Obesity Facts and Their Influence on Renal Function Across the Life Span

Obesity is a chronic disease, with a rapidly increasing prevalence worldwide. Body mass index (BMI) provides the most useful population-level measure of overweight and obesity. For adults, overweight is defined as a BMI (Kg/m2) ≥ 25, and obesity as a BMI ≥ 30, for non-Asians and ≥ 27.5 for Asians. Abdominal obesity can be defined as a waist circumference equal to or higher than 102 cm for men and ≥88 cm for women. The definition of children and adolescents BMI changes with age and sex. Obesity may be exogenous or endogenous obesity, the latter is multifactorial and predominantly manifested during childhood. Presently, overweight and obesity are linked to more deaths worldwide than underweight. The total kidney glomerular filtration rate (GFR) is determined by the sum of nephrons and the GFR within each nephron or single nephron GFR. In clinical practice, GFR is more frequently calculated by GFR estimating equations based upon the plasma levels of creatinine, cystatin C, or both. The measured value of plasma creatinine is strongly influenced by non-GFR factors, by its tubular and gastrointestinal secretion, and by the problems associated with the lack of standardization of creatinine's laboratory assay discrediting it as an ideal GFR biomarker. Unlike creatinine, cystatin C plasma levels are mainly determined by GFR. Obesity may affect the kidney, via development of systemic arterial hypertension and/or diabetes mellitus, or directly, by ectopic accumulation of adipose tissue in the kidney. As obesity is a clinical condition associated with altered body composition, creatinine may not be the ideal biomarker for GFR measurement in obese individuals.

Estimating Nephron Number from Biopsies: Impact on Clinical Studies

Background: Accumulating evidence supports an association between nephron number and susceptibility to kidney disease. However, it is not currently possible to directly measure nephron number in a clinical setting. Recent clinical studies have used glomerular density from a single biopsy and whole kidney cortical volume from imaging to estimate both nephron number and single nephron glomerular filtration rate. However, the accuracy of these estimates from individual subjects is unknown. Furthermore, it is not clear how sample size or biopsy location may influence these estimates. These questions are critical to study design and to the potential translation of these tools to estimate nephron number in individual subjects. Methods: We measured the variability in estimated nephron number derived from needle or virtual biopsies and cortical volume in human kidneys declined for transplantation. We performed multiple needle biopsies in the same kidney, and examined the three-dimensional spatial distribution of nephron density by magnetic resonance imaging. We determined the accuracy of a single kidney biopsy to predict the mean nephron number estimated from multiple biopsies from the same kidney. Results: A single needle biopsy had a 15% chance and virtual biopsy had a 60% chance of being within 20% of whole kidney nephron number. Single needle biopsies could be used to detect differences in nephron number between large cohorts of several hundred subjects. Conclusions: The number of subjects required to accurately detect differences in nephron number between populations can be predicted based on natural intra-kidney variability in glomerular density. A single biopsy is insufficient to accurately predict nephron number in individual subjects.

A new analysis approach for single nephron GFR in intravital microscopy of mice

Background: Intravital microscopy is an emerging technique in life science with applications in kidney research. Longitudinal observation of (patho-)physiological processes in living mice is possible in the smallest functional unit of the kidney, a single nephron (sn). In particular, effects on glomerular filtration rate (GFR) - a key parameter of renal function - can be assessed. Methods: After intravenous injection of a freely filtered, non-resorbable, fluorescent dye in C57BL/6 mice, a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Automatic modelling of actual PT volume in a 3D dataset replaced 2D volume estimation. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Repeated analysis of image data obtained in healthy mice showed a striking increase of reproducibility by reduction of user interaction. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function.

