Nephron Segments Research Articles

Familial Hyperkalemic Hypertension.

The rare disease Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the genes encoding Cullin 3 (CUL3), Kelch-Like 3 (KLHL3), and two members of the With-No-Lysine [K] (WNK) kinase family, WNK1 and WNK4. In the kidney, these mutations ultimately cause hyperactivation of NCC along the renal distal convoluted tubule. Hypertension results from increased NaCl retention, and hyperkalemia by impaired K + secretion by downstream nephron segments. CUL3 and KLHL3 are now known to form a ubiquitin ligase complex that promotes proteasomal degradation of WNK kinases, which activate downstream kinases that phosphorylate and thus activate NCC. For CUL3, potent effects on the vasculature that contribute to the more severe hypertensive phenotype have also been identified. Here we outline the in vitro and in vivo studies that led to the discovery of the molecular pathways regulating NCC and vascular tone, and how FHHt-causing mutations disrupt these pathways. Potential mechanisms for variability in disease severity related to differential effects of each mutation on the kidney and vasculature are described, and other possible effects of the mutant proteins beyond the kidney and vasculature are explored. © 2024 American Physiological Society. Compr Physiol 14:5839-5874, 2024.

Integrins in the kidney - beyond the matrix.

The development and proper functioning of the kidney is dependent on the interaction of kidney cells with the surrounding extracellular matrix (ECM). These interactions are mediated by heterodimeric membrane-bound receptors called integrins, which bind to the ECM via their extracellular domain and via their cytoplasmic tail to intracellular adaptor proteins, to assemble large macromolecular adhesion complexes. These interactions enable integrins to control cellular functions such as intracellular signalling and organization of the actin cytoskeleton and are therefore crucial to organ function. The different nephron segments and the collecting duct system have unique morphologies, functions and ECM environments and are thus equipped with unique sets of integrins with distinct specificities for the ECM with which they interact. These cell-type-specific functions are facilitated by specific intracellular integrin binding proteins, which are critical in determining the integrin activation status, ligand-binding affinity and the type of ECM signals that are relayed to the intracellular structures. The spatiotemporal expression of integrins and their specific interactions with binding partners underlie the proper development, function and repair processes of the kidney. This Review summarizes our current understanding of how integrins, their binding partners and the actin cytoskeleton regulate kidney development, physiology and pathology.

Immunohistochemical Localization and Expression of Aquaporin 3-2 (AQP3C1) in the Spiny Dogfish, Squalus acanthias

The previously available coding region for the spiny dogfish (Squalus acanthias) AQP3-2 gene was amplified from cDNAs using PCR. Agarose gel electrophoresis gave a band of the AQP3-2 coding region, as well as multiple smaller splice variant bands. The main AQP3-2 band and the largest and most fluorescently intense pair of these splice variant bands were cloned and sequenced. Amplifications were performed on a range of tissue cDNAs, but AQP3-2 was only expressed in the kidney and brain. Quantitative PCR amplifications using pre-existing kidney cDNA from an environmental salinity acclimation experiment showed that the abundance of mRNA from both the main AQP3-2 transcript and the largest splice variant (Splice Variant 1) was lower in 120% seawater (SW) acclimated fish, although only the values for Splice Variant 1 were statistically significant. A custom-made affinity-purified rabbit polyclonal AQP3-2 antibody was produced, and this gave four bands of around the correct sizes (which were 27 and 32 kDa) for the complete AQP3-2 and Splice Variant 1 proteins. Two of the bands may have been N-glycosylated forms of these proteins. Other bands were also present on the Western blot. No bands were present when the antibody was pre-blocked by the peptide antigen. In tissue sections of the dogfish kidney, immunohistochemical localization experiments showed that AQP3-2 was expressed in the early distal tubule (EDT) and late distal tubule (LDT) nephron segments. The results suggest that AQP3-2 may be involved in cell volume regulation in the EDT and water and urea absorption in the LDT nephron segment.

