**Author details**

**Figure 4.** Antibody-mediated rejection. Two of the three criteria required for AMR. (A to C) Microvascular inflammation: glomerulitis, peritubular capilaritis, and intimal arteritis. PAS and H&E 40×. (D) Linear staining of C4d in peritubular capillaries IP 40×. Courtesy of Dr. Claudia Mendoza-Cerpa, Laboratory of Pathology, IMSS-CMNO, Guadalajara; México.

antibodies directed primarily at the vasculature of the donor organ [72]. These antibodies activate the complement cascade and induce neutrophil infiltration, endothelial damage, interstitial hemorrhage (**Figure 3**), edema, fibrin deposition, platelet aggregation, and thrombosis; causing the organ to fail within a few hours after transplantation. Hyperacute rejection used to be a frequent occurrence in transplantation before cross-match tests were designed to screen potential recipients for circulating anti-HLA antibodies to the prospec-

Antibody-mediated rejection (ABMR) pathogenesis involves mechanisms of graft injury caused by donor-specific anti-HLA antibodies (DSAs) and non-HLA antibodies; and has been associated with progressive decline in graft function and poor transplantation outcomes [74]. Molecular changes in the microvasculature characteristic of tissue remodeling and repair are common manifestations of ABMR, as well as neutrophilic infiltration and fibrosis (**Figure 4**) [50]. ABMR can be acute or chronic, and can also manifest in cases of mixed TCMR/AMBR rejection [2]. It is estimated that close to 20% of renal allograft recipients will develop *de novo* DSAs within 10 years posttransplant [75]. DSAs bind to allogenic HLA and non-HLA targets

tive donor [73].

152 Pathophysiology - Altered Physiological States

Zesergio Melo1,2\*, Juan A. Ruiz-Pacheco1 , Claudia A. Mendoza-Cerpa2 and Raquel Echavarria<sup>1</sup>


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158 Pathophysiology - Altered Physiological States


**Chapter 9**

**Provisional chapter**

**The Way from Renal Calcifications and Urinary Crystals**

The formation of calcium (Ca) stones occurs in an initial phase by fixed growth on kidney calcifications consisting either of intratubular crystal accumulations protruding in renal calices (Randall's plugs) or of interstitial hydroxyapatite deposits (Randall's plaques) broken through the covering epithelial layers. Crystal aggregation (AGN) seems to be responsible for stone growth during crystalluria. This chapter reports on new aspects of the AGN of calcium oxalate being the most frequent stone compound and tries to explain why despite the widespread occurrence of kidney calcifications and crystalluria not everybody forms stones. Urinary crystals normally are protected from AGN by coats of urinary macromolecules (UMs) which by their identical electronegative charge create zones of electrostatic repulsion. At high urinary concentration or ionic strength respectively, these zones are compressed and can be bridged by self-aggregated UMs. Self-AGN occurs in concentrated urine by the adsorption of UMs on free surfaces like Randall's plugs or plaques. High oxalate excretion and high urine concentration favoring intratubular crystal accumulation, breaking of epithelial layers on Randall's plaques and self-AGN of UMs are most deleterious factors in Ca stone formation and have to be

**Crystals to Kidney Stones: An Important Aspect in the** 

DOI: 10.5772/intechopen.70598

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

The pathogenesis of kidney stones that often are accompanied by very painful colic and can lead to renal failure and even to the loss of a kidney is far from being clear. In Western

**Keywords:** calcium nephrolithiasis, crystalluria, Randall's plaques and plugs, urinary

**to Kidney Stones: An Important Aspect in the**

**The Way from Renal Calcifications and Urinary** 

**Pathogenesis of Calcium Nephrolithiasis**

**Pathogenesis of Calcium Nephrolithiasis**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70598

avoided by stone metaphylaxis.

macromolecules, calcium oxalate aggregation

Johannes M. Baumann

**Abstract**

**1. Introduction**

Johannes M. Baumann

**Provisional chapter**

#### **The Way from Renal Calcifications and Urinary Crystals to Kidney Stones: An Important Aspect in the Pathogenesis of Calcium Nephrolithiasis Crystals to Kidney Stones: An Important Aspect in the Pathogenesis of Calcium Nephrolithiasis**

**The Way from Renal Calcifications and Urinary** 

DOI: 10.5772/intechopen.70598

Johannes M. Baumann Additional information is available at the end of the chapter

