Rat Proximal Tubule Research Articles

Renal handling of albumin in rats with early stage diabetes: A theoretical analysis.

In early diabetic nephropathy (DN), recent studies have shown that albuminuria stems mostly from alterations in tubular function rather than from glomerular damage. Several factors in DN, including hyperfiltration, hypertrophy and reduced abundance of the albumin receptors megalin and cubilin, affect albumin endocytosis in the proximal tubule (PT). To assess their respective contribution, we developed a model of albumin handling in the rat PT that couples the transport of albumin to that of water and solutes. Our simulations suggest that, under basal conditions, ∼75% of albumin is retrieved in the S1segment. The model predicts negligible uptake in S3, as observed experimentally. It also accurately predicts the impact of acute hyperglycaemia on urinary albumin excretion. Simulations reproduce observed increases in albumin excretion in early DN by considering the combined effects of increased glomerular filtration rate (GFR), osmotic diuresis, hypertrophy, and megalin and cubilin downregulation, without stipulating changes in glomerular permselectivity. The results indicate that in isolation, glucose-elicited osmotic diuresis and glucose transporter upregulation raise albumin excretion only slightly. Enlargement of PT diameter not only augments uptake via surface area expansion, but also reduces fluid velocity and thus shear stress-induced stimulation of endocytosis. Overall, our model predicts that downregulation of megalin and cubilin and hyperfiltration both contribute significantly to increasing albumin excretion in rats with early-stage diabetes. The results also suggest that acute sodium-glucose cotransporter 2 inhibition lowers albumin excretion only if GFR decreases sufficiently, and that angiotensin II receptor blockers mitigate urinary albumin loss in early DN in large part by upregulating albumin receptor abundance. KEY POINTS: The urinary excretion of albumin is increased in early diabetic nephropathy (DN). It is difficult to experimentally disentangle the multiple factors that affect the renal handling of albumin in DN. We developed a mathematical model of albumin transport in the rat proximal tubule (PT) to examine the impact of elevated plasma glucose, hyperfiltration, PT hypertrophy and reduced abundance of albumin receptors on albumin uptake and excretion in DN. Our model predicts that glucose-elicited osmotic diuresis per se raises albumin excretion only slightly. Conversely, increases in PT diameter and length favour reduced albumin excretion. Our results suggest that downregulation of the receptors megalin and cubilin in PT cells and hyperfiltration both contribute significantly to increasing albumin excretion in DN. The model helps to better understand the mechanisms underlying urinary loss of albumin in early-stage diabetes, and the impact of specific treatments thereupon.

Sexually Dimorphic Gene Expression in Rat Proximal Tubules Suggests Activation of the Intrarenal Renin Angiotensin System and Elevated Transport Rates in Males

Introduction: Sexual dimorphism affects various organ systems, including renal and cardiovascular. The renin-angiotensin system (RAS) plays a pivotal role in regulating fluid homeostasis and blood pressure, with males being generally more sensitive to the hypertensive actions of angiotensin II (AngII). Proximal tubules (PT)s reabsorb 60-70% of filtered water, sodium, and bicarbonate and present sexually dimorphic biology. In addition, PTs are a key element of the intrarenal RAS and the production site of angiotensinogen ( Agt, AGT) and derived bioactive peptides. These peptides exert autocrine, as well as paracrine effects in distal portions of the nephron, regulating salt and water reabsorption. Hypothesis: We hypothesize that the increased sensitivity to the hypertensive actions of AngII in males is due to activation of the intrarenal RAS. Methods: We used 3 male and 3 female pools of whole kidney single-cell RNAseq transcriptomes to study gene expression differences across sexes in different segments of the nephron. Results: We identified sex-differentially expressed genes (DEGs) in all segments of the nephron, with the majority of DEG mapping to the PT. Analyzing all three PT segments together, we found 86 upregulated genes in females and 104 in males. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted pathways associated with glutathione biosynthesis and metabolism in females, while males presented greater expression of genes associated with the tricarboxylic acid cycle and mineral absorption, including the γ-subunit of the Na,K-ATPase ( Fxyd2). Differential expression of genes on each sub-segment of the PTs, namely S1, S2, and S3, showed the following number of upregulated genes: Females: 39, 37, and 73 in S1, S2, and S3, respectively; and Males: 67, 57, and 105 in S1, S2, and S3, respectively. This data indicates that S3 exhibits the largest sexually dimorphic transcriptomes. Pathway enrichment on S3 DEG in both males and females showed similar enrichment terms as in whole PTs. However, at this resolution, males also present upregulation of RAS genes, including Agt, and two proteases involved in the metabolism of angiotensin peptides: aminopeptidase A ( Enpep, APA) and prolyl endopeptidase ( Prep, PEP). APA and PEP can both cleave AngII to bioactive peptides AngIII and Ang(1-7), respectively. Conclusion: We conclude that the intrarenal RAS is upregulated in male PT.S3. The increase in AGT and proteases capable of metabolizing AngII suggest that this peptide may be elevated. Analyzing the expression of RAS genes alone does not allow us to infer a resultant effect on Na handling, as AngII, AngIII, and Ang(1-7) could exert both natriuretic and anti-natriuretic effects. However, enrichment terms associated with mineral transport and energy demands suggest active mineral transport prevails in males. Funding sources: This work was supported in part by CWRU BGT670107 and NIH-NIDDK DK128304 grants to AGV. 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.

