Post-translational modifications by SIRT3 de-2-hydroxyisobutyrylase activity regulate glycolysis and enable nephrogenesis

Abnormal kidney development leads to lower nephron number, predisposing to renal diseases in adulthood. In embryonic kidneys, nephron endowment is dictated by the availability of nephron progenitors, whose self-renewal and differentiation require a relatively repressed chromatin state. More recently, NAD+-dependent deacetylase sirtuins (SIRTs) have emerged as possible regulators that link epigenetic processes to the metabolism. Here, we discovered a novel role for the NAD+-dependent deacylase SIRT3 in kidney development. In the embryonic kidney, SIRT3 was highly expressed only as a short isoform, with nuclear and extra-nuclear localisation. The nuclear SIRT3 did not act as deacetylase but exerted de-2-hydroxyisobutyrylase activity on lysine residues of histone proteins. Extra-nuclear SIRT3 regulated lysine 2-hydroxyisobutyrylation (Khib) levels of phosphofructokinase (PFK) and Sirt3 deficiency increased PFK Khib levels, inducing a glycolysis boost. This altered Khib landscape in Sirt3−/− metanephroi was associated with decreased nephron progenitors, impaired nephrogenesis and a reduced number of nephrons. These data describe an unprecedented role of SIRT3 in controlling early renal development through the regulation of epigenetics and metabolic processes.

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.

Abstract 29: Acute Systemic Treatment With Sphingosine -1- Phosphate Receptor 1 Agonist Diminishes Sex-difference In Renal Function Seen In Intrauterine Growth Restricted Mice.

IUGR is a risk factor for the early development of cardiorenal diseases in life. We previously showed that our IUGR mouse model exhibits sex differences in blood pressure (BP) and kidney functions as males have elevated BP and impaired kidney function while females are not. Sphingosine-1-Phosphate Receptor 1 (S1PR1) is reported to be involved in developing and progressing several cardiorenal diseases. We found that acute activation of S1PR1 transiently reduced BP in male IUGR; however, the effects of SIPR1 on renal function in IUGR are still unknown. We hypothesize that S1PR1 plays a role in the sex differences of impaired kidney function in IUGR. Here we investigated the acute effects of a specific S1PR1 agonist (SEW2178) on kidney function in IUGR offspring generated through placental insufficiency. Methods: C57BL IUGR or control offspring were obtained from the Reduced Uterine Perfusion Pressure (RUPP) or sham surgeries on the 13 th day of gestation. At 6 months, nephron number was measured by analyzing kidney histology while Glomerular Filtration Rate (GFR) was measured using FITC-Anulin decay after retroorbital injection. Mice were treated with 3mg/kg SEW2871 i.p before GFR measurement. Results: Both male and female IUGR had a decrease in nephron number compared to same-sex control (371.8± 28.8 vs 284.0± 11.3, P<0.01, male control vs IUGR) and (333.6±13.3 vs 256± 13.0, P<0.05, female control vs IUGR). Furthermore, IUGR impaired kidney function in male but not female mice as indicated by reduced GFR (8.9 ±0.6 vs 13.9±1.1 μL/min/kg BW, P<0.05, IUGR vs control males) and (12.3±0.9 vs 15.9 ±1.9 L/min/kg BW, control females vs IUGR). Administration of SEW2871 diminished the difference of GFR between male control and IUGR (9.5±0.6 vs 9.7±1.1 μL/min/kg BW, P> 0.05 control vs IUGR). While in females, both IUGR and control had a similar response to SEW2178 (7.4±1.0 vs 10.6 ±1.9 μL/min/kg BW, control vs. IUGR). Conclusion: IUGR impairs renal function in male offspring but not in females, indicating sex differences in fetal programming of kidney disease. Male and female IUGR offspring respond differently to acute systemic activation of S1PR1. Further studies are required to investigate the kidney-specific and cell-specific of S1PR1 activation/inhibition.