A Computational Model of Kidney Function in a Patient with Diabetes

At the onset of diabetes, the kidney grows large and the glomerular filtration rate becomes abnormally high. These structural and hemodynamics changes affect kidney function and may contribute to the development of chronic kidney disease. The goal of this study is to analyze how kidney function is altered in patients with diabetes and the renal effects of an anti-hyperglyceamic therapy that inhibits the sodium-glucose cotransporter 2 (SGLT2) in the proximal convoluted tubules. To accomplish that goal, we have developed a computational model of kidney function in a patient with diabetes and conducted simulations to study the effects of diabetes and SGLT2 inhibition on solute and water transport along the nephrons. Simulation results indicate that diabetes-induced hyperfiltration and tubular hypertrophy enhances Na+ transport, especially along the proximal tubules and thick ascending limbs. These simulations suggest that SGLT2 inhibition may attenuate glomerular hyperfiltration by limiting Na+-glucose transport, raising luminal [Cl−] at the macula densa, restoring the tubuloglomerular feedback signal, thereby reducing single-nephron glomerular filtration rate.

MO429A NOVEL METHOD LINESCAN-DRIVEN FOR SNGFR MEASUREMENTS AS ALTERNATIVE TO HIGH FULL FRAME MULTIPHOTON MICROSCOPY ACQUISITION

Background and Aims Renal micropuncture, which requires the direct access to the renal tubules, has been for long time the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models, but this approach is challenging by virtue of complex animal preparation and numerous careful steps. The introduction of intravital multiphoton microscopy (MPM) permitted to improve the study of renal functions exploiting the high laser penetration and the optical sectioning capacity. Previous MPM studies measuring in vivo the SNGFR relied on fast full frame acquisition during the filtration process obtainable with microscope resonant scanners, which represent optional expensive equipment able to reach very high acquisition speed. In this work we propose an innovative linescan-based MPM method to calculate SNGFR in rodents doable without using the fast acquisition rate offered by resonant scanners. Method An in vivo MPM approach was used to measure the SNGFR in control Munich Wistar Frömter rats (MWF) and to test the feasibility of the innovative linescan approach. In order to validate this method in conditions known for reduced and increased SNGFR, it was applied to ischemia reperfusion injury (IRI) and low-dose dopamine treated conditions, respectively. Results The glomeruli connected to S1 proximal tubules extending at least 100 μm from the exit of the Bowman’s space were chosen for the measurement. A linescan path starting from the urinary pole and crossing many times the tubular lumen orthogonally to the cellular wall was hand drawn. The linescan was acquired soon after a i.v. bolus of low-molecular weight fluorescent marker was injected. The tubular length, the mean diameter and the transit time of the fluorescent marker within two lines of interest (called cross1 and cross2) were measured to obtain the SNGFR. SNGFR measured in control rats was comparable with previous reported data both at MPM and micropuncture. Significantly higher values compared to control were obtained in 3 μg/kg/min dopamine-treated rats. In IRI-treated rats the SNGFR was reduced about 35% compared to the controls. Conclusion The results achieved with our linescan method were quite similar to those obtained with conventional micropuncture, suggesting that the two methods overlap for the normal, dopamine and IRI treatment. Our results show that linescan approach is a promising and cheap alternative to the fast full frame acquisition for the investigation of SNGFR in health and disease, offering results comparable to conventional micropuncture with unprecedent temporal resolution.