Global Deletion of DPP4 Prevents the Development of Salt-sensitive Hypertension in Obese Mice via Regulation of Sodium and Water Excretion

Salt-sensitivity of hypertension (HTN) occurs in up to 50% of hypertensive patients and is more common in the older population, Blacks and the obesity/diabetes/hypertension subsets. The renin-angiotensin aldosterone system (RAAS), in particular Angiotensin II (Ang II) and aldosterone known to regulate the Na+/H+ exchanger isoform 3 (NHE3) and ENaC respectively, is thought to be activated in the obesity/diabetes/hypertension subsets and thereby plays an important role in salt-sensitive HTN. RAAS blockade is an acceptable treatment for salt-sensitive HTN along with the use of diuretics. However, not every patient can tolerate RAAS blockade. Recently, dipeptidyl peptidase 4 (DPP4) has been shown to bind to NHE3, colocalizes to the microvillar fractions with active NHE3 and DPP4 inhibition reduces sodium uptake into proximal tubule cells and isolated rat proximal tubules. In addition, DPP4 may act on more distal transporters. Furthermore, data from our lab has revealed that DPP4 knockout (DPP4KO) mice have a lower sodium balance both under conditions of high salt (1.23%) and low salt (0.02%) ingestion suggesting that DPP4 regulates sodium transport and/or balance under physiological conditions. However, whether DPP4 has any effects on salt-sensitive HTN and the underlying mechanisms remain unknown. Therefore, we hypothesized that the absence of DPP4 could mitigate the development of salt-sensitive HTN in an obesity model, by improving the natriuretic and diuretic response mediated by NHE3 and/or distal mechanisms. To this end, 6-8wk old DPP4KO and C57Bl/6 littermate wild-type (WT) mice were fed a control (CD) or Western diet (WD, high in refined fats and sugars) to induce obesity. Circulating (175%) and kidney (125%) DPP4 activity were both elevated in the obese WT mice compared to the CD-fed WT mice (100%) and DPP4KO mice had minimal DPP4 activity as expected. The lack of DPP4 prevented the increase in systolic blood pressure (~15 mmHg) in the obese animals, and the absence of the development of cardiac hypertrophy in this group corroborated the pressure effect. The development of salt-sensitive HTN in the obese WT mice was associated with a lower natriuretic and diuretic response assessed by the salt overload experiment, suggesting higher tubular reabsorption of sodium and water by the nephrons. In turn, the lack of DPP4 prevented the lowering of natriuretic and diuretic response in obese mice, indicating that the beneficial effect of the absence of DPP4 on the development of salt-sensitive HTN is closely related to effects on renal tubule transport. Importantly, a high salt diet increased sodium balance and blood pressure in the obese WT mice and DPP4 deletion mitigated this increase. Furthermore, on low salt diet, the absence of DPP4 led to “salt wasting” in the obese mice similar to the CD-fed mice. Surprisingly, gene and protein expression measurements showed involvement of distal tubule sodium transporters and aquaporins and not NHE3. In contrast, assessment of the DPP4 interactome in obese mice showed binding to Myosin 6 and APN, which may regulate proximal tubule transporters and hypertension. Transdermal measurements showed no changes in the glomerular filtration rate. In conclusion, deletion of DPP4 protects against the development of salt-sensitive hypertension in obese mice by improving the natriuretic and diuretic response likely via regulation of sodium and water transporters. FAPESP, Brazil NIH K08DK115886, USA. 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.

