Tubular Transport Research Articles

Oxygen sensing in the kidney.

The kidneys fulfil several essential homeostatic functions for the body. One of them is the maintenance of sufficient oxygen supply to the organs. For this purpose, the kidneys control the formation of red blood cells by the production of the hormone erythropoietin. This control of red cell formation is not only relevant to prevent states of oxygen deficiency but also to prevent an unwanted increase of red cell numbers causing thromboembolic risks. The adequate production of erythropoietin requires a sensing of the arterial oxygen content and transduction to hormone production. This oxygen sensing is a two-step process which includes a translation of the arterial oxygen content to respective oxygen tension in the tubulointerstitium and a perception of the resulting local interstitial oxygen tension to translate them into specific cellular responses such as the production of erythropoietin. This contribution will describe these steps of oxygen sensing for the healthy kidney and for the changes occurring during states of chronic renal disease, which are commonly associated with anemia. In this context a special focus will also be set on intrarenal hypoxia and oxygen sensing in the diabetic kidney including the treatment with tubular glucose transport (SGLT-2) inhibitors which might influence the oxygen sensing in the kidney. Finally, we will consider the effects of prolylhydroxylase inhibitors (PHD-I), which fundamentally interfere with the cellular oxygen sensing and which are meanwhile treatment options in renal anemia.

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

Interplay between the Redox System and Renal Tubular Transport.

The kidney plays a critical role in maintaining the homeostasis of body fluid by filtration of metabolic wastes and reabsorption of nutrients. Due to the overload, a vast of energy is required through aerobic metabolism, which inevitably leads to the generation of reactive oxygen species (ROS) in the kidney. Under unstressed conditions, ROS are counteracted by antioxidant systems and maintained at low levels, which are involved in signal transduction and physiological processes. Accumulating evidence indicates that the reduction-oxidation (redox) system interacts with renal tubular transport. Redox imbalance or dysfunction of tubular transport leads to renal disease. Here, we discuss the ROS and antioxidant systems in the kidney and outline the metabolic dysfunction that is a common feature of renal disease. Importantly, we describe the key molecules involved in renal tubular transport and their relationship to the redox system and, finally, summarize the impact of their dysregulation on the pathogenesis and progression of acute and chronic kidney disease.

Urinary Extracellular Vesicles for Non-Invasive Quantification of Principal Cell Damage in Kidney Transplant Recipients.

The objective of the present study was to compare principal cell-specific aquaporin-2 (AQP2) abundances in urinary extracellular vesicles (uEVs) on the first postoperative day in deceased-donor kidney transplant recipients without and with acute kidney injury. We measured uEV markers (CD9 and CD63) and the abundances of proximal tubular sodium-glucose transporter 2, distal tubular sodium/chloride cotransporter, and principal cell-specific aquaporin-2 using Western blotting of urine. uEV-AQP2 levels were normalized to living donor controls. The validation cohort consisted of 82 deceased-donor kidney transplant recipients who had a median age of 50 years (IQR 43 to 57 years). A total of 32% of recipients had acute kidney injury. The median uEV-AQP2 was significantly higher in recipients with acute kidney injury compared to immediate allograft function (2.05; IQR 0.87 to 2.83; vs. 0.81; IQR 0.44 to 1.78; p < 0.01). The Youden index indicated a uEV-AQP2 threshold of 2.00. Stratifying uEV-AQP2 into quartiles showed that recipients with higher uEV-AQP2 levels had higher rates of acute kidney injury (Cochran-Armitage, p = 0.001). The discovery cohort showed elevated CD9, CD63, and uEV-AQP2 levels in urine from recipients with acute kidney injury compared to immediate allograft function. We were able to quantify the damage of principal cells after kidney transplant to predict acute kidney injury using uEV-AQP2.

Urinary extracellular vesicles as a monitoring tool for renal damage in patients not meeting criteria for chronic kidney disease.