Tandem upregulation of ion transporters in thick ascending limb of Henle's loop of young Milan hypertensive strain of rats.

Milan hypertensive strain (MHS) of rat represents as one of the ideal rat models to study the genetic form of hypertension associated with aberrant renal salt reabsorption. In contrast to Milan normotensive strain (MNS), MHS rats possess missense mutations in three adducin genes and develop hypertension at 3 months old due to upregulation of sodium-chloride cotransporter (NCC). At pre-hypertensive stage (23-25 days old), MHS rats show enhanced protein abundance of Na+-K+-2Cl- cotransporter (NKCC2) but retain blood pressure comparable to MNS probably through enhanced GFR and reduced NCC and α-subunit of epithelial sodium channel (α-ENaC) expressed in distal convoluted tubule (DCT) and collecting duct (CD). In the present study, mRNA and protein expressions of ion transporters in thick ascending limb of Henle's loop (TAL) of young MHS rats were investigated. Protein abundance of core-glycosylated form of renal outer medullary potassium channel (ROMK) in inner stripe of outer medulla (ISOM) is remarkably increased in MHS rats at prehypertensive stage. Furthermore, basolaterally expressed Na+-K+-ATPase and Barttin were upregulated. These results may indicate that in TAL of MHS rats at this age, both total NKCC2 and core-glycosylated ROMK are upregulated in tandem potentially to balance the luminal potassium concentration. On the basolateral side, upregulation of Na+-K+-ATPase and CLC-Ka/b may energize the excretion of sodium and chloride out from the cells. These data may suggest the interplay of apical and basolateral ion transporters in TAL for the modulation of TAL function in favor of enhancing the transepithelial sodium reabsorption, although this seems compensated by NCC and ENaC expressed at the downstream nephron segments in young MHS rats.

GLUT1 and prorenin receptor mediate differential regulation of TGF-β and CTGF in renal inner medullary collecting duct cells during high glucose conditions

BackgroundDuring diabetes, prorenin is highly produced by the renal collecting ducts. The binding of prorenin to (pro)renin receptor (PRR) on the apical plasma membrane triggers intracellular profibrotic genes, including TGF-β and CTGF. However, the underlying mechanisms contributing to the stimulation of these pathways remain unclear. Hence, we hypothesize that the glucose transporter-1 (GLUT1) favors the PRR-dependent stimulation of TGF-β and CTGF in the distal nephron segments during high glucose (HG) conditions.MethodsTo test this hypothesis, primary cultured renal inner medullary collecting duct (IMCD) cells were treated with normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) for 48 h in the presence or absence of the GLUT1-specific inhibitor BAY 876 (2 nM). Additionally, IMCD cells were treated with the PRR antagonist PRO20. The expression of TGF-β and CTGF was quantified by immunoblot and qRT-PCR.ResultsHG increased GLUT1 mRNA and protein abundance, while BAY 876 inhibited these responses. HG treatment upregulated PRR, but the concomitant treatment with BAY 876 partially prevented this effect. TGF-β and CTGF expressions were augmented in IMCD cells treated with HG. However, PRO20 prevented the increases in TGF-β but not those of CTGF. GLUT1 inhibition partially prevented the increases in reactive oxygen species (ROS) during HG while PRO20 did not. ROS scavenging impaired CTGF upregulation during HG conditions. Additionally, long-term exposure to HG increases lipid peroxidation and reduced cell viability.ConclusionsThe data indicate that glucose transportation via GLUT1 is implicated in the PRR-dependent upregulation of TGF-β while CTGF is mediated mainly via a mechanism depending on ROS formation in renal medullary collecting duct cells.

Dynamic Kidney Organoid Microphysiological Analysis Platform.