Abstract 044: Knocking Out Sodium Glucose-Linked Transporter 5 Prevents Fructose-Induced Salt-Sensitive Hypertension

We showed: 1) a 20% fructose high-salt (3.7% NaCl; FHS) but not a 20% glucose high-salt diet increases systolic blood pressure (SBP) in a sex independent manner; 2) on normal salt, 20% fructose augments the ability of angiotensin II (Ang-II) to increase proximal tubule superoxide (O 2 - ); and 3) rat proximal tubule brush borders express sodium glucose-linked transporters (SGLT) 4 and SGLT5, which transport fructose. However, whether FHS enhances Ang II-stimulated proximal tubule O 2 - , its role in FISSH and the roles of SGLT4 and 5 are unknown. We hypothesized that fructose transport mediated by SGLT5 but not SGLT4 is required for FHS to enhance Ang II-stimulated proximal tubule O 2 - in males and females, and this is required for fructose-induced salt-sensitive hypertension (FISSH). We measured SBP in wild-type, SGLT4 knockout, and SGLT5 knockout male and female rats on a Sprague Dawley background fed FHS for 7 days. We also measured basal and Ang II-stimulated (37 nM) O 2 - in proximal tubule suspensions from these rats using the lucigenin assay. FHS increased SBP in wild type male rats 11 ± 2 mmHg (from 131 ± 4 to 142 ± 5 mmHg; n = 5, p < 0.01) and in female rats by 21 ± 5 mmHg (from 114 ± 4 to 130 ± 3 mmHg; n = 8; p < 0.01), not significantly different from males. FHS increased SBP in SGLT4 males by 22 ± 3 mmHg (from 126 ± 5 to 149 ± 6 mmHg; n = 8, p < 0.01) and in females 21 ± 5 mmHg (from 126 ± 3 to 138 ± 2 mmHg; n = 8; p < 0.01), not significantly different from males. In SGLT5 males SBP was 128 ± 6 on day 0 and 129 ± 8 mmHg on day 7 (n = 7). In SGLT5 females SBP was 114 ± 4 on day 0 and 117 ± 5 mmHg on day 7 (n = 7). Ang-II increased O 2 - by 11 ± 3 relative light units (RLU)/μg/s from 53 ± 9 to 64 ± 8 RLU/μg/s (n = 11, p < 0.01) in tubules from wild type males fed FHS for 7 days and by 10±3 RLU (n = 5; p < 0.02) in females fed FHS for 7 days. Ang-II increased O 2 - by 8 ± 2 RLU/μg/s in proximal tubules from FHS-fed SGLT4 males (n = 6, p < 0.02) and by 8 ± 2 in FHS-fed females (n = 6, p < 0.01). Ang-II did not significantly stimulate O 2 - in proximal tubules from FHS-fed SGTL5 male or female rats (Δ 3±7 and 0±3 RLU/μg/s, n = 5 and 3, respectively). We conclude that transport by SGLT 5 but not SGLT4 is required for FHS to augment Ang II-stimulated proximal tubule O 2 - in males and females, and that the increase in proximal tubule oxidative stress is required for FISSH.

Single-cell transcriptional phenotypes linked to anatomical localization of fructose transporters in rat proximal tubule segments

Kidneys of healthy individuals filter 4 to 25 g of fructose (FRU) a day, equivalent to ~10% of the filtered glucose. FRU reabsorbed by proximal tubules (PTs) is mostly used in gluconeogenesis. Elevated dietary FRU alters hormonal signaling in PTs and increases oxidative stress, which overtime, leads to salt-sensitive hypertension, tubulointerstitial injury, and glomerular damage. Four apical transporters could transport FRU at physiological concentrations: SGLT5 (km0.62 mM), NAGLT1 (km4.5 mM), GLUT5 (km12.6 mM) and SGLT4 (undetermined km). We hypothesize that single-cell (sc) transcriptional phenotypes correspond to anatomical features and could inform FRU transport in PTs. We used scRNAseq rat kidney transcriptomes (3 males & 3 females) to measure the expression of FRU transporters in cell clusters corresponding to the S1, S2, and S3 PT segments. We integrated cell transcriptional phenotypes with spatial and structural features using quantitative proteomics and transcriptomics from microdissected PT segments. Clusters S1, S2, and S3 had 1281, 2123 and 1441 cells, respectively. More than 90% of cells in all clusters expressed KHK, the first enzyme in FRU metabolism (expression: S1<S2<S3), showing high correlation with segment-proteomics (PCC (Pearson correlation coefficient) = 0.87) and transcriptomics (PCC = 0.65). In contrast, there were striking imbalances in transporters’ distributions. High-affinity SGLT5 was expressed by 3%, 24%, 75% cells in clusters S1, S2, and S3, with 4, 43, 230 aggregated counts per 100 cells (nCounts), respectively. SGLT5 showed a log2FC > 0.8 in S3 as compared to S1-S2 (p-adj < 1x10-300). SGLT5, PCC was 0.95 for quantitative proteomics and 0.48 for sequencing. Low-affinity NAGLT1 was expressed by 70%, 81%, 47% cells in clusters S1, S2, and S3, with 206, 306, 121 nCounts, respectively. These values were highly correlated with RNAseq in microdissected tubules (PCC = 0.99), but NAGLT1 was absent in the proteomics dataset. GLUT5 was expressed by 9%, 10%, 38% cells in clusters S1, S2 and S3, with 11, 11, 63 nCounts, respectively, and PCC > 0.95 for both quantitative proteomics and sequencing. Finally, SGLT4 was expressed by 4%, 6%, 8% cell in clusters S1, S2 and S3, with 5, 6, 8 nCounts, respectively. Due to the low signal in microdissected segments, we could not conduct a correlation analysis for SGLT4. Our data shows that SGLT5 is most abundant in the straight portion of PTs, particularly S3. GLUT5 and SGLT4 follow a similar distribution. In contrast, low-affinity NAGLT1 is expressed in all segments, most predominantly S1. However, as NAGLT1 affinity for glucose (km3.7 mM) is near that for FRU, it likely reabsorbs glucose in early PT. In the straight portion FRU will be predominantly reabsorbed by the most abundant higher-affinity SGLT5. Our analysis shows a high correlation between scRNAseq clusters and spatial proteomics data and highlights the relevance of SGLT5 as the main FRU transporter in PTs BGT670107 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.