Johannes M. Baumann

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70598

#### **Abstract**

The formation of calcium (Ca) stones occurs in an initial phase by fixed growth on kidney calcifications consisting either of intratubular crystal accumulations protruding in renal calices (Randall's plugs) or of interstitial hydroxyapatite deposits (Randall's plaques) broken through the covering epithelial layers. Crystal aggregation (AGN) seems to be responsible for stone growth during crystalluria. This chapter reports on new aspects of the AGN of calcium oxalate being the most frequent stone compound and tries to explain why despite the widespread occurrence of kidney calcifications and crystalluria not everybody forms stones. Urinary crystals normally are protected from AGN by coats of urinary macromolecules (UMs) which by their identical electronegative charge create zones of electrostatic repulsion. At high urinary concentration or ionic strength respectively, these zones are compressed and can be bridged by self-aggregated UMs. Self-AGN occurs in concentrated urine by the adsorption of UMs on free surfaces like Randall's plugs or plaques. High oxalate excretion and high urine concentration favoring intratubular crystal accumulation, breaking of epithelial layers on Randall's plaques and self-AGN of UMs are most deleterious factors in Ca stone formation and have to be avoided by stone metaphylaxis.

**Keywords:** calcium nephrolithiasis, crystalluria, Randall's plaques and plugs, urinary macromolecules, calcium oxalate aggregation

#### **1. Introduction**

The pathogenesis of kidney stones that often are accompanied by very painful colic and can lead to renal failure and even to the loss of a kidney is far from being clear. In Western

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

populations, nephrolithiasis has reached a prevalence up to 10% [1]. Kidney stones are composed of crystal aggregates within an organic matrix. Long times stone formation mainly was ascribed to a pathological excretion of substances being involved in crystal formation. Fourier transform infrared spectroscopy being now commonplace for stone analysis shows that calcium oxalate (CaOx) is the most frequent stone compound. For calcium nephrolithiasis, the most frequent stone disease and the topic of this chapter, the important substances are calcium, oxalic and citric acid the latter being a calcium chelator [2]. However, not all stone formers show a pathological excretion of these compounds, and some anomalies also are found in urine of people without stone formation. Later on, stone research was focused on urinary macromolecules (UMs, mainly proteins and some glycosaminoglycans) being an integral part of the stone matrix and some of them promoting or inhibiting the crystallization of stone minerals [3]. In the meantime, more than 70 of such substances were isolated [4, 5], and 11 of them containing anionic residues like carboxyglutamic acid, phosphate and sialic acid are thought to be relevant for stone formation [6]. But anomalies with respect to excretion or composition of these UMs were exclusively found in some small groups of patients [7].

protein-free solutions and with a long period of crystal ripening, CaOx was directly produced in urine by an oxalate titration [16]. Crystallization was followed monitoring optical density (OD) by a spectrophotometer. Typical crystallization curves are shown in **Figure 1A** and **C**. During a titration period of 15 min with the addition of 0.1 mM/min of sodium oxalate, a rapid increase of OD indicating crystal formation is observed. From the time lag of this increase, the critical oxalate addition to induce crystallization can be calculated and used as a measure for the metastability of urine with respect to CaOx crystallization. At the end of titration, maximal OD reflecting particle concentration in solutions [15] is determined, and magnetic stirring is stopped. Following the further course during at least 30 min, two different types of curves are observed. One type (**Figure 1A**) is characterized by a continuous slow OD decrease, which by scanning electron microscopy (SEM) of the sediment (**Figure 1B**) mainly can be attributed to the sedimentation of single crystals of CaOx monohydrate. From this low −mdOD/dt, the sedimentation rate of single crystals and an average particle size can be calculated [17]. The other type of curve is represented in **Figure 1C**. After an initial phase of slow OD decrease varying from 7 to 35 min and called suspensions stability, a rapid decline of OD is observed which by SEM of the sediment (**Figure 1D**) can be attributed to crystal AGN. Since OD mainly reflects particle concentration, the rapid OD decrease can be explained by an increased particle clearing in the observation field of the spectrophotometer. This high clearing bases on the one hand on an accelerated sedimentation of crystal aggregates (the sedimentation rate increases with particle diameter in square) and on the other hand on the diminution of particle concentration by the integration of a lot of single crystals into few large aggregates. Interestingly, the rapid

The Way from Renal Calcifications and Urinary Crystals to Kidney Stones: An Important Aspect...

http://dx.doi.org/10.5772/intechopen.70598

163

**Figure 1.** Crystallization curves (CC) of urine and scanning electron microscopy (SEM) of sediments: (A) urine with inhibition of CaOx AGN in CC (low maximal rate of OD decrease, −mdOD/dt) and (B) mainly with single crystals of CaOx monohydrate and only a few small aggregates in SEM, (C) urine with intensive AGN (high −mdOD/dt) in CC, and

(D) large aggregates predominating in SEM. Bars on SEM indicating 20 μm.