Kidney organoids, replicating human development, pathology, and drug responses, are a promising model for advancing bioscience and pharmaceutical innovation. However, reproducibility, accuracy, and quantification challenges hinder their broader utility for advanced biological and pharmaceutical applications. Herein, we present a dynamic kidney organoid microphysiological analysis platform (MAP), designed to enhance organoid modeling and assays within physiologically relevant environments, thereby expanding their utility in advancing kidney physiology and pathology research. First, precise control of the dynamic microenvironment in MAP enhances the ability to fine-tune nephrogenic intricacies, facilitating high-throughput and reproducible human kidney organoid development. Also, MAP's miniaturization of kidney organoids significantly advances pharmaceutical research by allowing for detailed analysis of entire nephron segments, which is crucial for assessing the nephrotoxicity and safety of drugs. Furthermore, the MAP's application in disease modeling faithfully recapitulates pathological development and functions as a valuable testbed for therapeutic exploration in polycystic kidney diseases. We envision the kidney organoid MAP enhancing pharmaceutical research, standardizing processes, and improving analytics, thereby elevating the quality and utility of organoids in biology, pharmacology, precision medicine, and education.

Beyond hemoglobin: Critical role of 2,3-bisphosphoglycerate mutase in kidney function and injury.

2,3-bisphosphoglycerate mutase (BPGM) is traditionally recognized for its role in modulating oxygen affinity to hemoglobin in erythrocytes. Recent transcriptomic analyses, however, have indicated a significant upregulation of BPGM in acutely injured murine and human kidneys, suggesting a potential renal function for this enzyme. Here we aim to explore the physiological role of BPGM in the kidney. A tubular-specific, doxycycline-inducible Bpgm-knockout mouse model was generated. Histological, immunofluorescence, and proteomic analyses were conducted to examine the localization of BPGM expression and the impact of its knockout on kidney structure and function. Invitro studies were performed to investigate the metabolic consequences of Bpgm knockdown under osmotic stress. BPGM expression was localized to the distal nephron and was absent in proximal tubules. Inducible knockout of Bpgm resulted in rapid kidney injury within 4 days, characterized by proximal tubular damage and tubulointerstitial fibrosis. Proteomic analyses revealed involvement of BPGM in key metabolic pathways, including glycolysis, oxidative stress response, and inflammation. Invitro, Bpgm knockdown led to enhanced glycolysis, decreased reactive oxygen species elimination capacity under osmotic stress, and increased apoptosis. Furthermore, interactions between nephron segments and immune cells in the kidney suggested a mechanism for propagating stress signals from distal to proximal tubules. BPGM fulfills critical functions beyond the erythrocyte in maintaining glucose metabolism in the distal nephron. Its absence leads to metabolic imbalances, increased oxidative stress, inflammation, and ultimately kidney injury.

Morphometric Analysis of Different Nephron Segments in Patients with Diabetic Nephropathy and Overt Proteinuria

Morphometric Analysis of Different Nephron Segments in Patients with Diabetic Nephropathy and Overt Proteinuria

UT-1 Transporter Expression in the Spiny Dogfish (Squalus acanthias): UT-1 Protein Shows a Different Localization in Comparison to That of Other Sharks.

The original UT-1 transporter gene was initially identified in the spiny dogfish (Squalus acanthias), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.

Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology. NEW & NOTEWORTHY This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.

Abstract P391: Role of Renal Dopamine D2 Receptor in Inverse Salt Sensitivity and Salt Sensitivity of Blood Pressure: Insights from Nephron Segment-Specific Deletion Models