MO431LIPIDOMIC ANALYSIS IN A NON-DIABETIC RAT MODEL WITH HYPERFILTRATION AND ALBUMINURIA REVEALS DYNAMIC CHANGES IN THE PROSTAGLANDIN E2 PATHWAY DURING ONSET OF ALBUMINURIA AS A POTENTIAL CAUSATIVE MECHANISM

Background and Aims We recently identified prostaglandin reductase 2 (Ptgr2) and the prostaglandin E2 (PGE2) pathway as potential causative mechanism in the Munich Wistar Frömter (MWF) non-diabetic rat model of chronic kidney disease. MWF is characterized by early onset of spontaneous albuminuria during a critical time window between 4 and 8 weeks of age, that associates with hyperfiltration, due to low nephron number, and podocyte injury. Ptgr2 plays an important role in the prostaglandin metabolism, in which PGE2 is metabolized by 15-prostaglandin dehydrogenase (15-PGDH) to 15-keto-PGE2. The latter is terminally degraded by prostaglandin reductases (PTGRs) 1, 2, and 3 to 13,14-dihydro-15-keto-PGE2. Recently, we detected elevated glomerular levels of PGE2 and 15-keto-PGE2 in MWF compared to spontaneously hypertensive rats (SHR) with no albuminuria. The aim of the present study was to characterize in detail the renal PGE2 metabolic pathway in MWF by lipidomic analysis during the time window of albuminuria onset. Method Male MWF and SHR rats were studied at week 4 and 8, respectively; 24 h-urine was collected in metabolic cages. In addition, plasma and kidney tissues including kidney cortex and isolated glomeruli were obtained from anesthetized rats. Lipidomic analysis was done by liquid chromatography electrospray ionization tandem mass spectrometry. Statistical analysis was performed by unpaired, two-tailed Student’s t-test, or otherwise the Mann-Whitney test. Results Urinary PGE2 was significantly lower (p&lt;0.01), while urinary 15-keto-PGE2 and 13,14-dihydro-15-keto PGE2 levels were significantly increased in MWF compared to SHR at week 4 (p&lt;0.01, respectively). All three analytes were significantly decreased in urine of MWF with increased albuminuria at week 8 (p&lt;0.05 vs. SHR). The urinary metabolic ratio of 15-keto-PGE2/13,14-dihydro-15-keto-PGE2 as a surrogate for PTGRs activities was significantly increased at week 4 (p&lt;0.01), whereas it was significantly decreased in MWF vs. SHR at week 8 (p&lt;0.05). Plasma levels of PGE2 and 13,14-dihydro-15-keto-PGE2 did not differ between strains at both time points, while 15-keto-PGE2 was below the detection limit. Glomerular levels of PGE2 and 15-keto-PGE2 were increased in MWF at week 4 and 8 (p&lt;0.01, respectively). In contrast, 13,14-dihydro-15-keto-PGE2 was only significantly higher at week 4 compared to SHR (p&lt;0.01). In isolated glomeruli, the metabolic ratios of PTGRs were similar between the strains at week 4, but significantly increased in MWF compared to SHR at week 8 (p&lt;0.01, respectively). In kidney cortex, 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2 were increased in MWF at week 4 (p&lt;0.05 and p&lt;0.01, respectively), whereas PGE2 levels were comparable to SHR. No difference for cortical levels of PGE2 and its metabolites was observed at week 8. Conclusion This study provides the first insights into age-dependent dynamic changes in the PGE2 pathway that support potential causality for the onset of albuminuria in the setting of non-diabetic hyperfiltration due to low nephron number. Notably, the increased glomerular levels of PGE2 and its downstream metabolites in 4-week-old MWF point towards an early activation of this pathway, i.e. before albuminuria occurs. This finding corroborates the hypothesis that glomerular PGE2 signaling is relevant in the early stages of hyperfiltration and suggests this pathway as a target for future therapeutics to modify the manifestation and progression of renal disease.

Gestational and Breastfeeding Low-Protein Intake on Blood Pressure, Kidney Structure, and Renal Function in Male Rat Offspring in Adulthood

Background: Our previous studies demonstrated that maternal protein-restricted (low-protein, LP) 16-week-old offspring had pronounced nephron number reduction and arterial hypertension associated with an unchanged glomerular filtration rate (GFR). An enhanced gomerular area may be related to increased glomerular filtration and overflow, which accounts for glomerular filtration barrier breakdown and early glomerulosclerosis. The effect of protein restriction during gestational and breastfeeding periods is unknown.Method: The functional e-structural kidney evaluation was obtained using lithium and creatinine clearance, kidney morphometry, immunoblotting, and immunostaining analysis in 16 and 24-week-old LP offspring compared to age-matched NP progeny.Results: Low protein rats’ progeny had significantly reduced birth weight, without previous catch-up growth phenomena, in parallel with a decreased adiposity index. Transforming growth factor-beta 1 (TGF-β1) glomerular expression was significantly enhanced in the LP group. Also, the LP offspring had a 38% lower nephron number and an increased glomerular volume. They also presented with a higher cardiac index and arterial blood pressure compared with age-matched NP offspring. The LP rats exhibited augmented Na+/K+-ATPase in the proximal segments, and NOS1 immunoreactivity in whole renal tissue was associated with sodium retention in the proximal nephron segments. We also found significantly enhanced collagen content associated with increased TGFβ1 and ZEB1/2 renal immunoreactivity in LP offspring compared with NP offspring. Increased hypertrophy markers in LP podocytes were associated with an amplified IL-6/STAT3 pathway activity.Conclusion: To our knowledge, these are the first data demonstrating renal functional and structural changes in protein restriction during gestation and lactation model of fetal programming. The fetal-programmed adult offspring showed pronounced structural glomerular disorders with an accentuated and advanced fibrosis stage, without a change in the GFR. These findings suggest that the glomerular enhanced TGF-β1 action may induce ZEB1/2 expression that may cause glomeruli epithelial-to-mesenchymal transition. Besides, decreased nephron number in the LP offspring with preserved glomerular function may be related to protective or even attenuate the activated IL-6/STAT3 pathway.