Ovarian Hormone‐Dependent miR‐146b‐5p Regulation of Plasminogen Activator Inhibitor‐1 in Renal Epithelial Cells

Chronic kidney disease (CKD) progression differs in males and females and the influence of gonadal hormones on pathology are incompletely understood. We utilized the 5/6 nephrectomy (5/6Nx) rat model, which recapitulates hallmarks of human CKD, to study differences in regulation of CKD pathology between sexes. We previously found that genomic deletion of microRNA 146b‐5p (miR‐146b) results in a sex‐dependent phenotype in 5/6Nx rats. Male miR‐146b‐/‐ rats develop renal pathology similar to their WT counterparts. However, female miR‐146b‐/‐ rats experience markedly exacerbated renal pathology relative to WT, 5/6Nx, females as well as WT and miR‐146b‐/‐, male, 5/6Nx rats. These sex differences are abolished by ovary removal, suggesting that renoprotective effects of female gonadal hormones are miR‐146b dependent. Our objective is to identify a molecular mechanism behind these effects. PAI‐1 has previously been shown to be regulated by miR‐146b‐5p isomiR, miR‐146a‐5p, which shares a common seed sequence. PAI‐1 expression is also known to be ovarian hormone dependent, its renal expression is low in health, but elevated in many disease settings, where it may contribute to remodeling. We hypothesized that PAI‐1 expression in rat proximal tubular epithelial cells (NRK‐52E) will be dependent upon (1) the presence of miR‐146b, (2) ovarian hormones, and (3) transforming growth factor beta (TGFβ), which is elevated in our 5/6Nx rats and known to contribute to renal pathology. To aid in testing our hypothesis we used CRISPR‐Cas9 technology to generate miR‐146b knockout rat proximal tubule epithelial (NRK‐52E) cells. Results. PAI‐1 mRNA was highly elevated in untreated miR‐146b KO rat NRK‐52E cells relative to WT NRK‐52E cell controls (11.25 ± 5.3 vs. 1.00 ± 0.68, P<0.05). Conversely, WT NRK‐52E cells treated with pre‐miR‐146b had significantly reduced PAI‐1 mRNA expression (0.137 ± 0.14 vs. 1.00 ± 0.45, P<0.05). Treatment of WT NRK‐52E cells with β‐estradiol did not significantly alter PAI‐1 mRNA expression relative to untreated controls, whereas treatment with progesterone (P4) significantly increased it. Treatment of WT NRK‐52E cells with TGFβ resulted in a significant increase of PAI‐1 mRNA expression. Lastly, treatment of WT NRK‐52E cells with P4 and TGFβ combined resulted in a significant increase in PAI‐1 mRNA expression, and this expression was significantly reduced by transfection with pre‐miR‐146b. Collectively, these observations demonstrate that miR‐146b, P4, and TGFβ regulate PAI‐1 expression in NRK‐52E cells and thereby suggest PAI‐1 as a mechanistic candidate behind the sex‐specific effects of miR‐146b in CKD.