The renal dopamine D2 receptor (D2R) is essential in renal and blood pressure (BP) homeostasis. In mice, global Drd2 deletion or renal-selective Drd2 silencing increases the renal expression of proinflammatory factors, renal injury markers, and BP. The D2R is expressed in several nephron segments, including the proximal and distal convoluted tubules. To study the differential effects of D2R in these segments, we generated two mouse models: one with Drd2 deletion only in the S1 and S2 segments of the proximal tubule P SGLT2 ::Cre+ Drd2 fl/fl ( Sglt2 cKO ) and their littermates as controls, P SGLT2 ::Cre- Drd2 fl/fl (Sglt2 WT) and another with deletion of the receptor in the whole nephron P CDHR16 ::Cre+ Drd2 fl/fl ( Cdhr16 cKO ) and their littermates as controls, P CDHR16 ::Cre- Drd2 fl/fl (Cdhr16 WT). In male Sglt2 cKO mice, compared with Sglt2 WT mice, D2R expression in the renal cortex was decreased by 25% (P<0.05, n=5-6/group). In Cdhr16 cKO mice, compared with Cdhr16 WT mice, D2R expression was decreased by 30% (P<0.05; n=3/group). In Sglt2 cKO mice, tubular lumen expansion, an indication of tubular damage, was higher on low salt (0.09%NaCl; LS) than on normal salt (0.4%NaCl; NS) (25.5±.8 vs. 11.4±.0.3; P<0.001) diet. In Cdhr16 cKO mice tubular lumen expansion was also higher on LS than NS (13.9±0.4 vs 8.7±0.2; P<0.001) diet. In Sglt2 cKO mice, BP (carotid artery under anesthesia) was higher on LS (130±2 mm Hg) than on NS (107±2 mm Hg) or HS (4% NaCl) (100±3 mm Hg) (n=10/group) (P<0.05) diet. In Cdhr16 cKO mice, BP (tail cuff, CODA under anesthesia, confirmed by telemetry) was higher on LS (110±8 mm Hg, P<0.02; n=4-5/group) and HS (119±9 mm Hg, P<0.05; n=4/group) diets than on NS diet (84±5 mmHg). On LS diet, UNaV was lower in Sglt2 cKO than Sglt2 WT (1±0.3 vs 7±2 µEq/24h; P< 0.05; n=4-6/group) mice. On LS diet, UNaV was also lower in Cdhr16 cKO than Cdhr16 WT (5±0.9 vs 34±14 µEq/24h; P<0.05; n=3-4/group) mice. These results indicate that a decrease in D2R expression only in the proximal tubule results in inverse salt sensitivity of BP, i.e., BP higher on LS than NS or HS diets, while downregulation of its expression in the whole nephron results in inverse salt sensitivity, as well as salt sensitivity.

Paradoxes in magnesium transport in type 1 Bartter's syndrome and Gitelman's syndrome: a modeling analysis.

Type 1 Bartter's syndrome and Gitelman's syndrome are characterized by mutations in two key renal Na+ transporters, Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC). Since these two transporters play an important role in regulating magnesium (Mg2+) and calcium (Ca2+) transport in the kidney, significant alterations in the transport of these two electrolytes are observed in type 1 Bartter's syndrome and Gitelman's syndrome. In this study, we used our sex-specific computational models of renal electrolyte transport in rats to understand the complex compensatory mechanisms, in terms of alterations in tubular dimensions and ion transporter activities, that lead to Mg2+ and Ca2+ preservation or wasting in these two genetic disorders. Given the sexual dimorphism in renal transporter patterns, we also assessed how the magnitude of these alterations may differ between males and females. Model simulations showed that in type 1 Bartter's syndrome, nephron adaptations prevent salt wasting and favor Mg2+ preservation but not Ca2+, whereas in Gitelman's syndrome, those adaptations favor Ca2+ preservation over Mg2+. In addition, our models predicted that the compensatory alterations in tubular dimensions and ion transporter activities are stronger in females than in males.NEW & NOTEWORTHY Although changes in Ca2+ excretion in type 1 Bartter's syndrome and Gitelman's syndrome are well understood, Mg2+ excretion displays an interesting paradox. This computational modeling study provides insights into how renal adaptations in these two disorders impact Ca2+ and Mg2+ transport along different nephron segments. Model simulations showed that nephron adaptations favor Mg2+ preservation over Ca2+ in Bartter's syndrome and Ca2+ preservation over Mg2+ in Gitelman's syndrome and are stronger in females than in males.

