Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Dietary sodium intake does not alter renal potassium handling and blood pressure in healthy young males.

BACKGROUND: The effects of sodium (Na+) intakes on renal handling of potassium (K+) are insufficiently studied. METHODS: We assessed the effect of Na+ on renal K+ handling in 16 healthy males assigned to three 7-day periods on low salt diet [LSD, 3 g sodium chloride (NaCl)/day], normal salt diet (NSD, 6 g NaCl/day) and high salt diet (HSD, 15 g NaCl/day), with constant K+ intake. Contributions of distal NaCl co-transporter and epithelial Na+ channel in the collecting system on K+ and Na+ handling were assessed at steady state by acute response to 100 mg oral hydrochlorothiazide and with addition of 10 mg of amiloride to hydrochlorothiazide, respectively. RESULTS: Diurnal blood pressure slightly increased from 119.30 ± 7.95 mmHg under LSD to 123.00 ± 7.50 mmHg (P = 0.02) under HSD, while estimated glomerular filtration rate increased from 133.20 ± 34.68 mL/min under LSD to 187.00 ± 49.10 under HSD (P = 0.005). The 24-h K+ excretion remained stable on all Na+ intakes (66.28 ± 19.12 mmol/24 h under LSD; 55.91 ± 21.17 mmol/24 h under NSD; and 66.81 ± 20.72 under HSD, P = 0.9). The hydrochlorothiazide-induced natriuresis was the highest under HSD (30.22 ± 12.53 mmol/h) and the lowest under LSD (15.38 ± 8.94 mmol/h, P = 0.02). Hydrochlorothiazide increased kaliuresis and amiloride decreased kaliuresis similarly on all three diets. CONCLUSIONS: Neither spontaneous nor diuretic-induced K+ excretion was influenced by Na+ intake in healthy male subjects. However, the respective contribution of the distal convoluted tubule and the collecting duct to renal Na+ handling was dependent on dietary Na+ intake.

Expression of claudin-8 is induced by aldosterone in renal collecting duct principal cells.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

Dietary sodium induces a redistribution of the tubular metabolic workload.

KEY POINTS: Body Na+ content is tightly controlled by regulated urinary Na+ excretion. The intrarenal mechanisms mediating adaptation to variations in dietary Na+ intake are incompletely characterized. We confirmed and expanded observations in mice that variations in dietary Na+ intake do not alter the glomerular filtration rate but alter the total and cell-surface expression of major Na+ transporters all along the kidney tubule. Low dietary Na+ intake increased Na+ reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments. High dietary Na+ intake decreased Na+ reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency. The abundance of apical transporters and Na+ delivery are the main determinants of Na+ reabsorption along the kidney tubule. Tubular O2 consumption and the efficiency of sodium reabsorption are dependent on sodium diet. ABSTRACT: Na+ excretion by the kidney varies according to dietary Na+ intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na+ reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na+ , a normal sodium diet (NSD) containing 0.18% Na+ and a moderately high sodium diet (HSD) containing 1.25% Na+ . As expected, LSD did not alter measured GFR and increased the abundance of total and cell-surface NHE3, NKCC2, NCC, α-ENaC and cleaved γ-ENaC compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na+ delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O2 consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) was related to the changes in tubular Na+ reabsorption. Our data show that fractional Na+ reabsorption is distributed differently according to dietary Na+ intake and induces changes in tubular O2 consumption and sodium transport efficiency.

Oxidative Stress and Nuclear Factor κB (NF-κB) Increase Peritoneal Filtration and Contribute to Ascites Formation in Nephrotic Syndrome.

Water accumulation in the interstitium (edema) and the peritoneum (ascites) of nephrotic patients is classically thought to stem from the prevailing low plasma albumin concentration and the decreased transcapillary oncotic pressure gradient. However, several clinical and experimental observations suggest that it might also stem from changes in capillary permeability. We addressed this hypothesis by studying the peritoneum permeability of rats with puromycin aminonucleoside-induced nephrotic syndrome. The peritoneum of puromycin aminonucleoside rats displayed an increase in the water filtration coefficient of paracellular and transcellular pathways, and a decrease in the reflection coefficient to proteins. It also displayed oxidative stress and subsequent activation of NF-κB. Scavenging of reactive oxygen species and inhibition of NF-κB prevented the changes in the water permeability and reflection coefficient to proteins and reduced the volume of ascites by over 50%. Changes in water permeability were associated with the overexpression of the water channel aquaporin 1, which was prevented by reactive oxygen species scavenging and inhibition of NF-κB. In conclusion, nephrotic syndrome is associated with an increased filtration coefficient of the peritoneum and a decreased reflection coefficient to proteins. These changes, which account for over half of ascite volume, are triggered by oxidative stress and subsequent activation of NF-κB.

