Protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell-derived kidney organoids using Seahorse XF96.

Metabolic derangement is a key culprit in kidney pathophysiology. Organoids have emerged as a promising in vitro tool for kidney research. Here, we present a fine-tuned protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell (iPSC)-derived kidney organoids using Seahorse XF96. We describe the generation of self-organized three-dimensional kidney organoids, followed by preparation of organoids for Seahorse XF96 analysis. We then detail how to carry out stress tests to determine mitochondrial and glycolytic rates in single kidney organoids.

Transcription Factor ß-Catenin Plays a Key Role in Fluid Flow Shear Stress-Mediated Glomerular Injury in Solitary Kidney.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3ß-ß-catenin signaling. The present study was undertaken to confirm (i) the activation of ß-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the ß-galactosidase reporter gene for ß-catenin activation, (ii) ß-catenin translocation in FFSS-treated mouse podocytes, and (iii) ß-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased ß-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-ß-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-ß-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-ß-catenin (Ser552) but not phospho-ß-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified ß-catenin as a key upstream regulator. We conclude that transcription factor ß-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

Nephron-deficient HSRA rats exhibit renal injury with age but have limited renal damage from streptozotocin-induced hyperglycemia.

Hypertension and diabetes are the greatest factors influencing the progression of chronic kidney disease (CKD). Investigation into the role of nephron number in CKD alone or with hypertension has revealed a strong inverse relationship between the two; however, not much is known about the connection between nephron number and diabetic kidney disease. The heterogeneous stock-derived model of unilateral renal agenesis (HSRA) rat, a novel model of nephron deficiency, provides a unique opportunity to study the association between nephron number and hypertension and diabetes on CKD. HSRA rats exhibit failure of one kidney to develop in 50-75% of offspring, whereas the remaining offspring are born with two kidneys. Rats born with one kidney (HSRA-S) develop significant renal injury with age compared with two-kidney littermates (HSRA-C). The induction of hypertension as a secondary stressor leads to significantly more renal injury in HSRA-S compared with HSRA-C rats and nephrectomized HSRA-C (HSRA-UNX) rats. The present study sought to address the hypothesis that nephron deficiency in the HSRA rat would hasten renal injury in the presence of a secondary stressor of hyperglycemia. HSRA animals did not exhibit diabetes-related traits at any age; thus, streptozotocin (STZ) was used to induce hyperglycemia in HSRA-S, HSRA-C, and HSRA-UNX rats. STZ- and vehicle-treated animals were followed for 15 wk. STZ-treated animals developed robust hyperglycemia, but in contrast to the response to hypertension, neither HSRA-S nor HSRA-UNX animals developed proteinuria compared with vehicle treatment. In total, our data indicate that hyperglycemia from STZ alone does not have a significant impact on the onset or progression of injury in young one-kidney HSRA animals.NEW & NOTEWORTHY The HSRA rat, a novel model of nephron deficiency, provides a unique opportunity to study the association between nephron number and confounding cardiovascular complications that impact kidney health. Although hypertension was previously shown to exacerbate renal injury in young HSRA animals, diabetic hyperglycemia did not lead to worse renal injury, suggesting that nephron number has limited impact on kidney injury, at least in this model.

Validation of non-invasive transcutaneous measurement for glomerular filtration rate in lean and obese C57BL/6J mice.

AIM: The measurement of glomerular filtration rate (GFR) in experimental rodents is pivotal to understanding the progression of kidney disease and benefits of treatment strategies. A non-invasive clearance device has been developed, which measures transcutaneous decay of injected FITC-sinistrin in conscious rodents. The technique was validated against the well-established plasma clearance method in the same mice, but on consecutive days, using only models of uninephrectomy and polycystic kidney disease. We aimed to validate this widely used technique in the same lean or obese mice, at the same time. METHODS: Five-week-old male C57BL/6J mice were randomised to a high fat diet (n = 12) or normal diet (n = 11) for 10 weeks. Transcutaneous and plasma clearance of FITC-sinistrin were measured simultaneously in each mouse. RESULTS: In lean mice, there was a positive correlation between transcutaneous and plasma derived GFR (P < .01, R2 = .704), although there was an approximate 40% underestimation by the transcutaneous method (P < .0001). In obese mice, no correlation was observed between transcutaneous and plasma derived GFR, nor elimination half-life which removes any effect of the conversion factor and injected dose. The limits of agreement in a Bland-Altman plot were narrower when we used new conversion factors derived from mice in the current study and, in lean mice, a generic conversion factor which assumes 20% extracellular volume. CONCLUSION: The non-invasive clearance device may be useful for serial GFR measurements in lean and healthy mice, provided validation studies have been carried out, but its utility in obesity requires further study.

