IntroductionPlasmin and its precursor, plasminogen, are detectable in urine from patients with glomerular disease. Urinary plasmin(ogen) levels correlate with blood pressure (BP) and may contribute to renal Na+ retention by activating the epithelial Na+ channel (ENaC). In a longitudinal nested-cohort study, we asked whether urinary plasmin(ogen) levels predict subsequent increase in BP, incident hypertension, or mortality in subjects with type I diabetes, who often develop proteinuria.MethodsThe Pittsburgh Epidemiology of Diabetes Complications (EDC) study followed up type I diabetic subjects for 25 years. Urine specimens from 70 subjects with a spectrum of baseline urinary albumin levels were examined. Outcomes included increased BP after 2 years (≥1 SD over baseline systolic or diastolic BP, examined via logistic regression), 25-year incident hypertension (≥140/90 mm Hg or initiating BP-lowering medications), and all-cause or cardiovascular mortality, examined using Cox regression.ResultsSubjects experiencing a 2-year increase in BP had higher baseline urinary plasmin(ogen)/creatinine levels (uPl/Cr) than other subjects (P = 0.04); the difference in baseline urinary albumin/creatinine levels (uAlb/Cr) was similar (P = 0.07). Baseline uPl/Cr was associated with increased 25-year hypertension incidence (hazard ratio = 2.05, P = 0.001), all-cause mortality (HR = 2.05, P = 0.01) and cardiovascular mortality (HR = 3.30, P = 0.005), although not independent of uAlb/Cr.ConclusionThis is the first long-term prospective study addressing clinical outcomes associated with increased urinary plasmin(ogen). Findings are consistent with a role for plasmin(ogen) in promoting increased BP, but also demonstrate the difficulty in distinguishing effects due to plasmin(ogen) from those of albuminuria.

Introduction Renal Na+ retention and extracellular fluid volume expansion are hallmarks of nephrotic syndrome, which occurs even in the absence of activation of hormones that stimulate renal Na+ transporters. Plasmin-dependent activation of the epithelial Na+ channel (ENaC) has been proposed to have a role in renal Na+ retention in the setting of nephrotic syndrome. We hypothesized that the ENaC inhibitor amiloride would be an effective therapeutic agent in inducing a natriuresis and lowering blood pressure in individuals with macroscopic proteinuria. Methods We conducted a pilot double-blind randomized cross-over study comparing the effects of daily administration of either oral amiloride or hydrochlorothiazide (HCTZ) to patients with type 2 diabetes and macroscopic proteinuria. Safety and efficacy were assessed by monitoring systolic blood pressure (SBP), kidney function, adherence, weight, urinary Na+ excretion and serum electrolytes. Nine subjects were enrolled in the trial. Results No significant difference in SBP or weight was seen between HCTZ and amiloride (p≥0.15). Amiloride induced differences in serum K+ (p<0.001), with a 0.88±0.30 mmol/L greater acute increase observed. Two subjects developed acute kidney injury and hyperkalemia when treated with amiloride. Four subjects had readily detectable levels of urinary plasminogen plus plasmin (uPl), and five did not. Changes in SBP in response to amiloride did not differ between individuals with vs. those without detectable uPl. Conclusion In summary, among patients with type 2 diabetes, normal renal function and proteinuria, there were reductions in SBP in groups treated with HCTZ or amiloride. Acute kidney injury and severe hyperkalemia were safety concerns with amiloride.

mTORC2 is a kinase complex that targets predominantly Akt, SGK1, and PKC, and has well characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular potassium (K+) concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of With No Lysine-1 (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2 resulting in enhanced SGK1 phosphorylation, and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1- dependent regulatory mechanism, which harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.

Well-designed research, with CVD as primary outcome, is needed to help bridge the gap in our knowledge on this topic. In the meantime, caution should be applied to avoid overdiagnosis and overtreatment of vitamin D deficiency.

II.

BACKGROUND: Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K+]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. METHODS: We used Cre-Lox-mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins following a K+ load via gavage. RESULTS: A K+ load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type but not in knockout mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type but not knockout mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K+] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). CONCLUSIONS: The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K+] in vivo. The effects of K+ on this signaling module are specific, in that other downstream mTORC2 targets such as PKC and Akt are not acutely affected, and ROMK and BK channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K+in vivo.

Women aged 51 years or older who have AGC are more likely to have AGC-EM and EC than women younger than 51 years. In women younger than age 51, AGC-EM is the subclass most associated with EC while compared to 2 other subclasses (AGC not otherwise specified and AGC-EC).

Methods: Rictor is a critical component of the mTORC2 complex. Tubule-specific Rictor knockout (TRKO) mice were made using doxycycline inducible Pax8-Cre Rictor-flox. Male and female TRKO mice and their control littermates were used for all experiments. Tolerance tests were performed with intraperitoneal glucose (1g/kg), insulin (0.75U/kg), and pyruvate (2g/kg) after overnight fasts. Refeeding at the time of sacrifice to induced insulin signaling; mice were fasted for 18 hours then refed 4 hours. Whole kidney relative mRNA was measured via RT-PCR. Kidney plasma membrane and cytosolic proteins were separated using the BioVision Plasma Membrane Protein Extraction Kit, and protein abundance was measured with western blotting. Results: There were no differences in serum glucose during refeeding experiments, glucose tolerance tests, or insulin tolerance tests between TRKO and control mice at any timepoints (n=9 per group). However, the mean ± standard error of mean (SEM) urine glucose concentration was 472.5±181.2mg/dL in TRKO mice compared to 30.8±5.0mg/dL in control animals during refeeding (n=9 per group; p<0.01). Serum glucose was higher in TRKO mice compared to controls (n=7 per group) after giving the gluconeogenic substrate pyruvate at 60 (234.4±15.0 vs 189.4±9.1mg/dL; p<0.01) and 90 minutes (194.3±7.4 vs 148.9±5.9mg/dL; p<0.01). TRKO mice (n=8) also had elevated hemoglobin A1c (HbA1c) compared to control mice (n=6 per group) after 3 months on a 0.5% K+ diet (6.00±0.21% vs 5.23±0.11%; p<0.01). Refed TRKO mice kidneys compared to controls had significantly higher relative mRNA of PEPCK (3.74±0.76 vs 1.18±0.15AU; p<0.01) and G6Pase (4.24±1.02 vs 1.37±0.29AU; p<0.01) (n=15 per group). Refed TRKO mice kidneys compared to controls also had elevated protein abundance of PEPCK (0.54±0.05 vs 0.28±0.03AU; p<0.001) but no difference in G6Pase (0.62±0.03 vs 0.49±0.05AU; ns). Kidneys from refed TRKO and control mice showed no differences in relative mRNA of SGLT2, SGLT1, or GLUT2 (n=15 per group). Kidneys from TRKO and control mice also showed no difference in plasma membrane protein abundance of SGLT2 and GLUT2 (n=8 per group). Conclusion: This study demonstrates that insulin signaling through mTORC2 is critical for suppression of renal GNG and complete reabsorption of glucose. Increased serum glucose during pyruvate tolerance testing, increased HbA1c, increased gluconeogenic gene transcription (i.e., PEPCK, G6Pase), and increased PEPCK protein abundance all support increased renal GNG in TRKO mice. Glycosuria was present in TRKO mice despite no difference in serum glucose between TRKO and control mice, suggesting that mTORC2 is important for both renal GNG and glucose reabsorption. Future studies will use TRKO mice to further evaluate glucose transporters and elucidate the mechanism of glycosuria. NIDDK Support, T32DK007219; Diacomp, 5U24DK115255-040 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.