Type IV Collagen Variants in CKD: Performance of Computational Predictions for Identifying Pathogenic Variants.

RATIONALE & OBJECTIVE: Pathogenic variants in type IV collagen have been reported to account for a significant proportion of chronic kidney disease. Accordingly, genetic testing is increasingly used to diagnose kidney diseases, but testing also may reveal rare missense variants that are of uncertain clinical significance. To aid in interpretation, computational prediction (called in silico) programs may be used to predict whether a variant is clinically important. We evaluate the performance of in silico programs for COL4A3/A4/A5 variants. STUDY DESIGN SETTING & PARTICIPANTS: Rare missense variants in COL4A3/A4/A5 were identified in disease cohorts, including a local focal segmental glomerulosclerosis (FSGS) cohort and publicly available disease databases, in which they are categorized as pathogenic or benign based on clinical criteria. TESTS COMPARED & OUTCOMES: All rare missense variants identified in the 4 disease cohorts were subjected to in silico predictions using 12 different programs. Comparisons between the predictions were compared with: (1) variant classification (pathogenic or benign) in the cohorts and (2) functional characterization in a randomly selected smaller number (17) of pathogenic or uncertain significance variants obtained from the local FSGS cohort. RESULTS: In silico predictions correctly classified 75% to 97% of pathogenic and 57% to 100% of benign COL4A3/A4/A5 variants in public disease databases. The congruency of in silico predictions was similar for variants categorized as pathogenic and benign, with the exception of benign COL4A5 variants, in which disease effects were overestimated. By contrast, in silico predictions and functional characterization classified all 9 pathogenic COL4A3/A4/A5 variants correctly that were obtained from a local FSGS cohort. However, these programs also overestimated the effects of genomic variants of uncertain significance when compared with functional characterization. Each of the 12 in silico programs used yielded similar results. LIMITATIONS: Overestimation of in silico program sensitivity given that they may have been used in the categorization of variants labeled as pathogenic in disease repositories. CONCLUSIONS: Our results suggest that in silico predictions are sensitive but not specific to assign COL4A3/A4/A5 variant pathogenicity, with misclassification of benign variants and variants of uncertain significance. Thus, we do not recommend in silico programs but instead recommend pursuing more objective levels of evidence suggested by medical genetics guidelines.

Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS.

BACKGROUND AND OBJECTIVES: FSGS and nephrotic syndrome studies have shown that single gene causes are more likely to be found in pediatric cases than adults. Consequently, many studies have examined limited gene panels in largely pediatric cohorts. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Whole-exome sequencing was performed in adults with FSGS diagnosed between 1976 and 2017 in the Toronto GN Registry. An expanded panel of 109 genes linked to FSGS, glomerular basement membrane abnormalities, as well as causes of pediatric ESKD including congenital abnormalities of the kidney and urinary tract (CAKUT) and nephronophthisis, were examined. RESULTS: The cohort was composed of 193 individuals from 179 families. Nearly half (49%) developed ESKD at a mean age of 47±17 years. The genetic diagnostic rate was 11%. Of definitely pathogenic variants, 55% were in COL4A (A3/A4/A5), 40% were in podocyte genes, and 5% were in CAKUT genes. Many, but not all individuals with COL4A definitely pathogenic variants had some evidence of glomerular basement membrane abnormalities. The estimated mean survival/age of kidney failure for individuals with COL4A definitely pathogenic variants was 58 years (95% confidence interval, 49 to 69), far later than what has been reported in the literature. Likely pathogenic variants were identified in an additional 9% of the cohort, with most in COL4A. Correlation with glomerular basement membrane morphology suggested a causal role for at least some of these likely pathogenic variants. CONCLUSIONS: Even with an expanded gene panel, we find that COL4A disorders are the leading monogenic cause in adults diagnosed with FSGS. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_01_15_CJASNPodcast_19_02_.mp3.