Evaluation of orellanine-induced toxicity from the mushroom Cortinarius orellanus and the antagonistic effect of Petroselinum crispum

Mushroom poisoning is a worldwide public health problem that may cause serious toxic consequences on renal functions. The study aimed to evaluate the acute toxicity (24 h) of orellanine (OR) from Cortinarius orellanus in rat kidney and the ameliorative effect of parsley ethanolic extract. Twelve adult male Wistar rats were used to determine intraperitoneal (ip) median lethal dose (LD50) of OR, and 32 rats were divided into 4 groups (n = 8): OR group had 500 mg OR per kg bwt; OR + parsley group had the same dose of OR and after 1 h had 500 mg/kg parsley orally; parsley group had parsley only; and control had the vehicle 0.1% DMSO. Blood and kidney samples were collected at Hour 48. The LD50 dose was 1430 mg/kg for an observation period of 24 h. There were significant reductions (p < 0.01) in the body weight, and relative kidney weight of intoxicated rats compared to parsley treated rats and to controls. Similarly, this group had significantly higher levels of creatinine (p < 0.001), uric acid and urea (p < 0.05). The antioxidant glutathione peroxidase activity was significantly reduced (p < 0.01), while Cystatin C serum levels were significantly higher (p < 0.001) in the intoxicated untreated rats compared to all groups. Histopathological examination indicated necrotic damage in glomeruli and proximal tubules of rats given OR, which was relieved by parsley extract. Overall, the study showed that parsley extract ameliorated OR-induced kidney toxicity. This could be utilized in future research on adjunct therapy for toxicity-induced renal injury.

Potential and Limits of Kidney Cells for Evaluation of Renal Excretion.

A large number of therapeutic drugs, herbal components and their metabolites are excreted by the kidneys. Therefore, generally applied models for estimating renal excretion, including freshly isolated rat proximal tubule cells, cultured tubule cells and immortalized kidney cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, were investigated regarding their predictive potential for active renal transport. Cultured proximal tubule cells showed an epithelial cell-like morphology and formed tight monolayers. However, mRNA expression analyses and immunohistochemical studies revealed patterns of tight junction proteins that were notably different from freshly isolated cells and distinct from those in vivo. High levels of mannitol permeation were found in NRK-52E, IHKE-1 and Caki-1 cells, suggesting that they are not suitable for bidirectional transport studies. Cultured cells and freshly isolated cells also differed in proximal tubule markers and transport proteins, indicating that cultured primary cells were in a state of dedifferentiation. Cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 did not accurately reflect the characteristics of proximal tubules. The expression patterns of marker and transport proteins differed from freshly isolated primary cells. In summary, each of these models has profound disadvantages to consider when adopting them reliable models for the in vivo situation. Thus, they should not be used alone but only in combination.

A well-developed endolysosomal system reflects protein reabsorption in segment 1 and 2 of rat proximal tubules

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and β2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress.

Abstract P129: Angiotensin II Does Not Directly Stimulate The TLR4-MD2-MyD88 Inflammatory Pathway

Current evidence suggests that the deleterious actions of Angiotensin II (Ang II) reflect activation of both innate and adaptive inflammatory pathways. Indeed, recent studies find that Ang II stimulates the TLR-4 signaling cascade to promote cytokine release and renal injury by directly binding to the TLR4 accessory protein MD2 to induce TLR-4 dimerization and internalization that is independent of the AT 1 receptor (AT 1 R). Moreover, knockdown of TLR4 or MD2 attenuates renal injury and expression of pro-inflammatory cytokines in a chronic Ang II infusion model and in rat proximal tubule NRK-52e cells treated with Ang II. In the present study, we evaluated the proposed Ang II-TLR4-MD2 pathway in the NRK-52e cells regarding the stimulation of the chemokine MCP-1 (CCL2) and activation of the TLR4-MyD88 complex. NRK-52e cells, maintained in antibiotic-free media, were transferred to serum-free media prior to stimulation with the TLR4 agonists LPS and palmitate or Ang II for 24 hrs; reported data are the means ± SEM, n=4. The NRK-52e cells were sensitive to a low dose of LPS (1 ng/ml) stimulating CCL2 release 20-fold [Basal: 24.3 ± 1.0 pg/ml vs. 504 ± 30.4 pg/ml, p&lt;0.001]. The LPS induced release of CCL2 was abolished by the specific TLR4 inhibitor Tak-242 [TAK: 23.9 ± 1.3 pg/ml] and was significantly reduced by the MD2 inhibitor L48H37 [135 ± 24.2 pg/ml]. Palmitate [100 μM] also stimulated CCL2 release [482 ± 21.0 pg/ml; p&lt;0.001] that was abolished by TAK [32.3 ± 6.3 pg/ml]. The overall effects are consistent with previously reported responses to LPS and palmitate in these cells, as well as TLR4 protein expression and TAK inhibition. However, Ang II treatment over a dose range of 0.1 to 10 μM for 24 hrs failed to elicit CCL2 release [24.3 ± 1.0, 22.6 ± 0.5, and 27.6 ± 2.3 pg/ml, respectively; P&gt;0.1 vs. Basal]. Ang II [1 μM] also failed to augment the CCL2 response to LPS [496 ± 6.5 pg/ml] or palmitate [511 ± 35.0 pg/ml]. Finally, pre-treatment of the NRK-52e cells with the AT 1 R antagonist candesartan [5 μM] failed to attenuate CCL2 release to LPS [512 ± 21 pg/ml]. We conclude that Ang II does not directly stimulate the TLR4-MD2 complex to induce CCL2 and that the inflammatory events associated with Ang II in vivo may reflect the release of other factors (LPS, AGEs, HMGB1) that serve as TLR-4 agonists.