Current definition of chronic kidney disease (CKD) identifies only advanced stages, but effective management demands early detection. Urinary albumin-to-creatinine ratio (ACR) 30mg/g is a cut-off point for CKD clinical diagnosis. Patients with lower values (normoalbuminuria) and eGFR>60mL/min/1.73m2 are considered at no increased cardiorenal risk. However, higher incidence of renal function decline and cardiovascular events have been shown within the normoalbuminuria range. Novel subclinical indicators may help to identify higher-risk patients. Urinary extracellular vesicles (uEVs) are sentinels of renal function non-invasively. Here we aimed to approach the early assessment of cardiorenal risk by investigating the protein cargo of uEVs. Hypertensive patients were classified in control group (C) with ACR<10mg/g, and high-normal group (HN) with ACR 10-30mg/g. Isolated uEVs were characterized by western blotting and electron microscopy and the protein cargo was analyzed by untargeted proteomics (LC-MS/MS) in a first discovery cohort. Protein confirmation was performed in a different cohort by ExoView. Immunohistochemistry of human kidney biopsies was also performed to evaluate the potential of uEVs to reflect renal damage. HN albuminuria does not affect the uEVs concentration, size, or tetraspanin profile. Among >6200 uEVs proteins identified, 43 define a panel significantly altered in HN patients without variation in urine, mostly annotated in the tubule (39 out of 43). The tubular transporter long-chain fatty acid transport protein 2 (SLC27A2) and the apical membrane protein amnionless (AMN) confirmed their alteration in HN patients evidencing impaired tubular reabsorption. SLC27A2 showed tubular expression and significantly reduced levels in patients with diagnostic criteria for CKD. Alterations in the EV-mediated molecular profile are evident before pathological ACR levels are reached. Direct quantitation of SLC27A2 and AMN in uEVs helps identifying normoalbuminuric subjects with higher cardiorenal risk in early monitoring of CKD.

Abstract MP33: A20 in the Kidney Epithelium Attenuates Angiotensin II-induced Hypertension by Constraining Renal Tubular NHE3 Expression

Whereas the ubiquitin editor A20 limits NF-κB-mediated signaling in myeloid leukocytes, the functions of A20 in renal epithelial cells during hypertension have not been described. We recently found that A20 in the kidney tubule ameliorates hypertension and cardiac hypertrophy induced by chronic angiotensin (Ang) II infusion (500ng/kg/min) in mice. Here we report that following 3 weeks of hypertension, the kidneys of mice with tubular deletion of A20 in the kidney epithelium (“A20 iKKO” = A20 flox/flox Pax8-rtTA TetO-Cre + ) have augmented protein expression of the proximal tubular sodium transporter NHE3 (1.5±0.1 vs. 1.0±0.1; p &lt;0.01) but similar levels of ENaC subunit expression in the collecting tubule ( p =NS for all 3 subunits) vs wild-type (WT) controls. Moreover, kidneys from A20 iKKO mice have upregulated mRNA levels for the T cell chemokine CCL5 (2.4±0.3 vs. 1.0±0.3; p &lt;0.01) and higher proportions of effector memory CD8 + T cells (51.2±2.3 vs. 39.3±2.9 % of CD8 + ; p =0.013) compared to WTs despite similar numbers of effector memory T cells in the spleen ( p =NS). To examine whether A20 in the kidney attenuates hypertension by constraining tubular NHE3 expression, we bred A20 iKKO mice with an NHE3 flox/flox line, thereby generating mice with double A20 and NHE3 deletion in the kidney tubule (“Dual KKO”) mice. At baseline, kidneys from Dual KKO mice showed robust reductions in mRNA for A20 (0.5±0.1 vs. 1.0±0.1; p =0.022) and NHE3 (0.2±0.05 vs. 1.0±0.15; p =0.002) compared to WT littermates. During the first ten days of Ang II infusion, mean arterial pressures (MAPs) in the Dual KKO mice were lower than the MAPs previously recorded in our A20 iKKO cohort (129±3 vs. 152±4 mmHg; p &lt;0.01). Moreover, during 3 weeks of Ang II infusion, Dual KKO and WT mice maintained similar MAPs (145±7 vs. 149±13 mmHg; p =0.78), and heart weight to body weight ratios (5.9±0.3 vs. 5.5±0.2 mg/g; p =0.37) in concomitant measurements. Thus, A20 in the kidney tubule blunts the chronic hypertensive response by curtailing NHE3 expression. Our data further suggest that A20 in the nephron can blunt renal inflammation without altering systemic T cell immunity. Stimulating the A20 signaling pathway in the kidney may thus attenuate NHE3-dependent hypertension without conferring attendant risks of global immunosuppression.