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 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 while remaining offspring are born with two kidneys. Rats born with one kidney (HSRA-S) develop significant renal injury with age compared to two-kidney littermates (HSRA-C). Induction of hypertension as a secondary stressor leads to significantly more renal injury in HSRA-S compared to HSRA-C and HSRA-UNX (nephrectomized HSRA-C rats). The current 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. STZ and vehicle-treated animals were followed for 15 weeks. STZ animals developed robust hyperglycemia, but in contrast to the response to hypertension, HSRA-S nor HSRA-UNX animals developed proteinuria compared to vehicle. In total, our data indicates that hyperglycemia from STZ alone does not have a significant impact on the onset or progression of injury in young one kidney HSRA animals.

Long-Term but Not Short-Term Maternal Fasting Reduces Nephron Number and Alters the Glomerular Filtration Barrier in Rat Offspring

The present study examined the effects of maternal Ramadan-type fasting during selected days in the first, second, or third trimester, or during the entire pregnancy, on the kidney structure of male rat offspring. Pregnant rats were provided with food ad libitum during pregnancy (control group, C), or they were exposed to 16 h of fasting/day for three consecutive days in the middle of the first (FT1), second (FT2), or third trimester (FT3), or during whole pregnancy (FWP). Our results showed that dams in the FWP group demonstrated lower food intake and body weight during gestation. Litter size was unaltered by fasting in all groups; however, litter weight was significantly reduced only in the FWP group. Nephron number was decreased in the FWP group, but it remained unchanged in the other fasting groups. The ultrastructure of the glomerular filtration barrier indicated that the kidneys of offspring of the FWP group demonstrated wider diameters of fenestrations and filtration slits and smaller diameters of basement membranes. This was reflected by a significant increase in proteinuria in FWP only. These results suggest that, unlike with short-term fasting, which seems to be safe, maternal long-term fasting induces structural changes that were non-reversible, and that may contribute to impaired renal function, leading to chronic diseases in later life.

Podometrics in Japanese Living Donor Kidneys: Associations with Nephron Number, Age, and Hypertension

BackgroundPodocyte depletion, low nephron number, aging, and hypertension are associated with glomerulosclerosis and CKD. However, the relationship between podometrics and nephron number has not previously been examined.MethodsTo investigate podometrics and nephron number in healthy Japanese individuals, a population characterized by a relatively low nephron number, we immunostained single paraffin sections from 30 Japanese living-kidney donors (median age, 57 years) with podocyte-specific markers and analyzed images obtained with confocal microscopy. We used model-based stereology to estimate podometrics, and a combined enhanced–computed tomography/biopsy-specimen stereology method to estimate nephron number.ResultsThe median number of nonsclerotic nephrons per kidney was 659,000 (interquartile range [IQR], 564,000–825,000). The median podocyte number and podocyte density were 518 (IQR, 428–601) per tuft and 219 (IQR, 180–253) per 106μm3, respectively; these values are similar to those previously reported for other races. Total podocyte number per kidney (obtained by multiplying the individual number of nonsclerotic glomeruli by podocyte number per glomerulus) was 376 million (IQR, 259–449 million) and ranged 7.4-fold between donors. On average, these healthy kidneys lost 5.63 million podocytes per kidney per year, with most of this loss associated with glomerular loss resulting from global glomerulosclerosis, rather than podocyte loss from healthy glomeruli. Hypertension was associated with lower podocyte density and larger podocyte volume, independent of age.ConclusionsEstimation of the number of nephrons, podocytes, and other podometric parameters in individual kidneys provides new insights into the relationships between these parameters, age, and hypertension in the kidney. This approach might be of considerable value in evaluating the kidney in health and disease.