Effect of URAT1 inhibition with verinurad on proximal tubule intracellular lactate: A mathematical modeling analysis and hypothesis for antiproteinuric effect

Verinurad (RDEA3170) lowers serum uric acid by inhibiting URAT1, a urate/carboxylate co‐transporter in the proximal tubule (PT) of the kidney. In a recent clinical trial, verinurad also reduced proteinuria, but the mechanism remains unknown. One proposed hypothesis is that the inhibition of URAT1 augments protein reabsorption by increasing intracellular lactate in proximal tubule cells. The increased availability of lactate may lead to a restoration of the reabsorption of protein by increasing ATP levels and/ or improving endocytosis of protein by lowering endosomal pH. The goal of this study was to mathematically evaluate the effect of URAT1 inhibition with verinurad on intracellular lactate concentration.A mathematical model of urate and lactate transport in the rat proximal tubule was adapted to simulate transport in humans. The transporter model was further adjusted to represent states of hyperuricemia and altered expression of uric acid transporters that likely occur in hyperuricemia. The transporter model was linked to a systems description of plasma uric acid and the pharmacokinetics and pharmacodynamics of verinurad were incorporated into the model.The model was able to reproduce observed verinurad pharmacokinetics, as well as the observed changes in plasma and urinary uric acid. The model predicted that URAT1 inhibition with verinurad increases intracellular lactate concentration in the human PT by 26% in normouricemic and 20% in hyperuricemic conditions.Model simulations indicate that verinurad may substantially increase the availability of lactate inside PT epithelial cells. Since lactate is used as a fuel by the PT, increased availability of lactate may potentially help restore protein reabsorption capacity in fuel‐deficient PT cells by increasing ATP levels. In addition, increased intracellular lactate could help maintain the low endosomal pH necessary for protein reabsorption. This hypothesis remains to be experimentally proven; further non‐clinical/clinical data would be valuable to support model predictions.Urate and lactate proximal tubule transport model.Figure 1

Urolithin A, a Product of the Microbiome, Attenuates the Palmitate‐TLR4 Inflammatory Pathway in Renal Tubule Cells

Obesity is a major risk factor for cardiovascular disease and chronic renal injury. Tubulointerstial inflammation may reflect increased exposure of saturated fatty acids (sFA) to the renal epithelium and activation of the toll‐like receptor 4 (TLR4). Obesity‐induced inflammation may also facilitate gut microbiome dysbiosis and potentially alter the processing of exogenous polyphenolic compounds to metabolites with enhanced protective effects. Indeed, Urolithin A, the primary product of the microbiome metabolism of ellagitanins, promotes gut barrier function and attenuates colonic inflammation. However, the role of Urolithin A to attenuate tubular injury and inflammation has not been assessed. We propose that Urolithin A has anti‐inflammatory actions in rat proximal tubule cells (PTCs) chronically exposed to the sFA palmitate.PTCs (NRK‐52e cells) were exposed to palmitate [200 μM] for a 24 hours in the presence or absence of Urolithin A [50 μM], the TLR4 inhibitor TAK242 [1 μM], the EGFR inhibitor AG1478 [1 μM] and cSRC kinase inhibitor Dasatinib [10 μM] as both EGFR and cSRC may be integral to TLR4 activation. Cytokine expression in the PTCs was quantified by separate ELISAs.Palmitate exposure stimulated the robust release of PAI‐1 6‐fold [4.3 ± 0.3 vs. 24.2 ± 0.4 ng/ml; P&lt;0.001, n=3], CCL2 150‐fold [0.04 ± 0.01 vs. 6.2 ± 0.4 ng/ml; P&lt;0.001, n=3] and CCL5 10‐fold [23.4 ± 0.1 vs. 255 ± 11 pg/ml; P&lt;0.001, n=3]. The TLR4 inhibitor abolished the increase in CCL2 and CCL5, and markedly attenuated PAI‐1 release [70%, P&lt;0.01]. The EGFR inhibitor partially inhibited all three cytokines [40–50%, P&lt;0.01] while the cSRC inhibitor abolished PAI‐1 and partially inhibited CCL5, but failed to inhibit CCL2 release. Urolithin A essentially abolished the increase in PAI‐1 and CCL5, and markedly inhibited CCL2 release to palmitate [70%, P&lt;0.01].We conclude that Urolithin A may be renoprotective in obesity‐induced injury by targeting activation of TLR4 within the renal epithelium to attenuate the cytokine release. Moreover, a deficit in the microbiome‐dependent generation of Urolithin A may potentially promote renal inflammation.Support or Funding InformationChronic Disease Research Fund, AHA Transformative Grant 18TPA34170522; NIH R01HL146818, the Farley Hudson Foundation, the Hypertension &amp; Vascular Research Center