The influence of insulin and incretin-based therapies on renal tubular transport.

The tubular function of the kidney is very complex and is finely regulated by many factors. These include a variety of hormonal signaling pathways which are involved in the expression, activation and regulation of renal transporters responsible for the handling of electrolytes. Glucose-lowering drugs such as insulin and incretin-based therapies, exert a well-known renal protective role in diabetic kidney disease, mainly acting at the glomerular level. In the literature, several studies have described the effect of insulin and the incretin hormones on tubular transport. Most of these studies focused on the variations in excretion and clearance of sodium but did not extensively and systematically investigate the possible variations that these hormones may induce in the tubular regulation of all the other electrolytes, urea metabolism, acid-base balance and urinary pH. While insulin action on the kidney is very well-described, the renal tubular impact of incretin-based therapies is less consistent and the results available are scarce. To our knowledge, this is the first review summarizing the effects induced on renal tubules by insulin, glucagon-like peptide-1 (GLP-1) receptor agonists and serine protease dipeptidyl peptidase-4 (DPP4) inhibitors in both healthy and diabetic human subjects. This is significant because it highlights the existence of a renal-gut and pancreas axis which also has a direct tubular effect and enables a deeper understanding of renal physiology.

Modeling Metformin and Dapagliflozin Pharmacokinetics in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a complication of diabetes that affects circulating drug concentrations and elimination of drugs from the body. Multiple drugs may be prescribed for treatment of diabetes and co-morbidities, and CKD complicates the pharmacotherapy selection and dosing regimen. Characterizing variations in renal drug clearance using models requires large clinical datasets that are costly and time-consuming to collect. We propose a flexible approach to incorporate impaired renal clearance in pharmacokinetic (PK) models using descriptive statistics and secondary data with mechanistic models and PK first principles. Probability density functions were generated for various drug clearance mechanisms based on the degree of renal impairment and used to estimate the total clearance starting from glomerular filtration for metformin (MET) and dapagliflozin (DAPA). These estimates were integrated with PK models of MET and DAPA for simulations. MET renal clearance decreased proportionally with a reduction in estimated glomerular filtration rate (eGFR) and estimated net tubular transport rates. DAPA total clearance varied little with renal impairment and decreased proportionally to reported non-renal clearance rates. Net tubular transport rates were negative to partially account for low renal clearance compared with eGFR. The estimated clearance values and trends were consistent with MET and DAPA PK characteristics in the literature. Dose adjustment based on reduced clearance levels estimated correspondingly lower doses for MET and DAPA while maintaining desired dose exposure. Estimation of drug clearance rates using descriptive statistics and secondary data with mechanistic models and PK first principles improves modeling of CKD in diabetes and can guide treatment selection.

Sex-Specific Differences in Kidney Function and Blood Pressure Regulation.

Premenopausal women generally exhibit lower blood pressure and a lower prevalence of hypertension than men of the same age, but these differences reverse postmenopause due to estrogen withdrawal. Sexual dimorphism has been described in different components of kidney physiology and pathophysiology, including the renin-angiotensin-aldosterone system, endothelin system, and tubular transporters. This review explores the sex-specific differences in kidney function and blood pressure regulation. Understanding these differences provides insights into potential therapeutic targets for managing hypertension and kidney diseases, considering the patient's sex and hormonal status.