Abstract 04: Renal- and TAL-Specific Inhibition of Traf2 and NCK Interacting Kinase (TNIK) Decreases NKKC2 Phosphorylation and Blunts Salt-Sensitive Hypertension in Dahl Salt-Sensitive Rats

Up to 50% of hypertensive patients are salt-sensitive. Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) in the kidney contributes to the development of salt-sensitivity. NaCl reabsorption in the TAL is entirely mediated by the apical Na/K/2Cl cotransporter NKCC2. We showed that NKCC2 phosphorylation at Thr-96,101 is increased in TALs from Dahl salt-sensitive (DSS) rats. We discovered a new kinase, TNIK (Traf2-and-NCK-interacting kinase), that binds the amino-terminus of NKCC2 and phosphorylates NKCC2 at Thr-96,101. TNIK is abundantly expressed in TALs and other nephron segments. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 phosphorylation in TALs from DSS rats. First, we measured TNIK expression in isolated TALs. TNIK expression was 8±4-fold higher in TALs from DSS compared to SD rats (n=5, p<0.05). Then, we used a pharmacological specific inhibitor of TNIK, NCB-0846. We implanted renal medullary catheters connected to minipumps to directly infuse NCB-0846 or vehicle into the left kidney of uninephrectomized DSS rats. After recovery rats were fed 4% NaCl diet and measured systolic blood pressure (SBP) via tail-cuff. NCB-0846 decreased SBP by 21±6 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs vehicle). To specifically decrease TNIK gene expression in medullary TALs, we used Cas9-mediated gene editing. We injected the renal medulla of the left kidney with AAV-gRNA-TNIK simultaneously with AV-NKCC2promoter-Cas9 (drives Cas9 expression in TALs) or AV-NKCC2promoter-Cas9 alone (AV-control) in DSS rats. After 10 days, rats were fed a 4% NaCl diet and SBP was measured. Rats transduced with AAV-gRNA-TNIK had a 18±5 mmHg lower BP compared to AV-control group (AV-control:147 ± 4.7 vs AAV-gRNA-TNIK:129 ± 5.6 mmHg n=5, p<0.03 vs control) which remained lower after 14 days (AV-control:163 ± 4.8 vs AAV-gRNA-TNIK:140 ± 4.9 mmHg, n=5, p<0.01). After 14 days on high salt, TNIK expression was decreased by 30 ± 4% (n=4, p<0.05), and pThr-96,101-NKCC2 decreased by 43 ± 11% in AAV-gRNA-TNIK transduced TALs compared to AV-control transduced TALs (n=4, p<0.02). We conclude that hypertension and enhanced NKCC2 phosphorylation in the DSS rat is partly due to higher TNIK expression in TALs and that inhibition of this novel kinase could be a target for salt-sensitive hypertension.

Abstract 60: Atlas of Distal Nephron Mineralocorticoid Receptor-Dependent Transcriptome Reveals Novel Aldosterone Actions

