicate that the balance between the three macronutrients in the maternal diet, both pre- and postnatally, is crucial for nephron endowment.

Purpose: The total number of glomeruli is an important indicator of kidney function and susceptibility to disease. Glomeruli, however, are difficult to image using MRI without contrast enhancement. For this reason, previous studies have used cationic ferritin as a contrast agent to detect glomeruli in ex vivo and in vivo perfused kidneys. This study aimed to develop a new technique using MR microimaging at ultrahigh field, without contrast agents, to visualize and quantify glomeruli in human nephrectomy samples.Methods: MRI was performed at 16.4 T using three‐dimensional T₁/‐weighted gradient‐echo imaging at 30 to 200 μm isotropic resolution on cortical samples with volumes ranging from 155 to 1384 mm³. A segmentation pipeline based on shape recognition and clustering was developed to count glomeruli on MR images at 30‐μm isotropic resolution. Glomerular number and density were estimated after MRI using unbiased stereology, and glomerular distribution was visualized using a subset of samples imaged using confocal microscopy.Results: Glomeruli were identified in the MR images as hypointense spheres. High spatial concordance between glomeruli identified by MRI, histology, and confocal microscopy was observed. Statistical analyses showed that glomerular counts from stereology and MR images were similar, with Pearson correlation and intraclass correlation coefficients of 0.98.Conclusion: High‐resolution microimaging at 16.4 T combined with automated segmentation allowed identification and quantitation of human glomeruli ex vivo without the need for contrast agents. The results showed strong agreement with the gold‐standard stereological method for glomerular counting.

Antigen presentation on major histocompatibility complex (MHC) molecules is central to the initiation of immune responses, and a lot of our understanding about the antigen processing and presentation pathway has been gained through studies in mice. MHC molecules are the most genetically diverse genes; consequently, mouse strains differ substantially in their MHC make up and resulting antigen presentation. Swiss mice are commonly used in pharmacological research, yet our understanding of antigen presentation in this strain is surprisingly limited. Here, we have tested a range of anti-MHC antibodies and present a range of clones suitable to analyse MHC class I and class II molecules in Swiss mice who have the H2-q MHC haplotype. Moreover, we demonstrate using immunopeptidomics that clones 28-12-8, 34-1-2, MKD6, and N22 are also suited to isolate MHC class I and class II ligands in this mouse strain. Thus, this work also establishes a first experimental account of the H2-q-derived thymus and spleen immunopeptidome in Swiss mice which bears strong resemblance with ligands isolated from the H2-d MHC haplotype of Balb/C mice. The analysis of source proteins shows common but also organ- and function-specific antigen presentation in line with the involvement of the thymus in tolerance induction and the function of the spleen as a site of immune responses.

Antigen presentation on major histocompatibility complex (MHC) molecules is central to the initiation of immune responses and a lot of our understanding about the antigen processing and presentation pathway has been gained through studies in mice. MHC molecules are the most genetically diverse genes, consequently mouse strains differ substantially in their MHC make up and resulting antigen presentation. Swiss mice are commonly used in pharmacological research, yet our understanding of antigen presentation in this strain is surprisingly limited. Here, we have tested a range of anti-MHC antibodies and present a range of clones suitable to analyse MHC class I and II molecules in Swiss mice who have the H2-q MHC haplotype. Moreover, we demonstrate using immunopeptidomics that clones 28-12-8, 34-1-2, MKD6 and N22 are also suited to isolate MHC class I and class II ligands in this mouse strain. Thus, this work also establishes a first experimental account of the H2-q derived thymus and spleen immunopeptidome in Swiss mice which bears strong resemblance with ligands isolated from the H2-d MHC haplotype of Balb/C mice.

Fibrosis and loss of functional capillary surface area may contribute to renal tissue hypoxia in a range of kidney diseases. However, there is limited quantitative information on the impact of kidney disease on the barriers to oxygen diffusion from cortical peritubular capillaries (PTCs) to kidney epithelial tubules. Here, we used stereological methods to quantify changes in total cortical PTC length and surface area, PTC length and surface densities, and diffusion distances between PTCs and kidney tubules in adenine‐induced kidney injury. After 7 days of oral gavage of adenine (100 mg), plasma creatinine was 3.5‐fold greater than in vehicle‐treated rats, while total kidney weight was 83% greater. The total length of PTCs was similar in adenine‐treated (1.47 ± 0.23 km (mean ± standard deviation)) to vehicle‐treated (1.24 ± 0.24 km) rats, as was the surface density of PTCs (0.025 ± 0.002 vs. 0.024 ± 0.004 μm²/μm³). The total surface area of PTCs was 69% greater in adenine‐treated than vehicle‐treated rats. However, the length density of PTCs was 28% less in adenine‐treated than vehicle‐treated rats. Diffusion distances, from PTCs to the basal membrane of the nearest renal tubule (108%), and to the mid‐point of the cytoplasmic height of the nearest tubular epithelial cell (57%), were markedly increased. These findings indicate that, in adenine‐induced kidney injury, expansion of the renal cortical interstitium increases the distance required for diffusion of oxygen from PTCs to tubules, rendering the kidney cortex susceptible to hypoxia.