Identification of a substrate of the renal tubular transporters for detecting drug-induced early acute kidney injury.

Early identification of drug-induced acute kidney injury (AKI) is essential to prevent renal damage. The renal tubules are typically the first to exhibit damage, frequently accompanied by changes in renal tubular transporters. With this in mind, we have identified an endogenous substrate of the renal tubular transporters that may serve as a biomarker for early detection of drug-induced AKI. Using gentamicin- and vancomycin-induced AKI models, we found that traumatic acid (TA), an end metabolite, was rapidly increased in both AKI models. TA, a highly albumin-bound compound (96% to 100%), could not be filtered by the glomerulus and was predominantly eliminated by renal tubules via the OAT1, OAT3, OATP4C1, and P-gp transporters. Importantly, there is a correlation between elevated serum TA levels and reduced OAT1 and OAT3 levels. A clinical study showed that serum TA levels rose before an increase in serum creatinine in 13 out of 20 AKI patients in an intensive care unit setting. In addition, there was a notable rise in TA levels in the serum of individuals suffering from nephrotic syndrome, chronic renal failure, and acute renal failure. These results indicate that the decrease in renal tubular transporter expression during drug-induced AKI leads to an increase in the serum TA level, and the change in TA may serve as a monitor for renal tubular injury. Acute kidney injury (AKI) has a high clinical incidence, and if patients do not receive timely treatment and intervention, it can lead to severe consequences. During AKI, tubular damage is often the primary issue. Endogenous biomarkers of tubular damage are critical for the early diagnosis and treatment of AKI. However, there is currently a lack of reliable endogenous biomarkers for diagnosing tubular damage in clinical practice. Tubular secretion is primarily mediated by renal tubular transporters (channels), which are also impaired during tubular damage. Therefore, we aim to identify endogenous biomarkers of tubular damage from the perspective of renal tubular transporters, providing support for the early detection and intervention of AKI. TA is a substrate of multiple channels, including OAT1, OAT3, OATP4C1, and P-gp, and is primarily secreted by the renal tubules. In the early stages of rat AKI induced by GEN and VCA, serum TA levels are significantly elevated, occurring earlier than the rise in serum creatinine (SCr). Thus, TA is expected to become a potential endogenous biomarker for the early diagnosis of tubular damage.

Primary Distal Renal Tubular Acidosis: Toward an Optimal Correction of Metabolic Acidosis.

The term classic, type 1 renal tubular acidosis or primary distal renal tubular acidosis is used to designate patients with impaired ability to excrete acid normally in the urine as a result of tubular transport defects involving type A intercalated cells in the collecting duct. The clinical phenotype is largely characterized by the complications of chronic metabolic acidosis (MA): stunted growth, bone abnormalities, and nephrocalcinosis and nephrolithiasis that develop as the consequence of hypercalciuria and hypocitraturia. All these manifestations are preventable with early and sustained correction of MA with alkali therapy. The optimal target for plasma bicarbonate should be as close as possible to the range considered normal by current standards (between 23 and 28 mEq/L.). Most of the benefits of alkali therapy are tangible early in the course of the disease in childhood, but life-long treatment is required to prevent the vast array of complications attributable to chronic MA.

Bone mineral density assessment in patients with cystinuria.

Cystinuria is a rare genetic disease characterized by impaired tubular transport of cystine. Clinical features of cystinuria mainlyinclude nephrolithiasis and its complications, although cystinuric patients may present with other comorbidities. There are currently no data on bone features of patients with cystinuria. Our aim is to characterize bone mineral density (BMD) in cystinuria. Our study included adult cystinuric patients with estimated glomerular filtration rate (eGFR) ≥ 60mL/min/1.73m2 followed at 3 specialized outpatient clinics in Italy (Rome, Naples and Verona). Markers of bone turnover were analyzed in a centralized laboratory. Clinical, biochemical and dual-energy X-ray absorptiometry (DEXA) data were collected from September 2021 to December 2022. Linear regression models were used to evaluate statistically significant deviations from zero of Z-scores. Twenty-seven patients were included in the study. Mean (SD) age was 37 (15) years, 41% were women. Mean estimated glomerular filtration rate was 99mL/min/1.73m2. Serum parameters associated with bone turnover (parathyroid hormone, FGF23, calcium and phosphate) were all in the normal range, with only 4 patients showing mild hypophosphatemia. Prevalence of low bone mineral density, defined as Z-score ≤ - 2 at any site, was 15%. Average Z-scores were negative across most sites. Our study suggests that cystinuric patients have lower bone mineral density compared with individuals of the same sex and age, even when theirkidney functionis normal.