Aldosterone regulation of Na+ and K+ balance and blood pressure are mediated by the mineralocorticoid receptor (MR, Nr3c2). Although several aldosterone/MR-dependent genes have been identified, their regulation cannot fully explain how aldosterone activates electrogenic Na+- K+ exchange in the aldosterone-sensitive distal nephron (ASDN). Here, we apply RNA-Seq and comprehensive downstream analysis to define the complete inventory of MR-regulated genes. MRfl/fl/Pax8,rtTA/LC1 mice were used to knock out the Nr3c2 gene in an inducible and kidney-specific manner. RNA-Seq analysis was conducted in the micro-dissected connecting tubule and cortical collecting duct tubule segments, followed by multimodal bioinformatic analyses. Direct MR-dependent gene regulation was validated in vitro on immortalized collecting duct cells, ex vivo with isolated distal nephron segments and gene reporter assays. All known aldosterone-response genes, including Sgk1, Scnn1a, Ndrg2, Per3, Tsc22d3, Zbtb16, MLPH, and Atp1a1 were significantly decreased in MR KO mice. 1193 additional genes were down-regulated in the MR KO, and 526 of these have a Mineralocorticoid Responsive Element (MRE), revealing the MR-dependent transcriptome is much larger than previously imagined. MREs were found in most genes with closed chromatin regions, suggesting MR binding might pioneer transcription for most of its regulated genes. The architecture of some MR-regulated genes, highlighted by the co-regulation of 7 Kallikrein genes, points to a novel mechanism of MR-regulation involving epigenetic remodeling in topology-associated domains. Pathway enrichment analysis revealed that MR-regulated genes are highly enriched in mitochondria-associated metabolic processes, and direct MR regulation of Klf15 was identified as a key transcriptional driver. In summary, the inventory of MR-regulated genes in the ASDN is much larger than previously imagined, involving new mechanisms of gene regulation. In addition to pathways that directly up-regulate transport, the data suggest that aldosterone-MR may directly influence metabolism to make energy-consuming transport highly efficient.

Abstract 08: Genome-wide Maps of Human Kidney Proximal Tubules and Thick Ascending Limbs of Henle reveal an Enrichment for Human Blood Pressure associated Polymorphisms

GWAS has identified >1,000 blood pressure (BP)-associated sentinel SNPs, most of which (>90%) are intronic and intergenic. How they regulate BP-relevant genes remains an open question. We hypothesized many SNPs would reside within cis -regulatory elements specific to nephron segments critical to sodium resorption such as proximal tubule (PT) and thick ascending limb (TAL). To address this, we developed chromatin state (ATAC-seq) and transcriptome (RNA-seq) maps from manually dissected human PT and medullary TAL (mTAL) segments. We identified 216,168 and 179,467 open regions in PT and mTAL, respectively, with the majority (70%) being unique to one tissue. Interestingly, peaks shared between the two segments resided more frequently in promoters, whereas unique peaks were more often found in distal introns or intergenic regions, suggesting they lie within enhancers ( Figure 1A ). Both chromatin state and transcriptomic data demonstrated a correlation between open chromatin peaks within gene promoters and mRNA levels (r=0.11; p<4.6e-16). This correlation was weaker at open regions distal to the promoter, illustrating the difficulty of mapping distal open regions to the gene(s) they regulate. We hypothesized these open regions would be enriched for BP-relevant SNPs. We curated 1,071 bp-associated SNPs and found 87 and 67 in mTAL and PT, open regions respectively, many of which are near BP-relevant genes. This enrichment was significant based on 10 4 randomizations (pValue 10 -4 ) ( Figure 1B ). We conclude that these novel tissue-specific chromatin maps can be used to identify potential mechanisms by which SNPs influence gene expression and BP. Additional studies will be needed to demonstrate both causality and molecular mechanisms.

The association between renal medullary and cortical fibrosis, stiffness, and concentrating capacity: an observational, single-center cross-sectional study.

Fibrosis is a common final pathway leading to end-stage renal failure. As the renal medulla and cortex contain different nephron segments, we analyzed the factors associated with the progression of renal medullary and cortical fibrosis. A total of 120 patients who underwent renal biopsy at Kawashima Hospital between May 2019 and October 2022 were enrolled in this retrospective study. Renal medullary and cortical fibrosis and stiffness were evaluated using Masson's trichrome staining and shear wave elastography, respectively. Maximum urine osmolality in the Fishberg concentration test was also examined. Medullary fibrosis was positively correlated with cortical fibrosis (p < 0.0001) and log-converted urinary β2-microglobulin (MG) (log urinary β2-MG) (p = 0.022) and negatively correlated with estimated glomerular filtration rate (eGFR) (p = 0.0002). Cortical fibrosis also correlated with log urinary β2-MG, eGFR, and maximum urine osmolality. Multivariate analysis revealed that cortical fibrosis levels (odds ratio [OR]: 1.063) and medullary stiffness (OR: 1.089) were significantly associated with medullar fibrosis (≧45%). The severe fibrosis group with both medullary fibrosis (≧45%) and cortical fibrosis (≧25%) had lower eGFR and maximum urine osmolality values and higher urinary β2-MG levels than the other groups. Patients with disorders involving both renal medullary and cortical fibrosis had decreased maximum urine osmolality but had no abnormalities in the urinary concentrating capacities with either condition. Renal medullary and cortical fibrosis were positively correlated with urinary β2-MG, but not with urinary N-acetyl-beta-D-glucosaminidase.