Long-term outcomes of ultrasound-guided percutaneous nephrolithotomy in patients with solitary kidneys: a single-center experience.

PURPOSE: To report our experience with total ultrasound-guided percutaneous nephrolithotomy (PCNL) in the management of patients with solitary kidney, and evaluate the safety and feasibility of this technique. MATERIALS AND METHODS: Between October 2014 and December 2016, 48 patients with solitary kidneys underwent total ultrasound-guided PCNL at our institution. Stone-free rate (SFR), auxiliary procedures, and complications were recorded. Changes in renal function were evaluated by comparing preoperative and postoperative estimated glomerular filtration rates (eGFRs). Perioperative factors that may affect renal function were analyzed to define factors predicting renal function improvement on long-term follow-up. Of 48 patients, 44 were followed at least 6 months, whereas four patients were lost to follow-up. RESULTS: Among all patients, staghorn calculi were found in 18 (37.5%) patients. 14 (29.2%) patients required a two-stage PCNL. Struvite was found in six (12.5%) patients. Complications were reported in eight (16.7%) patients. Severe bleeding was noticed in three patients; no angioembolization was required. After a median follow-up of 12 (6-26) months, the final SFR was 81.8% after auxiliary treatments. There was a significant improvement of eGFR from 53.9 ± 24.0 to 61.3 ± 25.4 mL/min/1.73 m2 (P < 0.01). Renal function was stable, improved and worse in 65.9% (n = 29), 27.3% (n = 12), and 6.8% (n = 3) of patients, respectively, compared with preoperative levels. CONCLUSIONS: Ultrasound-guided PCNL is a safe and feasible procedure with an acceptably low complication rate in patients with solitary kidneys. At long-term follow-up, the renal function in more than 90% of the patients with solitary kidneys can be improved or stabilized after ultrasound-guided PCNL.

Renal Nitric Oxide Deficiency and Chronic Kidney Disease in Young Sheep Born with a Solitary Functioning Kidney.

Previously, we demonstrated that renal hemodynamic responses to nitric oxide (NO) inhibition were attenuated in aged, hypertensive sheep born with a solitary functioning kidney (SFK). NO is an important regulator of renal function, particularly, in the postnatal period. We hypothesized that the onset of renal dysfunction and hypertension in individuals with a SFK is associated with NO deficiency early in life. In this study, renal and cardiovascular responses to L-NAME infusion (N(w)-nitro-L-arginine methyl ester) were examined in 6-month old lambs born with a SFK, induced by fetal unilateral nephrectomy (uni-x). Renal responses to L-NAME were attenuated in uni-x sheep with the fall in glomerular filtration rate (GFR) and urinary sodium excretion (UNaV) being less in the uni-x compared to sham lambs (%ΔGFR; -41 ± 3 vs -54 ± 4: P = 0.03, %ΔUNaV; -48 ± 5 vs -76 ± 3, P = 0.0008). 24 hour-basal urinary nitrate and nitrite (NOx) excretion was less in the uni-x animals compared to the sham (NOx excretion µM/min/kg; sham: 57 ± 7; uni-x: 38 ± 4, P = 0.02). L-NAME treatment reduced urinary NOx to undetectable levels in both groups. A reduction in NO bioavailability in early life may contribute to the initiation of glomerular and tubular dysfunction that promotes development and progression of hypertension in offspring with a congenital nephron deficit, including those with a SFK.