Proximal tubule hypertrophy and hyperfunction: a novel pathophysiological feature in disease states.

The role of proximal tubules (PTs), a major component of the renal tubular structure in the renal cortex, has been examined extensively. Along with its physiological role in the reabsorption of various molecules, including electrolytes, amino acids and monosaccharides, transcellular transport of different hormones and regulation of homeostasis, pathological events affecting PTs may underlie multiple disease states. PT hypertrophy or a hyperfunctioning state, despite being a compensatory mechanism at first in response to various stimuli or alterations at tubular transport proteins, have been shown to be critical pathophysiological events leading to multiple disorders, including diabetes mellitus, obesity, metabolic syndrome and congestive heart failure. Moreover, pharmacotherapeutic agents have primarily targeted PTs, including sodium-glucose cotransporter 2, urate transporters and carbonic anhydrase enzymes. In this narrative review, we focus on the physiological role of PTs in healthy states and the current understanding of the PT pathologies leading to disease states and potential therapeutic targets.

#2565 Urine amino acid profiles enlighten new prediction models for renal Fanconi syndrome and its association with eGFR level

Abstract Background and Aims Renal Fanconi syndrome (FS) presented dysfunctions of renal proximal tubular (PT) transport with or without excessive urine amino acids (AAs) excretion, which might contribute to the progression of eGFR decline. This study aimed to explore the clinical benefit of 21 urinary AAs excretion patterns in renal FS and eGFR decline. Method Liquid chromatography-tandem Mass spectrometry (LC-MS-MS) is currently quantitative and detects over 21 types of amino acids in 157 patients with renal proximal tubular dysfunctions. To evaluate the diagnostic values of urine AAs in renal FS, we established a machine learning algorithm to perform binary classification using Random Forest Regressor in Python (version 3.8.8) with a training set and a testing set (8:2). We used Randomized search to find out the best model hyperparameters, calculated feature importance for contribution evaluation, and Receiver Operating Characteristic (ROC) curves to evaluate the performance of classification algorithms. Results A model based on a machine learning algorithm of all urine AAs to diagnose renal FS established with sensitivity = 0.71, specificity = 0.73, and AUC = 0.83 (95% CI 0.648-0.957) (Fig. 1), with similar performance in combined six urine AAs (citrulline, asparagine, threonine, phenylalanine, ornithine, and proline) models. The urine proline level independently correlated with eGFR &amp;lt; 60 ml/min/1.73 m2 (bias regression coefficient 0.84, P = 0.001, OR = 2.31, 95% CI 1.41-3.80) when adjusted by age, etiology, gender, and blood urea nitrogen level. For the primary diseased diagnosis, the most susceptible urine AAs were citrulline (56.7%), proline (56.0%,) threonine (52.2%), and ornithine (45.9%) and the less susceptible ones were histidine (24.8%), aspartate (16.6%), and arginine (13.4%). Accordingly, the most common affected AA transporters were SLC6A19 (B0AT1) and etiology-specific urine AA atlas major in the eight urine AAs (isoleucine, glutamine, methionine, proline, alanine, ornithine, lysine, and arginine). Different from primary Sjӧgren syndrome patients (n = 26) with less susceptible, the patients with plasmacyte diseases (n = 8) showed elevations of almost all the urine AAs except glycine and aspartate. The connection between urine AAs excretion and hypouricemia showed gender differences, with 20 urine AAs significantly negatively correlating with serum uric acid levels in males but only five in females. Conclusion We first established a model based on the urine AA atlas detected by (LC-MS-MS) which contributed to differentiating the etiologies of renal tubular dysfunctions and associated well with eGFR. It shed light on the clinical practice in precision evaluation of tubule injury.