Three Dimensional Multiscalar Neurovascular Nephron Connectivity Map of the Human Kidney Across the Lifespan.

The human kidney is a vital organ with a remarkable ability to coordinate the activity of up to a million nephrons, its main functional tissue unit (FTU), and maintain homeostasis. We developed tissue processing and analytical methods to construct a 3D map of neurovascular nephron connectivity of the human kidney and glean insights into how this structural organization enables coordination of various functions of the nephron, such as glomerular filtration, solute and water absorption, secretion by the tubules, and regulation of blood flow and pressure by the juxtaglomerular apparatus, in addition to how these functions change across disease and lifespans. Using light sheet fluorescence microscopy (LSFM) and morphometric analysis we discovered changes in anatomical orientation of the vascular pole, glomerular density, volume, and innervation through postnatal development and ageing. The extensive nerve network exists from cortex FTUs to medullary loop of Henle, providing connectivity within segments of the same nephron, and between separate nephrons. The nerves organize glomeruli into discreet communities (in the same network of nerves). Adjacent glomerular communities are connected to intercommunal "mother glomeruli" by nerves, a pattern repeating throughout the cortex. These neuro-nephron networks are not developed in postnatal kidneys and are disrupted in diseased kidneys (diabetic or hydronephrosis). This structural organization likely poises the entire glomerular and juxtaglomerular FTUs to synchronize responses to perturbations in fluid homeostasis, utilizing mother glomeruli as network control centers.

Galectin-3 protects distal convoluted tubules in rhabdomyolysis-induced kidney injury

Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.

Spatial proteomics of human diabetic kidney disease, from health to class III.

Diabetic kidney disease (DKD) is the leading cause of chronic and end-stage kidney disease in the USA and worldwide. Animal models have taught us much about DKD mechanisms, but translation of this knowledge into treatments for human disease has been slowed by the lag in our molecular understanding of human DKD. Using our Spatial TissuE Proteomics (STEP) pipeline (comprising curated human kidney tissues, multiplexed immunofluorescence and powerful analysis tools), we imaged and analysed the expression of 21 proteins in 23 tissue sections from individuals with diabetes and healthy kidneys (n=5), compared to those with DKDIIA, IIA-B and IIB (n=2 each) and DKDIII (n=1). These analyses revealed the existence of 11 cellular clusters (kidney compartments/cell types): podocytes, glomerular endothelial cells, proximal tubules, distal nephron, peritubular capillaries, blood vessels (endothelial cells and vascular smooth muscle cells), macrophages, myeloid cells, other CD45+ inflammatory cells, basement membrane and the interstitium. DKD progression was associated with co-localised increases in inflammatory cells and collagen IV deposition, with concomitant loss of native proteins of each nephron segment. Cell-type frequency and neighbourhood analyses highlighted a significant increase in inflammatory cells and their adjacency to tubular and αSMA+ (α-smooth muscle actin-positive) cells in DKD. Finally, DKD progression showed marked regional variability within single tissue sections, as well as inter-individual variability within each DKD class. Using the STEP pipeline, we found alterations in protein expression, cellular phenotypic composition and microenvironment structure with DKD progression, demonstrating the power of this pipeline to reveal the pathophysiology of human DKD.

Cryopreservation of human kidney organoids

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1’s efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.