Transcriptional Regulation of Transforming Growth Factor β1 by Glucose: Investigation into the Role of the Hexosamine Biosynthesis Pathway

The hexosamine biosynthesis pathway (HBP) is hypothesized to mediate many of the adverse effects of hyperglycemia. We have shown previously that increased flux through this pathway leads to induction of the growth factor transforming growth factor-α (TGF-α) and to insulin resistance in cultured cells and transgenic mice. TGF-β is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore hypothesized that the HBP was involved in the regulation of TGF-β by glucose in rat vascular and kidney cells. A plasmid containing the promoter region of TGF-β1 cloned upstream of the firefly luciferase gene was electroporated into rat aortic smooth muscle, mesangial, and proximal tubule cells. Luciferase activity was measured in cellular extracts from cells cultured in varying concentrations of glucose and glucosamine. Glucose treatment of all cultured cells led to a time- and dose-dependent stimulation in TGF-β1 transcriptional activity, with high (20 mM) glucose causing a 1.4- to 2.0-fold increase. Glucose stimulation did not occur until after 12 hours and disappeared after 72 hours of treatment. Glucosamine was more potent than glucose, with 3 mM stimulating up to a 4-fold increase in TGFβ1-transcriptional activity. The stimulatory effect of glucosamine was also dose-dependent but was slower to develop and longer lasting than that of glucose. The metabolism of glucose through the HBP mediates extracellular matrix production, possibly via the stimulation of TGF-β in kidney cells. Hexosamine metabolism therefore, may play a role in the development of diabetic nephropathy.

A model of mitochondrial O2 consumption and ATP generation in rat proximal tubule cells.

Oxygen tension in the kidney is mostly determined by O2 consumption (Qo2), which is, in turn, closely linked to tubular Na+ reabsorption. The objective of the present study was to develop a model of mitochondrial function in the proximal tubule (PT) cells of the rat renal cortex to gain more insight into the coupling between Qo2, ATP formation (GATP), ATP hydrolysis (QATP), and Na+ transport in the PT. The present model correctly predicts in vitro and in vivo measurements of Qo2, GATP, and ATP and Pi concentrations in PT cells. Our simulations suggest that O2 levels are not rate limiting in the proximal convoluted tubule, absent large metabolic perturbations. The model predicts that the rate of ATP hydrolysis and cytoplasmic pH each substantially regulate the GATP-to-Qo2 ratio, a key determinant of the number of Na+ moles actively reabsorbed per mole of O2 consumed. An isolated increase in QATP or in cytoplasmic pH raises the GATP-to-Qo2 ratio. Thus, variations in Na+ reabsorption and pH along the PT may, per se, generate axial heterogeneities in the efficiency of mitochondrial metabolism and Na+ transport. Our results also indicate that the GATP-to-Qo2 ratio is strongly impacted not only by H+ leak permeability, which reflects mitochondrial uncoupling, but also by K+ leak pathways. Simulations suggest that the negative impact of increased uncoupling in the diabetic kidney on mitochondrial metabolic efficiency is partly counterbalanced by increased rates of Na+ transport and ATP consumption. This model provides a framework to investigate the role of mitochondrial dysfunction in acute and chronic renal diseases.

Insulin promotes sodium transport but suppresses gluconeogenesis via distinct cellular pathways in human and rat renal proximal tubules