Corticosteroid therapy, often in combination with inhibition of the renin-angiotensin system, is first-line therapy for primary focal and segmental glomerulosclerosis (FSGS) with nephrotic-range proteinuria. However, the response to treatment is variable, and therefore new approaches to indicate the response to therapy are required. Podocyte depletion is a hallmark of early FSGS, and here we investigated whether podocyte number, density and/or size in diagnostic biopsies and/or the degree of glomerulosclerosis could indicate the clinical response to first-line therapy. In this retrospective single center cohort study, 19 participants (13 responders, 6 non-responders) were included. Biopsies obtained at diagnosis were prepared for analysis of podocyte number, density and size using design-based stereology. Renal function and proteinuria were assessed 6 months after therapy commenced. Responders and non-responders had similar levels of proteinuria at the time of biopsy and similar kidney function. Patients who did not respond to treatment at 6 months had a significantly higher percentage of glomeruli with global sclerosis than responders (p < 0.05) and glomerulosclerotic index (p < 0.05). Podocyte number per glomerulus in responders was 279 (203–507; median, IQR), 50% greater than that of non-responders (186, 118–310; p < 0.05). These findings suggest that primary FSGS patients with higher podocyte number per glomerulus and less advanced glomerulosclerosis are more likely to respond to first-line therapy at 6 months. A podocyte number less than approximately 216 per glomerulus, a GSI greater than 1 and percentage global sclerosis greater than approximately 20% are associated with a lack of response to therapy. Larger, prospective studies are warranted to confirm whether these parameters may help inform therapeutic decision making at the time of diagnosis of primary FSGS.

Low nephron number correlates with the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development. Differences in the expression of genes involved in metabolism were identified in most cell types we analyzed, yielding imbalances and shifts in cellular energy production. We further demonstrate that LPD impedes branching morphogenesis and significantly reduces the number of pretubular aggregates – the initial precursors to nephron formation. The most striking observation was that LPD changes the developmental trajectory of nephron progenitor cells, driving the formation of a partially committed cell population which likely reflects a failure of cells to commit to nephron formation and which ultimately reduces endowment. This unique profile of a fetal programming defect demonstrates that low nephron endowment arises from the pleiotropic impact of changes in branching morphogenesis and nephron progenitor cell commitment, the latter of which highlights a critical role for nutrition in regulating the cell fate decisions underpinning nephron endowment. Significance Statement While a mother’s diet and behavior can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

Podocytes are terminally differentiated epithelial cells in glomeruli. Podocyte injury and loss are features of many diseases leading to chronic kidney disease (CKD). The developmental origins of health and disease hypothesis propose an adverse intrauterine environment can lead to CKD later in life, especially when a second postnatal challenge is experienced. The aim of this study was to examine whether a suboptimal maternal environment would result in reduced podocyte endowment, increasing susceptibility to diabetes‐induced renal injury. Female C57BL/6 mice were fed a low protein diet (LPD) to induce growth restriction or a normal protein diet (NPD) from 3 weeks before mating until weaning (postnatal Day 21, P21) when nephron and podocyte endowment were assessed in one male and one female offspring per litter. Littermates were administered streptozotocin or vehicle at 6 weeks of age. Urinary albumin excretion, glomerular size, and podometrics were assessed following 18 weeks of hyperglycemia. LPD offspring were growth restricted and had lower nephron and podocyte number at P21. However, by 24 weeks the podocyte deficit was no longer evident and despite low nephron endowment neither albuminuria nor glomerulosclerosis were observed. Podocyte number was unaffected by 18 weeks of hyperglycemia in NPD and LPD offspring. Diabetes increased glomerular volume reducing podocyte density, with more pronounced effects in LPD offspring. LPD and NPD diabetic offspring developed mild albuminuria with LPD demonstrating an earlier onset. LPD offspring also developed glomerular pathology. These findings indicate that growth‐restricted LPD offspring with low nephron number and normalized podocyte endowment were more susceptible to alterations in glomerular volume and podocyte density leading to more rapid onset of albuminuria and renal injury than NPD offspring.

Progressive podocyte loss is a feature of healthy ageing. While previous studies have reported age-related changes in podocyte number, density and size and associations with proteinuria and glomerulosclerosis, few studies have examined how the response of remaining podocytes to podocyte depletion changes with age. Mild podocyte depletion was induced in Pod^CreiDTR mice aged 1, 6, 12 and 18 months via intraperitoneal administration of diphtheria toxin. Control mice received intraperitoneal vehicle. Podometrics, proteinuria and glomerular pathology were assessed, together with podocyte expression of p-rp-S6, a phosphorylation target that represents activity of the mammalian target of rapamycin (mTOR). Podocyte number per glomerulus did not change in control mice in the 18-month time period examined. However, control mice at 18 months had the largest podocytes and the lowest podocyte density. Podocyte depletion at 1, 6 and 12 months resulted in mild albuminuria but no glomerulosclerosis, whereas similar levels of podocyte depletion at 18 months resulted in both albuminuria and glomerulosclerosis. Following podocyte depletion at 6 and 12 months, the number of p-rp-S6 positive podocytes increased significantly, and this was associated with an adaptive increase in podocyte volume. However, at 18 months of age, remaining podocytes were unable to further elevate mTOR expression or undergo hypertrophic adaptation in response to mild podocyte depletion, resulting in marked glomerular pathology. These findings demonstrate the importance of mTORC1-mediated podocyte hypertrophy in both physiological (ageing) and adaptive settings, highlighting a functional limit to podocyte hypertrophy reached under physiological conditions.