Modern urological endoscopy which allows the inspection of the whole renal cavity showed that calcium oxalate and phosphate stones comprising about 80% of all concretions [8] generally start by a fixed growth on papillary calcifications, which were already described as potential source of nephrolithiasis in 1937 by Randall [9]. Recently much work was done to elucidate the pathogenesis of these calcifications being present either as interstitial deposits of calcium phosphate (Randall's plaques) or as intratubular accumulation of calcium oxalate crystals (Randall's plugs) [10–12]. These calcifications when protruding into renal calyces can induce stone formation either by heterogeneous nucleation of new crystals or more probably by the aggregation (AGN) of crystals during crystalluria. The initial fixation on kidney calcifications allows stones to grow to a critical size where they cannot be washed out anymore by the urine flow and where they become symptomatic. However, the finding of kidney calcifications and crystalluria is much more frequent than stone disease [3], and Randall's plaques can persist during some decades without or with only minimal stone formation [13]. Therefore, the question raises whether special crystallization conditions in urine might be responsible for stone formation by the apposition of new crystals on Randall's plaques and plugs. This question stimulated us to an intensive study of the formation and especially the AGN of calcium oxalate crystals being with 60% the most frequent stone compound [8]. Experiments were directly performed in urine where like in other biological mediums, crystals as well as Randall's plaques and plugs always are coated by proteins [14]. Instead of the study of individual compounds thought to be involved in stone formation, the overall effect of UMs was compared to that one of urine and urinary ultrafiltrate. UMs were isolated either by a hemofiltration procedure or by temporary adsorption on Ca phosphate to which urinary proteins have a high affinity.

#### **2. Measurement of calcium oxalate (CaOx) crystallization in urine**

An approved test system which uses an increase of the rate of particle sedimentation as measure for crystal AGN was modified [15]. Contrary to current crystal production in standardized and protein-free solutions and with a long period of crystal ripening, CaOx was directly produced in urine by an oxalate titration [16]. Crystallization was followed monitoring optical density (OD) by a spectrophotometer. Typical crystallization curves are shown in **Figure 1A** and **C**. During a titration period of 15 min with the addition of 0.1 mM/min of sodium oxalate, a rapid increase of OD indicating crystal formation is observed. From the time lag of this increase, the critical oxalate addition to induce crystallization can be calculated and used as a measure for the metastability of urine with respect to CaOx crystallization. At the end of titration, maximal OD reflecting particle concentration in solutions [15] is determined, and magnetic stirring is stopped. Following the further course during at least 30 min, two different types of curves are observed. One type (**Figure 1A**) is characterized by a continuous slow OD decrease, which by scanning electron microscopy (SEM) of the sediment (**Figure 1B**) mainly can be attributed to the sedimentation of single crystals of CaOx monohydrate. From this low −mdOD/dt, the sedimentation rate of single crystals and an average particle size can be calculated [17]. The other type of curve is represented in **Figure 1C**. After an initial phase of slow OD decrease varying from 7 to 35 min and called suspensions stability, a rapid decline of OD is observed which by SEM of the sediment (**Figure 1D**) can be attributed to crystal AGN. Since OD mainly reflects particle concentration, the rapid OD decrease can be explained by an increased particle clearing in the observation field of the spectrophotometer. This high clearing bases on the one hand on an accelerated sedimentation of crystal aggregates (the sedimentation rate increases with particle diameter in square) and on the other hand on the diminution of particle concentration by the integration of a lot of single crystals into few large aggregates. Interestingly, the rapid

populations, nephrolithiasis has reached a prevalence up to 10% [1]. Kidney stones are composed of crystal aggregates within an organic matrix. Long times stone formation mainly was ascribed to a pathological excretion of substances being involved in crystal formation. Fourier transform infrared spectroscopy being now commonplace for stone analysis shows that calcium oxalate (CaOx) is the most frequent stone compound. For calcium nephrolithiasis, the most frequent stone disease and the topic of this chapter, the important substances are calcium, oxalic and citric acid the latter being a calcium chelator [2]. However, not all stone formers show a pathological excretion of these compounds, and some anomalies also are found in urine of people without stone formation. Later on, stone research was focused on urinary macromolecules (UMs, mainly proteins and some glycosaminoglycans) being an integral part of the stone matrix and some of them promoting or inhibiting the crystallization of stone minerals [3]. In the meantime, more than 70 of such substances were isolated [4, 5], and 11 of them containing anionic residues like carboxyglutamic acid, phosphate and sialic acid are thought to be relevant for stone formation [6]. But anomalies with respect to excretion or composition