Calcineurin inhibitor effects on kidney electrolyte handling and blood pressure: tacrolimus versus voclosporin.

Calcineurin inhibitors affect kidney electrolyte handling and blood pressure through an effect on the distal tubule. The second generation calcineurin inhibitor voclosporin causes hypomagnesemia and hypercalciuria less often than tacrolimus. This suggests different effects on the distal tubule, but this has not yet been investigated experimentally. Rats were treated with voclosporin, tacrolimus or vehicle for 28 days. Dosing was based on a pilot experiment to achieve clinically therapeutic concentrations. Drug effects were assessed by electrolyte handling at day 18 and 28, thiazide testing at day 20, telemetric blood pressure recordings, and analysis of mRNA and protein levels of distal tubular transporters at day 28. Compared to vehicle, tacrolimus but not voclosporin significantly increased the fractional excretions of calcium (>4-fold), magnesium and chloride (both 1.5-fold) and caused hypomagnesemia. Tacrolimus but not voclosporin significantly reduced distal tubular transporters at mRNA and/or protein level, including the sodium-chloride cotransporter, transient receptor melastatin 6, transient receptor potential vanilloid 5, cyclin M2, sodium-calcium exchanger and calbindin-D28K. Tacrolimus but not voclosporin reduced the mRNA level and urinary excretion of epidermal growth factor. The saluretic response to hydrochlorothiazide at day 20 was similar in the voclosporin and vehicle groups, whereas it was lower in the tacrolimus group. The phosphorylated form of the sodium-chloride cotransporter was significantly higher at day 28 in rats treated with voclosporin than in those treated with tacrolimus. Tacrolimus transiently increased blood pressure, whereas voclosporin caused a gradual but persistent increase in blood pressure which was further characterized by high renin, normal aldosterone, and low endothelin-1. In contrast to tacrolimus, voclosporin does not cause hypercalciuria and hypomagnesemia, but similarly causes hypertension. Our data reveal differences between the distal tubular effects of tacrolimus and voclosporin and provide a pathophysiological basis for the clinically observed differences between the two calcineurin inhibitors.

Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive rats

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in hypertensive patients. However, the underlying molecular mechanisms remain largely unknown. Proximal tubule (PT) NHE3-mediated sodium reabsorption is inhibited by SGLT2i in normotensive rodents, yet no BP-lowering effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. Administration of EMPA for 14 days reduced BP in 12-week-old SHRs but not in age-matched Wistar rats. PT NHE3 was inhibited by EMPA treatment in both Wistar and SHRs. EMPA increased NCC expression and phosphorylation levels in Wistar but not SHRs. SHRs exhibited higher cortical and medullary NKCC2 and β-ENaC subunit mRNA and higher medullary α-ENaC expression levels than Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP was reduced in response to EMPA and HCTZ combined but not by EMPA or HCTZ alone. Altogether, these data suggest that upregulation of NCC counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, thereby maintaining their baseline BP. Moreover, reducing NHE3 activity without further upregulation of the main apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension. FAPESP 2016/22140-7 and 2021/14534-3. This is the full abstract presented at the American Physiology Summit 2024 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.