MO467SINGLE NEPHRON GFR AND GLOMERULAR HYDROSTATIC PRESSURE IN JAPANESE HEALTHY SUBJECTS

Background and Aims Hemodynamic abnormalities such as intraglomerular hypertension and glomerular hyperfiltration associated with the nephron loss are deeply involved in renal damage progression. Recent studies in humans have described a new method to calculate single nephron GFR (SNGFR), which is defined as GFR divided by the estimated total number of non-sclerosed glomeruli. We have previously shown that a higher SNGFR, which indicates glomerular hyperfiltration, is associated with CKD risk factors. However, it is still unknown that the hemodynamic changes in humans within the glomerulus in response to SNGFR. In the present study, we analysed the renal microcirculatory dynamics with SNGFR in healthy individuals without CKD. Method We retrospectively identified 51 living kidney donors who underwent enhanced computed tomography and kidney biopsy at the time of donation from January 2007 till December 2020. Nglom was calculated as the cortical volume of both kidneys assessed on computed tomography times the 1-hour posttransplant renal biopsy-determined glomerular density. SNGFR was calculated by dividing GFR (calculated from the corrected creatinine clearance) by the non-sclerosed Nglom. Glomerular hydrostatic pressure (Pglom) and afferent / efferent arteriolar pressure were calculated from the Gomez / Ohm's formula. Results Age was 57.0 ± 10.4 years, mean blood pressure was 88.7 ± 13.2 mmHg, GFR was 73.3 ± 16.7 mL/min, and Nglom was 807,774 ± 401,0352 / kidney. SNGFR was significantly associated with renal blood flow and Pglom (p for trend = 0.02, and 0.02, respectively), but not with age, mean blood pressure, renal plasma flow, filtration fraction, afferent / efferent arteriolar pressure (Table1). Conclusion Our results showed that the increase in SNGFR is associated with elevated Pglom, indicating that glomerular hyperfiltration may lead to intraglomerular hypertension.

A new analysis approach for single nephron GFR in intravital microscopy of mice

Background: Intravital microscopy is an emerging technique in life science with applications in kidney research. Longitudinal observation of (patho-)physiological processes in living mice is possible in the smallest functional unit of the kidney, a single nephron (sn). In particular, effects on glomerular filtration rate (GFR) - a key parameter of renal function - can be assessed. Methods: After intravenous injection of a freely filtered, non-resorbable, fluorescent dye in C57BL/6 mice, a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Automatic modelling of actual PT volume in a 3D dataset replaced 2D volume estimation. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Repeated analysis of image data obtained in healthy mice showed a striking increase of reproducibility by reduction of user interaction. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function.

Total Nephron Number and Single-Nephron Parameters in Patients with IgA Nephropathy

Background: Single nephron dynamics in progressive IgA nephropathy (IgAN) have not been studied. We applied novel methodology to explore single nephron parameters in IgAN. Methods: Non-globally sclerotic glomeruli (NSG) and globally sclerotic glomeruli (GSG) per kidney were estimated using cortical volume assessment via unenhanced computed tomography and biopsy-based stereology. Estimated single-nephron GFR (eSNGFR) and urine protein excretion (SNUPE) were calculated by dividing eGFR and UPE by the number of NSG. Associations with CKD stage and clinicopathologic findings were cross-sectionally investigated. Results: This study included 245 IgAN patients (mean age 43 y, 62% male, 45% on renin-angiotensin aldosterone system [RAAS] inhibitors pre-biopsy) evaluated at kidney biopsy. CKD stages were 10% CKD1, 43% CKD2, 19% CKD3a, 14% CKD3b and 14% CKD4-5. With advancing CKD stage, NSG decreased from mean 992,000 to 300,000 per kidney whereas GSG increased from median 64,000 to 202,000 per kidney. In multivariable models, advancing CKD stage associated with lower numbers of NSG, higher numbers of GSG, and lower numbers of GSG+NSG, indicating potential resorption of sclerosed glomeruli. In contrast to the higher mean glomerular volume and markedly elevated SNUPE in advanced CKD, the eSNGFR was largely unaffected by CKD stage. Lower SNGFR associated with Oxford scores for endocapillary hypercellularity and crescents whereas higher SNUPE associated with segmental glomerulosclerosis and tubulointerstitial scarring. Conclusions: SNUPE emerged as a sensitive biomarker of advancing IgAN. The failure of eSNGFR to increase in response to reduced number of functioning nephrons suggests limited capacity for compensatory hyperfiltration by diseased glomeruli with intrinsic lesions.

mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants

BackgroundAlthough zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.MethodsTranscription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67, the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity.ResultsAdult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants.ConclusionsAdult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.