Modern urological endoscopy which allows the inspection of the whole renal cavity showed that calcium oxalate and phosphate stones comprising about 80% of all concretions [8] generally start by a fixed growth on papillary calcifications, which were already described as potential source of nephrolithiasis in 1937 by Randall [9]. Recently much work was done to elucidate the pathogenesis of these calcifications being present either as interstitial deposits of calcium phosphate (Randall's plaques) or as intratubular accumulation of calcium oxalate crystals (Randall's plugs) [10–12]. These calcifications when protruding into renal calyces can induce stone formation either by heterogeneous nucleation of new crystals or more probably by the aggregation (AGN) of crystals during crystalluria. The initial fixation on kidney calcifications allows stones to grow to a critical size where they cannot be washed out anymore by the urine flow and where they become symptomatic. However, the finding of kidney calcifications and crystalluria is much more frequent than stone disease [3], and Randall's plaques can persist during some decades without or with only minimal stone formation [13]. Therefore, the question raises whether special crystallization conditions in urine might be responsible for stone formation by the apposition of new crystals on Randall's plaques and plugs. This question stimulated us to an intensive study of the formation and especially the AGN of calcium oxalate crystals being with 60% the most frequent stone compound [8]. Experiments were directly performed in urine where like in other biological mediums, crystals as well as Randall's plaques and plugs always are coated by proteins [14]. Instead of the study of individual compounds thought to be involved in stone formation, the overall effect of UMs was compared to that one of urine and urinary ultrafiltrate. UMs were isolated either by a hemofiltration procedure or by temporary adsorption on Ca phosphate to which urinary proteins

of these UMs were exclusively found in some small groups of patients [7].

**2. Measurement of calcium oxalate (CaOx) crystallization in urine**

An approved test system which uses an increase of the rate of particle sedimentation as measure for crystal AGN was modified [15]. Contrary to current crystal production in standardized and

have a high affinity.

162 Pathophysiology - Altered Physiological States

**Figure 1.** Crystallization curves (CC) of urine and scanning electron microscopy (SEM) of sediments: (A) urine with inhibition of CaOx AGN in CC (low maximal rate of OD decrease, −mdOD/dt) and (B) mainly with single crystals of CaOx monohydrate and only a few small aggregates in SEM, (C) urine with intensive AGN (high −mdOD/dt) in CC, and (D) large aggregates predominating in SEM. Bars on SEM indicating 20 μm.

OD decrease reflecting AGN stops after on average 30% of OD has disappeared by AGN [16]. Therefore, AGN in crystal suspensions seems to be limited to a critical OD or particle concentration. The maximal OD decrease observed in our experiments is expressed as maximal OD change per minute (−mdOD/dt).

For stone formation, crystals have to be retained in the kidney. This seems for single crystals hardly to be possible. In the nephron, Ox normally reaches a sufficient concentration for CaOx crystallization at the end of collecting ducts (CD), but at extremely high Ox concentrations, crystallization already can occur in the descending loop of Henle (DLH) [26]. Both situations are schematically indicated in **Figure 2**. Even at the high Ox concentrations necessary to induce crystal formation in DLH crystallization is a relative slow process compared to urinary transit time (UT) through the nephron. Following CaOx crystallization in urine by repeated measurement of the ionic Ca decay by a ion selective electrode shows that even after an extreme Ox addition of 1 mM crystallization reaches during an average transit time through the nephron of 3 min only about half of its final value (**Figure 3**). The figure demonstrates that the study of crystals in urine which previously has remained several times in the bladder hardly can be representative for the situation in the kidney. For the end of the nephron when crystals have passed inner tubular diameters of minimal 15–60 μm, maximal crystal diameters of 4 μm were calculated [27]. The discrepancy of crystals size and tubular dimensions is even more pronounced when crystallization starts at the end of collecting ducts where crystals only can grow during a few seconds until they reach the renal papilla (**Figure 2**). Therefore, for the formation of obstructing plugs, crystals have to aggregate as demonstrated

The Way from Renal Calcifications and Urinary Crystals to Kidney Stones: An Important Aspect...

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**Figure 2.** Localization of CaOx crystallization in the nephron (hatched areas): descending loop of Henle (DLH), collecting

), at RP (<sup>b</sup>

), minimal inner

duct (CD), renal papilla (RP), maximal crystal diameter (DCr) expected at nucleation site (<sup>a</sup>

tubular diameter (DT), urinary transit time (UT) to RP.

UM solutions and urinary ultrafiltrate (UF) were obtained using a hemofilter the excluding limit of the dialysis membrane being 5 kDa [18]. To gain UF urine was placed on one side of the membrane and the filtrate collected on the other side. UMs were isolated by dialyzing urine against bi distilled water. This procedure showed a volume recovery of 96% and thus allowed the isolation of UMs in their almost original concentration. UM solutions also were prepared by Ca phosphate precipitation in urine, which was induced by the addition of sodium hydrogen phosphate at pH 7.0 [19]. After centrifugation and discharge of the supernatant, the precipitate was dissolved in distilled water buffered to pH 5.0 and with a volume corresponding to the urine volume used for precipitation. To obtain comparable results experiments in urine, ultrafiltrate and UM solutions were performed at identical pH, Na, and Ca concentration.