Locally produced nerve growth factor is a novel regulator of (juxta)glomerular and collecting duct functions in the kidney

The dense network of sympathetic efferent and sensory afferent nerves innervating the kidney is well recognized, however the elements and the functional importance of neurotrophin signaling in renal tissues are much less known. Peripherally innervated tissues are known to produce and release nerve growth factor (NGF) which belongs to the neurotrophin family of neuronal signaling proteins and is essential for the growth and survival of peripheral sensory and sympathetic nerves. This study aimed to characterize the expression and role of NGF in the kidney. We hypothesized that NGF is produced by multiple renal tubular and vascular cell types and helps to maintain hemodynamic and tubular transport functions. Local NGF expression was studied using histological sections of kidneys harvested from NGF-GFP reporter mice. To address the functional importance of NGF signaling, a new mouse model was generated by crossing Ren1d-Cre and NGF-floxed mice and Cre/lox-mediated knockout of NGF from cells of the renin lineage co-labeled with the multicolor reporter Confetti (Ren1d-Confetti-NGF KO). WT and NGF KO mice were placed in metabolic cages for urine collections and analysis, and systolic blood pressure (SBP) was measured with tail-cuff pletysmography. Intercalated cells of the collecting duct (CD) showed by far the highest NGF expression among all renal cell types based on the reporter signal in NGF-GFP mice, in addition to select cells in the renal interstitium including juxtaglomerular (JG) renin cells. Since both CD cells and JG cells are part of the renin lineage, the applied genetic strategy using Ren1d-Confetti-NGF KO mice was ideal, and showed diminished NGF expression compared to WT. Urine excretion rate and albuminuria were 3-fold increased in Ren1d-Confetti-NGF KO vs. WT mice, while there was no difference in SBP between WT and KO groups. Immunohistochemistry showed aberrant CD structure with multiple small, undifferentiated cells with apparently lost polarity and reduced AQP2 labeling in CD principal cells in Ren1d-Confetti-NGF KO mice compared to WT. In addition, the characteristic strong apical membrane AQP2 (in principal cells) and H+-ATPase (in intercalated cells) labeling in WT mice was diminished in Ren1d-Confetti-NGF KO mice. JG renin cell number per nephron was significantly reduced in Ren1d-Confetti-NGF KO (2.5±1.0) vs. WT mice (6.2±1.5, n=4 mice each). In summary, our results suggest high level of NGF expression in the adult kidney that plays important physiological functional roles in maintaining collecting duct water and electrolyte reabsorption and acid secretion, and intrarenal renin production. Deficient intrarenal NGF production may cause kidney disease, suggesting the importance of altered renal neurotrophin signaling in pathology development. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 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.

Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive male rat.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve invivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

How can inhibition of glucose and sodium transport in the early proximal tubule protect the cardiorenal system?

What mechanisms can link the inhibition of sodium-glucose cotransporter 2 (SGLT2) in the early proximal tubule to kidney and heart protection in patients with and without type 2 diabetes? Due to physical and functional coupling of SGLT2 to other sodium and metabolite transporters in the early proximal tubule (including NHE3, URAT1), inhibitors of SGLT2 (SGLT2i) reduce reabsorption not only of glucose, inducing osmotic diuresis, but of other metabolites plus of a larger amount of sodium than expected based on SGLT2 inhibition alone, thereby reducing volume retention, hypertension and hyperuricemia. Metabolic adaptations to SGLT2i include a fasting-like response, with enhanced lipolysis and formation of ketone bodies that serve as additional fuel for kidneys and heart. Making use of the physiology of tubulo-glomerular communication, SGLT2i functionally lower glomerular capillary pressure and filtration rate, thereby reducing physical stress on the glomerular filtration barrier, tubular exposure to albumin and nephrotoxic compounds, and the oxygen demand for reabsorbing the filtered load. Together with reduced gluco-toxicity in the early proximal tubule and better distribution of transport work along the nephron, SGLT2i can preserve tubular integrity and transport function and, thereby, glomerular filtration rate in the long-term. By shifting transport downstream, SGLT2i may simulate systemic hypoxia at the oxygen sensors in the deep cortex/outer medulla, which stimulates erythropoiesis and, together with osmotic diuresis, enhances hematocrit and thereby improves oxygen delivery to all organs. The described SGLT2-dependent effects may be complemented by off-target effects of SGLT2i on the heart itself and on the microbiome formation of cardiovascular-effective uremic toxins.