Insulin is known to promote sodium transport and regulate gluconeogenesis in renal proximal tubules. Although protein kinase B (also known as Akt) and mammalian target of rapamycin complexes (mTORC) have been established as key regulators in the insulin signaling pathway, their roles in proximal tubules are poorly understood. To help define this, we examined the components of insulin signaling in sodium transport and gluconeogenesis in isolated human and rat proximal tubules, and also investigated the role of insulin in sodium handling and mTORC1 in insulin signaling invivo. In isolated human and rat proximal tubules, Akt and mTORC1/2 inhibition suppressed insulin-stimulated sodium-bicarbonate co-transporter 1 (NBCe1) activity, whereas mTORC1 inhibition had no effect. Akt2 and mTORC2 gene silencing largely inhibited insulin-stimulated NBCe1 activity, whereas silencing of Akt1 and mTORC1 had no effect. Furthermore, insulin decreased sodium excretion, and this effect depended on phosphoinositide 3 kinase invivo. Moreover, insulin reduced glucose production in rat proximal tubules and the expression of gluconeogenic genes in human and rat proximal tubules. Akt and mTORC1 inhibition largely abolished the observed insulin-mediated inhibitory effects. Gene silencing of insulin receptor substrate 1 (IRS1), Akt2, mTORC1, and mTORC2 also abolished insulin-mediated inhibition of gluconeogenesis. Additionally, invivo, mTORC1 inhibition abolished insulin-mediated inhibitory effects in rat proximal tubules, although not in liver. These results indicate that insulin-stimulated proximal tubule sodium transport is mediated via the Akt2/mTORC2 pathway, whereas insulin-suppressed proximal tubule gluconeogenesis is mediated via the IRS1/Akt2/mTORC1/2 pathway. Thus, distinct pathways may play important roles in hypertension and hyperglycemia in metabolic syndrome and diabetes.

Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule.

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-β2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.

A model of uric acid transport in the rat proximal tubule.

The objective of the present study was to theoretically investigate the mechanisms underlying uric acid transport in the proximal tubule (PT) of rat kidneys, and their modulation by factors, including Na+, parathyroid hormone, ANG II, and Na+-glucose cotransporter-2 inhibitors. To that end, we incorporated the transport of uric acid and its conjugate anion urate in our mathematical model of water and solute transport in the rat PT. The model accounts for parallel urate reabsorption and secretion pathways on apical and basolateral membranes and their coupling to lactate and α-ketoglutarate transport. Model results agree with experimental findings at the segment level. Net reabsorption of urate by the rat PT is predicted to be ~70% of the filtered load, with a rate of urate removal from the lumen that is 50% higher than the rate of urate secretion. The model suggests that apical URAT1 deletion significantly reduces net urate reabsorption across the PT, whereas ATP-binding cassette subfamily G member 2 dysfunction affects it only slightly. Inactivation of basolateral glucose transporter-9 raises fractional urate excretion above 100%, as observed in patients with renal familial hypouricemia. Furthermore, our results suggest that reducing Na+ reabsorption across Na+/H+ exchangers or Na+-glucose cotransporters augments net urate reabsorption. The model predicts that parathyroid hormone reduces urate excretion, whereas ANG II increases it. In conclusion, we have developed the first model of uric acid transport in the rat PT; this model provides a framework to gain greater insight into the numerous solutes and coupling mechanisms that affect the renal handing of uric acid.

Fructose reabsorption by rat proximal tubules: role of Na+-linked cotransporters and the effect of dietary fructose.

Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructose in the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructose by proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructose in Controls was 14.1 ± 1.5 pmol·mm-1·min-1. SGLT inhibition with phlorizin reduced Jfructose to 4.9 ± 1.4 pmol·mm-1·min-1 ( P < 0.008), whereas removal of Na+ diminished Jfructose by 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructose from 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm-1·min-1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructose by proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.

Cloning, function, and localization of human, canine, and Drosophila ZIP10 (SLC39A10), a Zn2+ transporter.

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

Signaling pathways involved in the rapid biphasic effect of aldosterone on Na+/H+ exchanger in rat proximal tubule cells

The receptors and signaling pathways for nongenomic effects of aldosterone (Aldo) on the proximal Na+/H+ exchanger are still unknown; therefore, the aim of this study was to investigate the mineralocorticoid receptor (MR) and/or glucocorticoid receptor (GR) participation in rapid Aldo effects on NHE1 (basolateral Na+/H+ exchanger isoform) and cytosolic calcium concentration ([Ca2+]i). In addition, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase kinase (MEK) involvement in signaling pathways of such effects was evaluated, using immortalized proximal tubule cells of rat (IRPTC) as an experimental model. MR and GR expression was investigated using reverse transcription polymerase chain reaction and immunoblotting. The intracellular pH recovery rate (after acid loading) and [Ca2+]i were determined by the probes BCECF-AM and FURA 2-AM, respectively. Aldo (10−12 M) promoted a moderate increase in [Ca2+]i and stimulation of NHE1, whereas Aldo (10−6 M) greatly increased the [Ca2+]i, but inhibited the NHE1. BAPTA-AM (a calcium chelator), GR antagonism and inhibition of PLC, PKC and MEK pathway abolished the biphasic and dose-dependent effect of Aldo on NHE1 and decreased the [Ca2+]i; whereas MR do not appear to participate in this rapid signaling in IRPTC cells. The reduction of GR content, by gene silencing, abolished the Aldo effect on NHE1, in low concentration, confirming the importance of this receptor in the rapid modulation of proximal sodium and hydrogen transports.